Patent Grant
11248269
The invention relates, in part, to methods and compositions for identifying pathogenic Vibrio parahaemolyticus contamination and infections.
Rare pathogenic variants of Vibrio parahaemolyticus, a ubiquitous yet typically harmless estuarine bacterium, can cause human gastric infections most often from the consumption of raw or improperly handled seafood, and wound infections from recreational aquatic activities [Daniels N A, et al. (2000) J Infect Dis 181: 1661-1666; Scallan E, et al. (2011) Emerg Infect Dis 2011 January DOI:10.3201/eid1701.P11101]. A better understanding of conditions that promote emergence and relative abundance of pathogens is necessary to develop appropriate strategies for disease prevention but an obstacle for the study of emergent pathogenic strains of V. parahaemolyticus is a lack of understanding of factors that define virulence and that could be used to detect pathogens within mostly non-pathogenic populations
According to one aspect of the invention, methods for identifying the status of a pathogenic V. parahaemolyticus in a sample are provided. The methods include: (a) detecting in a sample, a level of at least one ST36prp polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 21, 22, 23, 24, 25 or 26, or a variant thereof; (b) comparing the level of the detected ST36prp polynucleotide to a control level of the detected ST36prp polynucleotide; and (c) identifying the status of the pathogenic V. parahaemolyticus in the sample, based at least in part on a difference between the level of the detected ST36prp polynucleotide and the control level of the detected ST36prp polynucleotide. In some embodiments, a higher level of the detected ST36prp polynucleotide compared to the control level of the detected ST36prp polynucleotide identifies the status of the pathogenic V. parahaemolyticus as present in the sample. In certain embodiments, the control level of the detected ST36prp polynucleotide is zero. In some embodiments, the method also includes: (d) detecting in the sample a level of at least one of a ST36cps polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO:8, 9, 10, 27 or 28, or a variant thereof; (e) comparing the level of the detected ST36cps polynucleotide to a control level of the detected ST36cps polynucleotide; and (f) identifying the status of the pathogenic V. parahaemolyticus in the sample, based at least in part on the difference between the level of the detected ST36cps polynucleotide and the control level of the detected ST36cps polynucleotide. In some embodiments, a higher level of the detected ST36cps polynucleotide compared to the control level of the detected ST36cps polynucleotide identifies the status of the pathogenic V. parahaemolyticus as present in the sample. In some embodiments, the control level of the detected ST36cps polynucleotide is zero. In certain embodiments, the method also includes (g) detecting in the sample a level of at least one of a tlh polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO:11, 12, 13, 14, 29, 30 or 31, or a variant thereof; and (h) comparing the level of the detected tlh polynucleotide to a control level of the detected tlh polynucleotide. In some embodiments, a higher level of the detected tlh polynucleotide compared to the control level of the detected tlh polynucleotide aids in identifying the status of the pathogenic V. parahaemolyticus in the sample. In certain embodiments, the control level of the detected tlh polynucleotide is zero. In some embodiments, the method also includes (i) detecting in the sample a level of at least one of a tdh and trh polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO:15, 16, 17, 18, 19, 20, 32, 33, 34 or 35, or a variant thereof; and (j) comparing the level of the detected polynucleotide to a control level of the detected polynucleotide. In some embodiments, a higher level of the detected tdh or trh polynucleotide compared to the control level of the detected polynucleotide aids in identifying the status of the pathogenic V. parahaemolyticus in the sample. In some embodiments, the control level of the detected tdh or trh polynucleotide is zero. In certain embodiments, the method also includes: (k) detecting in the sample a level of at least one of a ST36flp polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO:36, 37, 38, 39 or 40, or a variant thereof; (1) comparing the level of the detected ST36flp polynucleotide to a control level of the detected ST36flp polynucleotide; and (m) identifying the status of the pathogenic V. parahaemolyticus in the sample, based at least in part on difference between the level of the detected ST36flp polynucleotide and the control level of the detected ST36flp polynucleotide. In some embodiments, a higher level of the detected ST36flp polynucleotide compared to the control level of the detected ST36flp polynucleotide aids in identifying the status of the pathogenic V. parahaemolyticus in the sample. In some embodiments, the control level of the detected ST36flp polynucleotide is zero. In certain embodiments, the variant of the SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 21, 22, 23, 24, 25 or 26 nucleic acid sequence has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 21, 22, 23, 24, 25 or 26, respectively. In some embodiments, the variant of the SEQ ID NO:8, 9, 10, 27 or 28 nucleic acid sequence has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:8, 9, 10, 27 or 28, respectively. In some embodiments, the variant of the SEQ ID NO:11, 12, 13, 14, 29, 30 or 31 nucleic acid sequence has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:11, 12, 13, 14, 29, 30 or 31, respectively. In some embodiments, the variant of the SEQ ID NO:15, 16, 17, 18, 19, 20, 32, 33, 34 or 35 nucleic acid sequence has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:15, 16, 17, 18, 19, 20, 32, 33, 34 or 35, respectively. In certain embodiments, the variant of the SEQ ID NO:36, 37, 38, 39 or 40 nucleic acid sequence has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:36, 37, 38, 39 or 40, respectively. In some embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence. In some embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence and the fragment has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the region of the nucleic acid sequence with which it aligns. In certain embodiments, the sample is obtained from a subject. In some embodiments, the sample is obtained from a substrate.
According to another aspect of the invention methods of identifying the status of a pathogenic V. parahaemolyticus in a sample are provided. The methods include: (a) detecting in a sample a level of at least one ST36cps polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO:8, 9, 10, 27 or 28, or a variant thereof; (b) comparing the level of the detected ST36cps polynucleotide to a control level of the detected ST36cps polynucleotide; and (c) identifying the status of a pathogenic V. parahaemolyticus in the sample, based at least in part on a difference in the level of the detected ST36cps polynucleotide and the control level of the detected ST36cps polynucleotide. In some embodiments, a higher level of the detected ST36cps polynucleotide compared to the control level of the detected ST36cps polynucleotide identifies the status of the pathogenic V. parahaemolyticus as present in the sample. In certain embodiments, the control level of the detected ST36cps polynucleotide is zero. In some embodiments, the method also includes detecting in the sample a level of at least one of a polynucleotide comprising a ST36prp nucleic acid sequence or variant thereof, a tlh nucleic acid sequence or variant thereof, a tdh nucleic acid sequence or variant thereof, a trh nucleic acid sequence or variant thereof, and an ST36flp nucleic acid sequence or variant thereof. In some embodiments, the variant of the SEQ ID NO: 8, 9, 10, 27, or 28 nucleic acid sequence has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:8, 9, 10, 27 or 28, respectively. In certain embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence. In some embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence and the fragment has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the region of the nucleic acid sequence with which it aligns. In some embodiments, the sample is obtained from a subject. In some embodiments, the sample is obtained from a substrate.
According to yet another aspect of the invention, methods of identifying the presence of a pathogenic V. parahaemolyticus in a sample are provided. The methods include (a) detecting in a sample a level of at least one ST36flp polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO: 36, 37, 38, 39 or 40, or a variant thereof; (b) comparing the level of the detected ST36flp polynucleotide to a control level of the detected ST36flp polynucleotide; and (c) identifying the status of a pathogenic V. parahaemolyticus in the sample, based at least in part on the difference between the level of the detected ST36flp polynucleotide and the control level of the detected ST36flp polynucleotide. In certain embodiments, a higher level of the detected ST36flp polynucleotide compared to the control level of the detected ST36flp polynucleotide identifies the status of the pathogenic V. parahaemolyticus as present in the sample. In some embodiments, the control level of the detected ST36flp polynucleotide is zero. In some embodiments, the method also includes detecting in the sample a level of at least one of a polynucleotide comprising a ST36prp nucleic acid sequence or variant thereof, a tlh nucleic acid sequence or variant thereof, a tdh nucleic acid sequence or variant thereof, a trh nucleic acid sequence or variant thereof, and an ST36cps nucleic acid sequence or variant thereof. In certain embodiments, the variant of the SEQ ID NO:36, 37, 38, 39 or 40 nucleic acid sequence has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:36, 37, 38, 39 or 40, respectively. In some embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence. In some embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence and the fragment has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the region of the nucleic acid sequence with which it aligns. In some embodiments, the sample is obtained from a subject. In certain embodiments, the sample is obtained from a substrate.
According to another aspect of the invention, methods of assaying a sample are provided. The methods include (a) detecting in a sample determined to have at least one tdh and trh polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO: 15, 16, 17, 18, 19, 20, 32, 33, 34, 35, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 or 121, or a variant thereof, and a level of at least one: (i) ST36prp polynucleotide comprising at least one of the nucleic acid sequences set forth as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 21, 22, 23, 24, 25, 26, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 or 139, or a variant thereof; (ii) ST36flp polynucleotide comprising at least one of the nucleic acid sequences set forth as SEQ ID NO: 36, 37, 38, 39, 40, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, or a variant thereof; (iii) ST631end polynucleotide comprising at least one of the nucleic acid sequences set forth as: SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162 or 163, or a variant thereof; and (iv) 631-ENV polynucleotide comprising at least one of the nucleic acid sequences set forth as: SEQ ID NO: 59, 60, 61, 62, 170, 171, 172, 173, 174 or 175, or a variant thereof; and (b) comparing the level of the detected polynucleotide in at least one of steps (i), (ii), (iii), and (iv) to a control level of the detected polynucleotide in steps (i), (ii), (iii), and (iv), respectively. In some embodiments, the method also includes (c) determining the presence of a difference between the detected polynucleotide level and the control level of the detected polynucleotide as compared in (b); and (d) identifying the presence or absence of the pathogenic V. parahaemolyticus in the sample, based at least in part on difference between the level of the detected polynucleotide of at least one of steps (i), (ii), (iii), and (iii) and the respective control level of the detected polynucleotide. In some embodiments, assaying the sample comprises identifying the presence of a pathogenic V. parahaemolyticus in a sample. In some embodiments, the method also includes detecting an additional amplification control polynucleotide in the sample. In certain embodiments, the nucleic acid sequence of the variant of the tdh, trh, ST36prp, ST36fl, ST631end and ST631-ENV polynucleotide has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of the polynucleotide of which it is a variant thereof. In some embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence. In some embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence and the fragment has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the region of the nucleic acid sequence with which it aligns. In some embodiments, the sample is obtained from at subject. In certain embodiments, the sample is obtained from a substrate.
According to another aspect of the invention, methods of identifying the status of a pathogenic V. parahaemolyticus in a sample are provided. The methods including: (a) detecting in a sample a level of at least one of (a) a ST36Phage polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO 81, 82, 83 or 84, or a or a variant thereof; (b) a ST36PhHypD-Orf9 polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO:94, 95, 96 or 97, or a variant thereof; (c) a ST36NEOrf10-Hyp polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO: 87, 88, 89 or 90, or a variant thereof; and (d) a tlh polynucleotide comprising a nucleic acid sequence set forth as SEQ ID NO: 12 or 14, or a variant thereof; (b) comparing the level of the detected ST36Phage polynucleotide, ST36PhHypD-Orf9 polynucleotide, ST36NEOrf10-Hyp polynucleotide, and tlh polynucleotide to a control level of the detected ST36Phage polynucleotide, ST36PhHypD-Or19 polynucleotide, ST36NEOrf10-Hyp polynucleotide, and tlh polynucleotide, respectively; and (c) identifying the status of a pathogenic V. parahaemolyticus in the sample, based at least in part on the difference between the level of the detected ST36Phage polynucleotide, ST36PhHypD-Or19 polynucleotide, ST36NEOrf10-Hyp polynucleotide, and tlh polynucleotide, and the control level of ST36Phage polynucleotide, ST36PhHypD-Orf9 polynucleotide, ST36NEOrf10-Hyp polynucleotide, and tlh polynucleotide, respectively. In some embodiments, a higher level of the detected ST36Phage polynucleotide compared to the control level of the detected ST36Phage polynucleotide and the presence of a higher level of one or more of the ST36PhHypD-Orf9 polynucleotide and the ST36NEOrf10-Hyp polynucleotide identifies the status of the pathogenic V. parahaemolyticus as present in the sample. In some embodiments, the control level of one or more of the detected ST36Phage polynucleotide, ST36PhHypD-Orf9 polynucleotide, and the ST36NEOrf10-Hyp polynucleotide is zero. In some embodiments, the method also includes detecting in the sample a level of at least one of a polynucleotide comprising a ST36prp nucleic acid sequence or variant thereof, a tlh nucleic acid sequence or variant thereof, a tdh nucleic acid sequence or variant thereof, a trh nucleic acid sequence or variant thereof, and an ST36cps nucleic acid sequence or variant thereof. In certain embodiments, the nucleic acid sequence of the variant of the SEQ ID NO: 81, 82, 83, 84, 94, 95, 96, 97, 87, 88, 89, 90, 12 or 14 nucleic acid sequence has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of which it is a variant. In some embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence. In some embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence and the fragment has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the region of the nucleic acid sequence with which it aligns. In some embodiments, the sample is obtained from a subject. In certain embodiments, the sample is obtained from a substrate.
According to an aspect of the invention, methods of identifying the status of a pathogenic V. parahaemolyticus in a sample are provided, the methods including: (a) detecting in the sample, the level of at least one ST631end polynucleotide comprising at least one nucleic acid sequence set forth as SEQ ID NO: 53, 54, 55, 56, 57, 58, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162 or 163, or a variant thereof; (b) comparing the level of the detected ST631end polynucleotide to a control level of the detected ST631end polynucleotide; and (c) identifying the presence of the pathogenic V. parahaemolyticus in the sample, based at least in part on a difference between the level of the detected ST631end polynucleotide and the control level of the detected ST631end polynucleotide. In some embodiments, a higher level of the detected ST631end polynucleotide compared to the control level of the detected ST631end polynucleotide identifies the status of the pathogenic V. parahaemolyticus as present in the sample. In some embodiments, the control level of the detected ST631end polynucleotide is zero. In some embodiments, the method also includes detecting in the sample a level of at least one of a polynucleotide comprising an ST631-ENV nucleic acid sequence or variant thereof, an ST34reg nucleic acid sequence or variant thereof, a ST674hyp nucleic acid sequence or variant thereof, a ST1127hyp nucleic acid sequence or variant thereof, a ST36Phage nucleic acid sequence or variant thereof, an ST36NEOrf1O-Hyp nucleic acid sequence or variant thereof, a ST36PhHypD-Orf9 nucleic acid sequence or variant thereof, a TdhUreG nucleic acid sequence or variant thereof, a ST36cps nucleic acid sequence or variant thereof, a ST36prp nucleic acid sequence or variant thereof, a tlh nucleic acid sequence or variant thereof, a tdh nucleic acid sequence or variant thereof, a trh nucleic acid sequence or variant thereof, a ST36flp nucleic acid sequence or variant thereof, and an ORF8 nucleic acid sequence or variant thereof. In some embodiments, the nucleic acid sequence of the variant of the ST631end, ST631-ENV, ST34reg, ST674hyp, ST1127hyp, ST36Phage, ST36NEOrf10-Hyp, ST36PhHypD-Or19, TdhUreG, ST36cps, ST36prp, tlh, tdh, trh, ST36flp, ORF8 polynucleotide has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of the polynucleotide of which it is a variant thereof. In certain embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence. In some embodiments, the variant of the nucleic acid sequence comprises a fragment of the nucleic acid sequence and the fragment has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the region of the nucleic acid sequence with which it aligns. In some embodiments, the sample is obtained from a subject. In certain embodiments, the sample is obtained from a substrate. In some embodiments of any of the aforementioned aspects of the invention, detecting comprises performing a polymerase chain reaction (PCR) to detect the level of the least one polynucleotide comprising an ST36prp nucleic acid sequence or variant thereof, an ST36cps nucleic acid sequence or variant thereof, a ST36flp nucleic acid sequence or variant thereof, ST631end nucleic acid sequence or variant thereof, ST631-ENV nucleic acid sequence or variant thereof, a ST34reg nucleic acid sequence or variant thereof, a ST674hyp nucleic acid sequence or variant thereof, a ST1127hyp nucleic acid sequence or variant thereof, a ST36Phage nucleic acid sequence or variant thereof, a ST36NEOrf1O-Hyp nucleic acid sequence or variant thereof, a ST36PhHypD-Orf9 nucleic acid sequence or variant thereof, and a TdhUreG nucleic acid sequence or variant thereof. In some embodiments, the PCR comprises qPCR. In certain embodiments, an oligonucleotide probe in the qPCR comprises a detectable label. In some embodiments, the oligonucleotide probe comprises a nucleic acid sequence set forth herein as: SEQ ID NO: 127, 128, 129, 130, 143, 144, 155, 156 or 170, or a variant thereof, wherein the variant has an 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of the polynucleotide of which it is a variant thereof. In some embodiments of any of the aforementioned aspects of the invention, detecting includes performing in situ hybridization to measure the level of the at least one polynucleotide comprising an ST36prp nucleic acid sequence or variant thereof, an ST36cps nucleic acid sequence or variant thereof, a ST36flp nucleic acid sequence or variant thereof, ST631end nucleic acid sequence or variant thereof, ST631-ENV nucleic acid sequence or variant thereof, a ST34reg nucleic acid sequence or variant thereof, a ST674hyp nucleic acid sequence or variant thereof, a ST1127hyp nucleic acid sequence or variant thereof, a ST36Phage nucleic acid sequence or variant thereof, a ST36NEOrf1O-Hyp nucleic acid sequence or variant thereof, a ST36PhHypD-Orf9 nucleic acid sequence or variant thereof, and a TdhUreG nucleic acid sequence or variant thereof. In some embodiments of any of the aforementioned aspects of the invention, detecting includes performing DNA hybridization to measure the level of the at least one polynucleotide comprising a ST36prp nucleic acid sequence or variant thereof, an ST36cps nucleic acid sequence or variant thereof, a ST36flp nucleic acid sequence or variant thereof, ST631 end nucleic acid sequence or variant thereof, ST631-ENV nucleic acid sequence or variant thereof, a ST34reg nucleic acid sequence or variant thereof, a ST674hyp nucleic acid sequence or variant thereof, a ST1127hyp nucleic acid sequence or variant thereof, a ST36Phage nucleic acid sequence or variant thereof, a ST36NEOrf1O-Hyp nucleic acid sequence or variant thereof, a ST36PhHypD-Orf9 nucleic acid sequence or variant thereof, and a TdhUreG nucleic acid sequence or variant thereof. In certain embodiments, the DNA hybridization is DNA microarray hybridization. In some embodiments of any of the aforementioned aspects of the invention, the method also includes one or more of selecting a therapeutic agent to reduce a pathogenic V. parahaemolyticus infection in the subject; and administering a therapeutic agent to treat a pathogenic V. parahaemolyticus infection in the subject, based at least in part on the identified status of the pathogenic V. parahaemolyticus in the sample. In some embodiments of any of the aforementioned aspects of the invention, the method also includes one or more of selecting an agent to reduce a pathogenic V. parahaemolyticus contamination of the substrate; and contacting the substrate with an agent selected to reduce a pathogenic V. parahaemolyticus contamination of the substrate, based at least in part on the identified status of the pathogenic V. parahaemolyticus in the sample obtained from the substrate. In certain embodiments of any of the aforementioned aspects of the invention, the presence of the pathogenic V. parahaemolyticus in the sample comprises the presence of one or more pathogenic V. parahaemolyticus bacteria in the sample. In some embodiments, the agent is an anti-V. parahaemolyticus agent. In some embodiments of any of the aforementioned aspects of the invention, the subject is at least one of: suspected of having a pathogenic V. parahaemolyticus infection and diagnosed with a pathogenic V. parahaemolyticus infection. In some embodiments of any of the aforementioned aspects of the invention, the subject is a vertebrate, and optionally is a mammal. In certain embodiments of any of the aforementioned aspects of the invention, the subject is an invertebrate. In some embodiments of any of the aforementioned aspects of the invention, the sample comprises one or more of a skin sample, fluid sample, tissue sample, stool sample, pus sample, gastric sample, emesis sample, inflammatory exudate sample, blood sample, or lymph sample. In some embodiments of any of the aforementioned aspects of the invention, the sample is a cultured sample. In certain embodiments of any of the aforementioned aspects of the invention, the substrate is at least one of: known to have been exposed to a V. parahaemolyticus bacteria, suspected of being contaminated with a V. parahaemolyticus bacteria; and identified as being contaminated with a V. parahaemolyticus bacteria. In some embodiments of any of the aforementioned aspects of the invention, reducing the pathogenic V. parahaemolyticus contamination of the substrate comprises reducing the level of the pathogenic V. parahaemolyticus bacteria on the substrate. In some embodiments of any of the aforementioned aspects of the invention, the substrate comprises a liquid and the liquid optionally comprises water. In certain embodiments of any of the aforementioned aspects of the invention, the substrate comprises one or more of a metal, wood, plastic, glass, cork, fiber, a polymer, or a fabric. In some embodiments of any of the aforementioned aspects of the invention, the substrate comprises a food substance, and wherein the food substance optionally comprises shellfish. In some embodiments of any of the aforementioned aspects of the invention, the substrate comprises at least a portion of a tool, work surface, a medical device, body of water, clothing, skin, tissue, an edible substance, a beverage, or a food. In some embodiments of any of the aforementioned aspects of the invention, the substrate is a contaminated substrate and comprises one or more pathogenic V. parahaemolyticus bacteria. In certain embodiments of any of the aforementioned aspects of the invention, the sample comprises a fluid sample, semisolid sample, aqueous sample, or tissue sample.
According to another aspect of the invention, kits for performing a method of any embodiment of any aforementioned aspect of the invention are provided, the kits including: at least one a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to at least one of SEQ ID NO: 2-7, 9, 10, 36, 37, 52, 53, 59, 60, 63, 64, 75, 76, 81, 82, 87, 88, 94, 95, 122-126, 140-142, 151-154, 170, 171 and 172; wherein the kit additionally comprises instructions for detecting a status of a V. parahaemolyticus polynucleotide in a sample. In some embodiments, the V. parahaemolyticus polynucleotide is a pathogenic V. parahaemolyticus polynucleotide. In certain embodiments, the status comprises one or more of: the presence or absence of the polynucleotide; and a level of the polynucleotide. In some embodiments, the kit comprises one or more of: (a) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:2 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:3; (b) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:4 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:5; (c) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:6 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:7; (d) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:9 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:10; (e) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:36 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:37; (f) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:53 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:54; (g) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:59 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:60; (h) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:63 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:64; (i) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:69 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:70; (j) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:75 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:76; (k) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:122 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:123; (1) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:124 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:125; (m) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:126; (n) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:140 and one or more of a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:141, and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:142; (o) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:151 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:152; (p) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:153 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:154; and (q) a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:171 and a container comprising an oligonucleotide primer the sequence of which has at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:172. In some embodiments, the kit also includes one or more oligonucleotide probes. In some embodiments, one or more of the oligonucleotide primers and probes is linked to one or more detectable labels. In certain embodiments, the detectable label comprises one or more of a radioactive molecule, a luminescent molecule, a chemiluminescent molecule, biotin, an enzyme, a His tag, or an exogenous nucleic acid sequence. In some embodiments, the link is an indirect link. In some embodiments, the detectable label comprises: Hexachloro-Fluorescein (HEX), VIC fluorescent dye, 4-5-Dichloro carboxy fluorescein (JOE), Cy3 fluorescent dye, or TexasRed (TxRed).
According to yet another aspect of the invention, compositions that include a polynucleotide having a sequence set forth as SEQ ID NO: 2-7, 9, 10, 12, 13, 21-30, 36, 37, 40, 53-56, 59, 60, 63-66, 69-72, 81-84, 87-91, 94-97, 100-103, 122-174, 175, or another polynucleotide sequence disclosed herein, or a variant of any thereof are provided. In certain embodiments, the polynucleotide is linked to one or more detectable labels. In some embodiments, the detectable label comprises one or more of a radioactive molecule, a luminescent molecule, a chemiluminescent molecule, biotin, an enzyme, a His tag, or an exogenous nucleic acid sequence. In some embodiments, the link is an indirect link. In certain embodiments, the detectable label comprises: Hexachloro-Fluorescein (HEX), VIC fluorescent dye, 4-5-Dichloro carboxy fluorescein (JOE), Cy3 fluorescent dye, or TexasRed (TxRed).
According to yet another aspect of the invention, methods of identifying a test compound as an agent to reduce one or both of pathogenic V. parahaemolyticus in subject and pathogenic V. parahaemolyticus contamination of a substrate are provided, the methods including: (a) identifying the level of a pathogenic V. parahaemolyticus bacteria in a first portion of a sample using a method of any embodiment of any of the aforementioned methods of the invention; (b) contacting a second portion of the sample of (a) with a test compound; (c) incubating the contacted second portion of the sample with the test compound; (d) identifying the level of the pathogenic V. parahaemolyticus bacteria in the incubated second portion of the sample; and (e) comparing the level of the pathogenic V. parahaemolyticus bacteria in the first portion of the sample to the level of the pathogenic V. parahaemolyticus bacteria in the incubated second portion of the sample, wherein a decrease in the level of the pathogenic V. parahaemolyticus bacteria in the incubated second portion of the sample compared to the control level identified in step (a) identifies the test compound as a candidate compound to treat a pathogenic V. parahaemolyticus infection in a subject or as a candidate compound to reduce a pathogenic V. parahaemolyticus contamination of a substrate. In some embodiments, the incubation is at least 1 min, 5 min, 30 min, 1 hour, 6 hours, 12 hours, or 18 hours in duration. In some embodiments, the method also includes: confirming efficacy of the candidate compound for treating a subject having or at risk of having the pathogenic V. parahaemolyticus infection or for reducing contamination of a substrate by the pathogenic V. parahaemolyticus bacteria. In certain embodiments, the control level detected in step (a) is greater than zero. In some embodiments, detecting the level of the pathogenic V. parahaemolyticus bacteria in the sample comprises detecting in the sample, the level of a pathogenic V. parahaemolyticus polynucleotide of the pathogenic V. parahaemolyticus bacteria.
According to yet another aspect of the invention, methods of identifying the status of a pathogenic V. parahaemolyticus infection in a subject are provided wherein the methods include any embodiment of any of the aforementioned methods to identify a V. parahaemolyticus in a sample. According to yet another aspect of the invention, methods of identifying the status of a pathogenic V. parahaemolyticus contamination of a substrate are provided wherein the methods include any embodiment of any of the aforementioned methods to identify a V. parahaemolyticus in a sample. Polynucleotides listed in Table 2, Table 3, and in the Brief Description of the sequences may be used in embodiments of the aforementioned methods, kits, and compositions.
The present invention is not intended to be limited to a system or method that must satisfy one or more of any stated objects or features of the invention. It is also important to note that the present invention is not limited to the exemplary or primary embodiments described herein. Modifications and substitutions by one of ordinary skill in the art are considered to be within the scope of the present invention.
Definitive markers of virulence have now been identified for V. parahaemolyticus, and assays have been developed that allow identification pathogenic V. parahaemolyticus known to cause infections in subjects and to contaminate substrates. In certain aspects of the invention, assays are provided that can be used to identify the status of infection of a subject with, or contamination of a substrate with V. parahaemolyticus bacteria. In some aspects of the invention, methods include determining one or more of the presence and level of a pathogenic V. parahaemolyticus bacterium in a sample. A sample assayed using methods and compositions of the invention may be obtained from a variety of different sources such as, but not limited to: cells, tissues, subjects, and substrates. In certain aspects of the invention, the identification of status of a pathogenic V. parahaemolyticus polynucleotide in a sample may be extrapolated to identify the presence of the pathogenic V. parahaemolyticus bacterium in the cell, tissue, or subject, or substrate from which the sample was obtained. The presence of pathogenic V. parahaemolyticus bacterium may indicate infection or contamination of the source of the sample such as the cell, tissue, subject or the substrate by the pathogenic V. parahaemolyticus bacterium. Thus, some embodiments of the invention include methods of determining whether a cell, tissue or subject has, or is at risk of having, a pathogenic V. parahaemolyticus infection and certain embodiments of the invention include methods of determining whether a substrate is contaminated by, or is a risk of contamination by a pathogenic V. parahaemolyticus bacterium.
The invention in some aspects also includes methods to monitor treatment of a pathogenic V. parahaemolyticus infection in a cell, tissue, or subject and to monitor decontamination of a substrate contaminated by a pathogenic V. parahaemolyticus bacterium. Thus, some aspects of the invention include methods such as assays to identify changes in a level of a pathogenic V. parahaemolyticus bacterium in a sample. In instances in which a sample is obtained from a cell, tissue, or subject and/or from a substrate, methods of the invention may be used to monitor treatment of infection or contamination of the cell, tissue, or subject or a substrate, respectively, by pathogenic V. parahaemolyticus bacterium. The invention also provides in some aspects, methods useful to characterize one or more compounds to determine whether or not they may be useful to treat a pathogenic V. parahaemolyticus infection or to reduce or eliminate contamination by a pathogenic V. parahaemolyticus bacterium. Certain methods of the invention may be used to determine efficacy, cell toxicity, and other characteristics of compounds that may be used to treat a pathogenic V. parahaemolyticus infection or to reduce or eliminate pathogenic V. parahaemolyticus contamination of a substrate.
Pathogenic V. parahaemolyticus bacteria are rod-shaped, gram-negative, motile bacteria that are found in brackish water and saltwater. They can be found in organisms such as seafood, shellfish that when eaten by a subject, can cause gastrointestinal illness. Entry of the bacteria into a subject may also occur through contact with water that contains the pathogenic V. parahaemolyticus bacteria and thus can cause infections via contact with open wounds and handling of contaminated substrates such as shellfish, materials, liquids, etc. Outbreaks of V. parahaemolyticus bacterial infections generally occur along coastal regions during times when the water temperature supports high bacterial levels. Infection in a subject may be indicated by severe diarrhea, nausea, vomiting, abdominal pain, and fever and can last from 2 to 10 days. Subjects or substrates that have infection by or contamination with pathogenic V. parahaemolyticus bacteria may spread the disease, via contact by another subject, for example, transmission from body fluids, diarrhea, or by contact with wounds, food, beverages, etc. Although most infections are not life-threatening, deaths do result from pathogenic V. parahaemolyticus infection.
Pathogenic V. parahaemolyticus bacteria are a cause of a growing number of cases of food-borne illnesses. Methods and tools of the invention that can be used to determine (also referred to herein as “to identify”) the status of a pathogenic V. parahaemolyticus infection or contamination may, in some embodiments include determining the presence, absence, and/or level or quantity of the pathogenic V. parahaemolyticus bacteria in or on a cell, tissue, subject, and/or substrate have now been identified. Such methods of the invention may be used in identify disease or risk of disease or contamination or risk of contamination by pathogenic V. parahaemolyticus. Disease or contamination by a pathogenic V. parahaemolyticus bacteria are conditions characterized by abnormal (e.g., increased) levels of the pathogenic V. parahaemolyticus bacteria. With respect to a pathogenic V. parahaemolyticus level in a sample, as used herein a “normal” level would be no detectable pathogenic V. parahaemolyticus bacteria as determined using a method of the invention, and an “abnormal” level would be a detectable level of pathogenic V. parahaemolyticus bacteria determined using a method of the invention. As used herein, with respect to the level of a pathogenic V. parahaemolyticus bacteria, the terms: “increased”, “elevated”, and “higher” are used interchangeably, and the terms “decrease”, “reduced”, and “lower” are used interchangeably.
Methods and assays set forth in certain embodiments of the invention can be used to identify the status of, and evaluate and compare levels of pathogenic V. parahaemolyticus bacteria in samples, or in some embodiments of the invention, in or on subjects and substrates. Methods in some aspects of the invention may include detecting in a sample the presence and/or level of one or more specific polynucleotides and/or polypeptides that have now been identified as biomarkers for pathogenic V. parahaemolyticus bacteria. Information obtained using methods of the invention can be used to identify the status of infection of a subject or contamination of a substrate by a pathogenic V. parahaemolyticus bacterium and may be used to select a treatment for a subject and to select an agent with which to reduce contamination of a substrate.
As used herein the term “status” with respect to a pathogenic V. parahaemolyticus in a sample, may mean presence, absence, or level of the pathogenic V. parahaemolyticus in the sample. In certain embodiments of the invention the status of a pathogenic V. parahaemolyticus in a sample is binary, and is determined to be either present or absent using a method of the invention. In certain embodiments of the invention, a status may be a level of a pathogenic V. parahaemolyticus bacteria in a sample, and may be a difference in a level between two or more samples, and or between a sample and a control. In some aspects of the invention, a determination of a level greater than zero of a pathogenic V. parahaemolyticus polynucleotide in a sample indicates the presence of a pathogenic V. parahaemolyticus in the sample and in the source from which the sample was obtained. In some embodiments the source from which a sample is obtained is a subject and in certain embodiments of the invention, the source is a substrate. In certain aspects of the invention, a status of a pathogenic V. parahaemolyticus in a sample may be used to determine a status of a pathogenic V. parahaemolyticus bacterial infection in a subject from whom the sample was obtained, or a pathogenic V. parahaemolyticus bacterial contamination of a substrate from which the sample was obtained.
As used herein the term “status” with respect to infection or contamination may mean presence, absence, onset, end, recurrence, progression, regression, increase, decrease, or other indication of the state of the infection or contamination by the pathogenic V. parahaemolyticus bacteria. For example, in certain embodiments of the invention the status of infection or contamination is binary, and is determined to be either present or absent using a method of the invention. In certain embodiments of the invention, a status may be the state of progression of infection or contamination, which can be determined by testing a first sample obtained from a subject or substrate and testing a second sample obtained from the subject or substrate, respectively, wherein the second sample is obtained from the subject or substrate at a later point in time. An increase in the level of a pathogenic V. parahaemolyticus bacteria determined in the second sample compared to the level determined in the first sample indicates the status of progression or increase of the infection or contamination of the subject or substrate by the pathogenic V. parahaemolyticus bacteria. A determination of a level of zero of a pathogenic V. parahaemolyticus bacteria in a first sample obtained from a subject or substrate followed by a determination of a level greater than zero of the pathogenic V. parahaemolyticus bacteria in a sample subsequently obtained from the subject or substrate, respectively, may indicate a status of onset of the infection or contamination of the subject or substrate, respectively, by the pathogenic V. parahaemolyticus bacteria. Similarly, a decrease in the level of a pathogenic V. parahaemolyticus bacteria determined in a sample obtained from a subject or substrate at a later time point compared to that determined in a sample obtained from the subject or substrate, respectively, at an earlier time point would indicate a status of regression or decrease of the infection or contamination with the pathogenic V. parahaemolyticus bacteria. A determination of a level of a pathogenic V. parahaemolyticus bacteria that is greater than zero in a first sample obtained from a subject or substrate followed by determination of a zero level of the pathogenic V. parahaemolyticus bacteria in a sample subsequently obtained from the subject or substrate, respectively, indicate a status of the infection or contamination of the subject or substrate, respectively, by the pathogenic V. parahaemolyticus bacteria as ended or resolved.
Some embodiments of the invention provide methods that may be diagnostic and/or prognostic for infection with or contamination by pathogenic V. parahaemolyticus bacteria. Methods of the invention, in some aspects, utilize detection of the identified nucleic acid and/or polypeptide biomarkers to determine the presence and/or level of the bacteria in biological samples obtained from subjects, culture, and/or substrates. It has now been identified that increased presence of signature pathogenic V. parahaemolyticus molecules in or on a cell, tissue, subject, or substrate is correlated with infection or contamination of the cell, tissue, subject, or substrate with the pathogenic V. parahaemolyticus bacteria.
As used herein, the term “V. parahaemolyticus molecule” means a polynucleotide or polypeptide naturally expressed by a V. parahaemolyticus bacterium and the term “pathogenic V. parahaemolyticus molecule” means a polynucleotide or polypeptide naturally expressed by a pathogenic V. parahaemolyticus bacterium. As used herein, a “signature” pathogenic V. parahaemolyticus molecule refers to a polynucleotide or polypeptide that is naturally expressed by a pathogenic V. parahaemolyticus bacterium and is not naturally expressed by a non-pathogenic V. parahaemolyticus bacterium.
In an infection of a cell, tissue, or subject with, or contamination of a substrate by, pathogenic V. parahaemolyticus bacteria, the level of the pathogenic bacteria is statistically significantly higher in that cell, tissue, subject, or substrate as compared to the level of the pathogenic bacteria in cells, tissues, subjects, or substrates that do not have the infection or contamination. It has now been identified that the presence and/or level of a signature pathogenic V. parahaemolyticus polynucleotide or polypeptide of the invention in a sample obtained from a subject or substrate correlates with the presence and/or level of the pathogenic V. parahaemolyticus bacteria in the subject or substrate, respectively. In an infection of a cell, tissue, or subject with a pathogenic V. parahaemolyticus bacteria, and in contamination of a substrate with a pathogenic V. parahaemolyticus bacteria, the level of a signature pathogenic V. parahaemolyticus polynucleotide or polypeptide of the invention is statistically significantly higher in or on the cell, tissue, subject, or substrate as compared to the level of the signature pathogenic V. parahaemolyticus polynucleotide or polypeptide in a cell, tissue, subject, or substrate not infected with or contaminated by the pathogenic V. parahaemolyticus bacteria. Thus, detecting a level of a signature pathogenic V. parahaemolyticus polynucleotide or polypeptide of the invention in a sample obtained from a subject or substrate permits identification of the status of the infection of the subject with the pathogenic V. parahaemolyticus bacteria, and permits identification of the status of the contamination of the substrate with the pathogenic V. parahaemolyticus bacteria, respectively.
In some embodiments of the invention detection of an amount greater than zero and/or the presence of a signature pathogenic V. parahaemolyticus polynucleotide or polypeptide in a sample identifies the status of the source (for example, the subject or substrate) of the sample as infected and/or contaminated with the pathogenic V. parahaemolyticus bacteria. In some embodiments of the invention, the level of a signature pathogenic V. parahaemolyticus polynucleotide or polypeptide in a control sample is zero, which means the absence of the pathogenic V. parahaemolyticus bacterium. In certain embodiments of the invention, any detected level of a signature pathogenic V. parahaemolyticus polynucleotide or polypeptide of the invention that is greater than zero in a sample need not be compared to a control because a level greater than zero confirms the presence of the pathogenic V. parahaemolyticus bacterium in the sample.
Methods of the invention, in some embodiments, may include detecting a level of a pathogenic V. parahaemolyticus polynucleotide (and hence bacteria) in a sample and comparing the detected level to one or more of: a level of zero, a control level, and a prior level in a biological sample from the same subject or substrate. For example in some aspects of the invention, a method may include detecting a level of a pathogenic V. parahaemolyticus bacterium in a sample and the sample was obtained from a subject that has, is suspected of having, is susceptible to having, or is at risk of having an infection by the pathogenic V. parahaemolyticus bacterium.
As used herein, the term “level” when used in reference to detecting a level of a V. parahaemolyticus polynucleotide, polypeptide, or bacterium means: (a) a quantitative value such as an amount, weight, or concentration of the polynucleotide, polypeptide, or bacterium; or (b) the “presence” or “absence” of the polynucleotide, polypeptide or bacterium. It will be understood that in certain embodiments of the invention, the presence or absence of a V. parahaemolyticus polynucleotide, polypeptide, or bacterium is detected as a binary determination, without quantification. Thus, detecting may include one or more of determining the presence or absence of a pathogenic V. parahaemolyticus bacterium polynucleotide or polypeptide molecule; and determining a quantity of a pathogenic V. parahaemolyticus bacterium polynucleotide or polypeptide molecule. Means for detecting levels of polypeptides and polynucleotides and for determining amino acid and nucleic acid sequences are known in the art and non-limiting examples of detection means are provided herein.
In certain aspects of the invention, a level of a pathogenic V. parahaemolyticus bacterium in/on a subject or substrate can be detected using methods of the invention to measure the pathogenic V. parahaemolyticus bacterium polynucleotide and/or polypeptide molecule (for example, one or more biomarker polynucleotides or polypeptides) in an in vitro assay of a biological sample obtained from the subject or substrate, respectively. As used herein, the term “measure” used in reference to measuring a level of a V. parahaemolyticus polynucleotide comprising a nucleic acid sequence means: (a) determining a quantitative value, such as an amount, weight, concentration, relative amount, relative concentration, etc., of a polynucleotide comprising the nucleic acid sequence; or (b) determining the “presence” or “absence” of a polynucleotide comprising the nucleic acid sequence. It will be understood that in certain embodiments of the invention, the presence or absence of a V. parahaemolyticus polynucleotide is measured as a binary determination, without quantitation. Thus, measuring may include one or more of determining the presence or absence of a pathogenic V. parahaemolyticus bacterium polynucleotide or polypeptide molecule; and determining a quantity of a pathogenic V. parahaemolyticus bacterium polynucleotide or polypeptide molecule. Means for measuring levels of polypeptides and polynucleotides and for determining amino acid and nucleic acid sequences are known in the art. Non-limiting examples of measuring means are provided herein.
Detection methods suitable for use in certain embodiments of methods of the present invention can be used to detect and measure levels of pathogenic V. parahaemolyticus bacterium polypeptide or nucleic acid molecules in samples (also interchangeably herein as biological samples) in vitro as well as in vivo. For example, in certain embodiments of the invention in vitro techniques for detecting and/or measuring levels of pathogenic V. parahaemolyticus polynucleotides (genes) include but are not limited to: polymerase chain reaction methods, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), Northern hybridizations, in situ hybridizations, DNA or oligonucleotide array, sequencing, etc. In certain aspects of the invention in vitro techniques for detecting and/or measuring levels of pathogenic V. parahaemolyticus bacterium DNA include, but are not limited to: polymerase chain reaction (PCR) and Southern hybridizations. In vitro techniques for detecting and/or measuring levels of pathogenic V. parahaemolyticus bacterium polypeptides in certain embodiments of the invention include, but are not limited to enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, immunofluorescence, and other known suitable techniques.
In some embodiments of the invention, methods may include comparing a level of a pathogenic V. parahaemolyticus polynucleotide or polypeptide molecule in a sample to a control value or level for the pathogenic V. parahaemolyticus polynucleotide or polypeptide molecule. As used herein a “control” may be a normal control or an infection or contamination control. Selection and use of appropriate controls in comparative, diagnostic, and assay methods are well known in the art. In some embodiments of the invention, a normal control level may zero and represent the amount in a subject or substrate free of the pathogenic V. parahaemolyticus bacterium. A control level of a pathogenic V. parahaemolyticus molecule can readily be determined by measuring a level of the pathogenic V. parahaemolyticus polynucleotide or polypeptide molecule using a method of the invention, as described herein. In some embodiments of the invention, an infection or contamination control level may be obtained from a sample from a subject or substrate known have infection with or contamination by, respectively, the pathogenic V. parahaemolyticus bacterium. In some embodiments, a disease control pathogenic V. parahaemolyticus bacterium level may be based on levels obtained from one or more subjects known to have the infection, or one or more surfaces known to have contamination with the pathogenic V. parahaemolyticus bacterium. In certain embodiments of the invention, the disease control may be a sample from a subject diagnosed with a pathogenic V. parahaemolyticus infection and the subject's disease control may be compared to another sample obtained from the subject at a different time. In certain embodiments of the invention, a disease control level of a pathogenic V. parahaemolyticus molecule can readily be determined by measuring levels of the pathogenic V. parahaemolyticus molecule in a sample obtained from a subject known to have a pathogenic V. parahaemolyticus infection, or obtained from a substrate known to have a pathogenic V. parahaemolyticus contamination.
In some embodiments of the invention, a control level of pathogenic V. parahaemolyticus is a level determined from samples, subjects, substrates, tissues, cells, etc. that do not have contamination by or infection by the pathogenic V. parahaemolyticus bacteria that is being tested for in the sample. For example, in some embodiments, a control level of pathogenic V. parahaemolyticus molecule is a level determined in a normal sample that does not have contamination by the pathogenic V. parahaemolyticus bacteria that is suspected to be in the sample obtained from the subject or substrate. In such a case, the presence of infection and/or contamination by the pathogenic V. parahaemolyticus can be determined based on an increase in the level of the pathogenic V. parahaemolyticus polynucleotide or polypeptide in the subject's or substrate's sample as compared to the control that is free of the pathogenic V. parahaemolyticus. In another embodiment of the invention, the control is from a normal subject or substrate and the test sample is from a subject or substrate that is suspected of having infection with or contamination by a pathogenic V. parahaemolyticus bacteria.
In some aspects of the invention methods are provided that include comparing a level of a pathogenic V. parahaemolyticus molecule measured a biological sample obtained from a subject, or substrate to a control value for determining infection or contamination status, stage, prognosis, etc. Changes over time in the presence and/or level of a pathogenic V. parahaemolyticus molecule in a subject or substrate can be assessed by determining levels in two or more samples obtained from a subject or substrate at different times. Values obtained from a sample obtained at one time can be compared to values obtained from samples obtained at other times. For example, a first level obtained from a subject may serve as a baseline level or control level for that subject, thus allowing comparison of the pathogenic V. parahaemolyticus molecule level and the determination of change or stability of the pathogenic V. parahaemolyticus infection over time. Levels of a pathogenic V. parahaemolyticus molecule may also be measured after a specific course of treatment of the pathogenic V. parahaemolyticus infection or contamination has been initiated, with the intent of determining the efficacy of that treatment of the subject or the decontamination of the substrate, or just the natural change of the level of the pathogenic V. parahaemolyticus bacteria in or on a subject or substrate over time. For example, though not intended to be limiting, samples may be obtained from a substrate such as a body of water, at different times of the year and the levels of a signature pathogenic V. parahaemolyticus polynucleotide or polypeptide determined to assess changes in the contamination of the substrate over time. Thus, the status of infection with or contamination by a pathogenic V. parahaemolyticus bacterium in a subject or substrate can be monitored over time using aspects of methods of the invention to assess change in levels of one or more pathogenic V. parahaemolyticus molecules.
In some aspects of the invention, assessing a change in at least one of the presence, absence, and level of a pathogenic V. parahaemolyticus molecule in a subject or substrate may be desirable, and methods of the invention may be used to monitor the level of the pathogenic V. parahaemolyticus bacteria in biological samples obtained from the subject or substrate over time to assess changes. Some aspects of the invention include methods to monitor the level of a pathogenic V. parahaemolyticus molecule over time to assess changes in the level. A decrease in a level of the pathogenic V. parahaemolyticus bacteria over time may be indicated by a level of the pathogenic V. parahaemolyticus molecule that, in a second biological sample is less than 100%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the level identified in a first biological sample obtained from the subject or substrate at a prior time point than the time at which the second sample was obtained. An increase in a level of a pathogenic V. parahaemolyticus bacterium over time may be indicated by a level of the pathogenic V. parahaemolyticus molecule that, in second biological sample is more than 100%, 101%, 105%, 110%, 115%, 120%, 125%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 500%, 1000%, 10,000% of the level identified in a first biological sample obtained from the subject or substrate at a prior time point, including all percentages in between those stated. A higher level of increase, for example 200%, 400%, 600%, 1000% or higher increase may indicate the severity of the infection or contamination with a higher percent increase corresponding to a more severe infection or contamination. An increase in a level of a pathogenic V. parahaemolyticus molecule between a first and second sample may also indicate the progression of a pathogenic V. parahaemolyticus infection or contamination. In embodiments where the level of a pathogenic V. parahaemolyticus molecule was zero in a first biological sample, any level over zero in the second biological sample indicates an increase in a level of the pathogenic V. parahaemolyticus bacteria and in some embodiments may indicate the onset of infection or contamination in a subject or substrate, respectively. It will be understood that if zero pathogenic V. parahaemolyticus molecules are detected in a sample, any level of detection in a subsequent sample that is greater than zero indicates an increase. Similarly, if any level of a pathogenic V. parahaemolyticus molecule is detected in an initial sample and no pathogenic V. parahaemolyticus molecules are detected in a subsequent sample, it indicates a decrease.
Samples (also referred to herein as biological samples) may be obtained from a subject or substrate at any desired interval such as at least once per day, every other day, once per week, every other week, once per month, every other month, every third month, every year, etc. It will be understood that interval testing using methods of the invention may be performed to assess the level of a pathogenic V. parahaemolyticus bacteria to determine information including, but not limited to: the likelihood of infection from a substrate, reinfection of a substrate, reinfection of a subject, decreased contamination of a substrate, carrier status of a subject, etc.
Assessment of efficacy of candidate agents to treat subjects or de-contaminate substrates that have been contacted with a pathogenic V. parahaemolyticus bacterium may also be done according to some embodiments of the invention, such as, in a non-limiting example, screening assays to assess candidate agents or compounds to reduce or eliminate pathogenic V. parahaemolyticus bacteria from a subject or substrate. It will be understood that a therapeutic regimen or decontamination regime may be either prophylactic or a treatment of an existing pathogenic V. parahaemolyticus infection or contamination of a subject or substrate, respectively. The invention in some aspects provides methods that may be used to monitor a subject's or substrate's response to prophylactic treatment provided to a subject or substrate. Some embodiments of the invention may include methods to assess treatments intended to reduce, prevent, or eliminate pathogenic V. parahaemolyticus bacteria and/or to provide an indicator of a status of a pathogenic V. parahaemolyticus infection or contamination, and in some embodiments of the invention, can be used to select one or more therapies for the subject, or one or more decontamination treatments for the substrate, for example, to select a drug therapy, disinfection protocol, etc. In some aspects, methods of the invention may include one or more of selecting a treatment for a subject or substrate and treating a subject or substrate for a pathogenic V. parahaemolyticus infection or contamination based at least in part on the results of an assay of the invention.
Assays for assessing pathogenic V. parahaemolyticus bacteria levels in certain embodiments of the invention may include determining one or more pathogenic V. parahaemolyticus molecule levels, including but not limited to determining levels of signature polynucleotides that encode pathogenic V. parahaemolyticus polypeptides and/determining levels of pathogenic V. parahaemolyticus polypeptides in cells, tissues, subjects, and substrates. Levels of pathogenic V. parahaemolyticus polynucleotides and polypeptides can be determined in a number of ways when carrying out various embodiments of methods of the invention. In some embodiments of the invention, a level of a pathogenic V. parahaemolyticus polynucleotide acid or polypeptide is measured in relation to a control level of the polynucleotide or polypeptide, respectively, in a biological sample from at least one of a cell, tissue, subject, and substrate. One possible measurement of the level of pathogenic V. parahaemolyticus polynucleotide or polypeptide is a measurement of an absolute level of the polynucleotide or polypeptide. This could be expressed, for example, in the level of the polynucleotide or polypeptide per unit of cells or tissue. Another measurement of a level of a pathogenic V. parahaemolyticus polynucleotide or polypeptide is a measurement of a change in the level of the polynucleotide or polypeptide over time. This may be expressed in an absolute amount or may be expressed in terms of a percentage increase or decrease over time. Methods disclosed herein may be used in embodiments of the invention to assess pathogenic V. parahaemolyticus polypeptide and polynucleotide molecules to assess the status of infection in a subject and contamination of a substrate and/or the efficacy of treatments for such infection and/or contamination.
As used herein a “subject” refers to an animal, such as, but not limited to a human, a non-human primate, a rodent, a dog, cat, a fish, a bird, or other animal. Thus, in addition to human medical application, some aspects of the invention include veterinary application of methods described herein. In certain aspects of the invention a subject is an invertebrate, including but not limited to a shellfish, including but not limited to a clam, oyster, or mussel. A subject may be suspected of being infected with a pathogenic V. parahaemolyticus bacterium and methods of the invention can be used to measure the pathogenic V. parahaemolyticus molecules in a biological sample from the subject, thus the subject may be diagnosed with the infection by the pathogenic V. parahaemolyticus bacteria. In some embodiments of the invention, a subject may not have been previously or be currently diagnosed with infection by a pathogenic V. parahaemolyticus bacteria, but may be considered at risk of having the infection, for example, a subject who may be free of symptoms, but who may have been exposed to, or be considered to be at risk of having been exposed to a pathogenic V. parahaemolyticus bacterium.
Similarly, a substrate may be suspected of having contamination with a pathogenic V. parahaemolyticus bacterium and methods of the invention can be used to measure the pathogenic V. parahaemolyticus in a biological sample from the substrate, thus the substrate may be determined to be contaminated with the pathogenic V. parahaemolyticus bacteria. In some embodiments, a substrate may not have been previously, or be currently identified as being contaminated with the pathogenic V. parahaemolyticus, but may be considered to be at risk of contamination by a pathogenic V. parahaemolyticus bacterium. In some embodiments of the invention, a substrate may have previously identified as having contamination by a pathogenic V. parahaemolyticus bacteria, but conditions such as temperature or environmental status may have changed and methods of the invention may be used to identify an increase, or decrease in contamination of the substrate.
In some embodiments of the invention, a sample comprises a cell or tissue or extracellular material from a subject or substrate. In some embodiments a sample comprises one or more of: a skin sample, fluid sample, tissue sample, stool sample, mucus sample, sweat sample, tear sample, urine sample, pus sample, tissue scraping, gastric sample, emesis sample, inflammatory exudate sample, blood sample, or lymph sample. It will be understood that additional types of samples may also be used in methods of the invention. In certain embodiments of the invention, a sample is a cultured sample.
In certain embodiments of the invention a sample is a sample obtained from a substrate. In some embodiments of the invention, a substrate comprises a liquid. A liquid component of a sample may, in some embodiments comprise water. In some embodiments of the invention a substrate is at least a portion of a tool, work surface, a medical device, body of water, clothing, skin, tissue, an edible substance (e.g., a beverage or food), etc. In certain embodiments, a substrate may comprise a solid, such as, but not limited to: metal, wood, dirt, rock, plastic, glass, cork, fiber, a polymer, stone, leather, plant matter, fiberglass, nylon, or a fabric. In some embodiments of the invention a substrate may comprise a food or beverage substance. Non-limiting examples of food substances that may be assessed using methods of the invention include shellfish, fish, water organisms, plants, dairy products, etc. In certain embodiments, a sample may be obtained from one or more of a plurality of substrates, for example from one or more individual shellfish that make up part of a plurality of shellfish, and results of an detection method of the invention may be used to identify whether or not the plurality of substrates is contaminated with a pathogenic V. parahaemolyticus bacteria.
As used herein, the term “isolated”, when used in the context of a sample, is intended to indicate that the sample has been removed from a subject, culture, or substrate. In some embodiments of the invention, a sample is isolated from a subject, culture, or substrate and is subjected to a method of the present invention without further processing or manipulation subsequent to its isolation. In some embodiments of the invention, a sample can be processed or manipulated subsequent to being isolated and prior to being subjected to a method of the invention. For example, a sample can be refrigerated (e.g., stored at 4° C.), frozen (e.g., stored at −20° C., stored at −135° C., frozen in liquid nitrogen, or cryopreserved using any one of many standard cryopreservation techniques known in the art). Furthermore, a sample may be purified subsequent to isolation from a subject, culture, or a substrate and prior to subjecting it to a method of the present invention.
As used herein, the term “purified” when used in the context of a sample, is intended to indicate that at least one component of the isolated biological sample has been removed from the sample such that fewer components, and consequently, purer components, remain following purification. For example, a serum sample can be separated into one or more components using centrifugation techniques known in the art to obtain partially-purified sample preparation. Furthermore, it is possible to purify a biological sample such that substantially only one component remains. For example, a sample can be purified such that substantially only the polypeptide or polynucleotide component of the sample remains. Thus it will be understood that a sample may originally comprise one or more of a pathogenic V. parahaemolyticus bacterium may be purified and processed prior to assay and thus may no longer contain the V. parahaemolyticus bacteria, but containing components of the pathogenic V. parahaemolyticus bacteria such as one or more pathogenic V. parahaemolyticus bacteria polynucleotides (also referred to herein as pathogenic V. parahaemolyticus polynucleotides). Therefore, identifying a status of a pathogenic V. parahaemolyticus polynucleotide in a sample that has been isolated or processed identifies that status in the original sample.
Furthermore, it may be desirable to amplify a component of a sample such that detection of the component is facilitated. For example, the mRNA component of a biological sample can be amplified (e.g., by RT-PCR) such that detection of V. parahaemolyticus mRNA is facilitated. As used herein, the term “RT-PCR” (an abbreviation for reverse transcriptase-polymerase chain reaction) involves subjecting mRNA to the reverse transcriptase enzyme, resulting in the production of a cDNA which is complementary to the base sequences of the mRNA. Large amounts of selected cDNA can then be produced by means of the polymerase chain reaction which relies on the action of heat-stable DNA polymerase for its amplification action. Alternative amplification methods include: self-sustained sequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizardi, P. M. et all, 1988, Bio/Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
V. parahaemolyticus Detection Techniques
V. parahaemolyticus molecules (for example, V. parahaemolyticus polypeptides and polynucleotides that encode V. parahaemolyticus polypeptides), can be detected and measured using any suitable means known in the art. In certain aspects of the invention a V. parahaemolyticus polypeptide and/or polynucleotide that encodes a V. parahaemolyticus polypeptide are molecules that are present in pathogenic V. parahaemolyticus bacteria, and are not present in non-pathogenic bacteria. Thus, in certain embodiments the polypeptide molecules and nucleic acid molecules that encode the polypeptides are signature molecules. In some embodiments of the invention, a detection or measurement means for V. parahaemolyticus molecules includes an immunological assay, nucleotide determination (mRNA or DNA), etc. Examples of immunological assays suitable for use in methods of the invention may include, but are not limited to ELISA assays, assays that utilize an anti-V. parahaemolyticus polypeptide antibody (or FV derivative) to which is conjugated a detectable label. A detectable label may be conjugated (also referred to herein as “linked”) either directly or indirectly. In some aspects of the invention, pathogenic V. parahaemolyticus presence and/or levels may be measured by a PCR reaction, in situ hybridization, DNA microarray hybridization, or other suitable method to identify the presence of a sequence present in a pathogenic V. parahaemolyticus bacterium. Methods of measuring levels of polynucleotides encoding pathogenic V. parahaemolyticus bacteria (i.e. pathogenic V. parahaemolyticus bacteria mRNA) may include, but are not limited to, real-time polymerase chain reaction (qRT-PCR), DNA array, and next generation sequencing methods. Application and optimization of such methods are known and practiced in the art.
The present invention features agents that are capable of detecting and/or quantitating a pathogenic V. parahaemolyticus bacteria-encoding polynucleotide such that the presence and/or level of the pathogenic V. parahaemolyticus bacteria are determined. As defined herein, an “agent” refers to a substance that is capable of identifying or detecting a pathogenic V. parahaemolyticus bacteria in a sample (e.g., identifies or detects pathogenic V. parahaemolyticus bacteria mRNA, DNA, polypeptide, activity, etc.). As used herein, the terms “labeled” or “labelable” refers to the attaching or including of a label (e.g., a marker or indicator) or ability to attach or include a label (e.g., a marker or indicator). A detectable label may be conjugated to (also referred to herein as “linked to”) a polynucleotide either directly or indirectly. For example, though not intended to be limiting: a direct link may be a be when a detectable label and a polynucleotide are physically attached to each other, and an indirect link may be when a detectable label is physically attached to an entity and the entity and not the detectable label is physically attached to a polynucleotide. Detectable labels, markers, or indicators useful in methods of the invention may include, but are not limited to, for example, radioactive molecules, colorimetric molecules, and enzymatic molecules that produce detectable changes in a substrate. A detectable label for use in methods, compositions, and kits of the invention may be a radioactive molecule, a luminescent molecule, a fluorescent molecule, a chemiluminescent molecule, biotin, an enzyme, a His tag, or an exogenous nucleic acid sequence, or other suitable detectable label known in the art. In some aspects of the invention, a detectable label comprises: Hexachloro-Fluorescein (HEX), VIC fluorescent dye, 4-5-Dichloro carboxy fluorescein (JOE), Cy3 fluorescent dye, or TexasRed (TxRed).
Methods of the invention for detecting the presence and/or quantity of a pathogenic V. parahaemolyticus bacterium may also include procedures such as an immunological assay, a polymerase chain reaction, real-time polymerase chain reaction (qRT-PCR), mass spectroscopy, etc. In addition, embodiments of the invention include may include nucleic “aptamers”, i.e. nucleic acids [DNA, RNA or peptide nucleic acids (PNAs)] that possess high affinity for polypeptides now identified as present in pathogenic V. parahaemolyticus bacteria (which may not be present in non-pathogenic V. parahaemolyticus bacteria) and can be readily labeled for high throughput binding assays. Aptamers can be produced by standard molecular biological techniques by those skilled in the art by repeated rounds of binding, selection, and affinity, and amplification [Hamaguchi, et al. Anal. Biochem. (2001) 294; pt 2, pages 126-131].
In some embodiments of the invention an agent is a labeled or labelable polynucleotide probe capable of hybridizing to a pathogenic V. parahaemolyticus polynucleotide, (e.g., a pathogenic V. parahaemolyticus RNA or DNA). For example, the agent can be an oligonucleotide primer for the polymerase chain reaction that flanks or lies within the nucleotide sequence encoding pathogenic V. parahaemolyticus polypeptides that may be present in pathogenic V. parahaemolyticus and may not be present in non-pathogenic V. parahaemolyticus bacteria. In some embodiments of the invention, the biological sample being tested is an isolate, for example, RNA. In yet another embodiment, the isolate (e.g., the RNA) is subjected to an amplification process that results in amplification of a pathogenic V. parahaemolyticus polynucleotide. As defined herein, an “amplification process” is designed to strengthen, increase, or augment a molecule within the isolate. For example, where the isolate is mRNA, an amplification process such as RT-PCR can be utilized to amplify the mRNA, such that a signal is detectable or detection is enhanced. Such an amplification process is beneficial particularly when the biological, tissue, or tumor sample is of a small size or volume.
Pathogenic V. parahaemolyticus Nucleic Acid Binding Agents
Types of agents that can be used to determine levels of pathogenic V. parahaemolyticus-encoding polynucleotides may include, but are not limited to cDNA, riboprobes, synthetic oligonucleotides, and genomic probes. Probes of the invention may also be used in real-time PCR (also referred to as qPCR) methods. It will be understood that primers listed herein are also probes in the sense that they bind, hybridize, anneal to a sequence. The type of agent (e.g. probe) used will generally be dictated by the particular situation, such as riboprobes for in situ hybridization, and cDNA for Northern blotting, for example. In certain aspects of the invention, the probe is directed to nucleotide regions unique to the polypeptide. Detection of the pathogenic V. parahaemolyticus-encoding gene, per se, will be useful for diagnostic methods of the invention and for screening for compounds to treat infection and/or contamination by the pathogenic V. parahaemolyticus bacteria. Other forms of assays to detect targets more readily associated with levels of expression—transcripts and other expression products—will generally be useful as well. A probe may be as short as is required to differentially recognize pathogenic V. parahaemolyticus mRNA transcripts, and may be as short as, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 bases, or more may be used. Non-limiting examples of polynucleotides that may be considered probes in certain instances are set forth herein as: SEQ ID NOs:2, 3, 4, 5, 6, 7, 9, 10, 12, 13, 14, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, and polynucleotides identified as primers and probes in Tables 2 and 3. One or more probes may also be reverse engineered by one skilled in the art, for example using the nucleic acid sequence of a pathogenic V. parahaemolyticus provided herein, such as SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:38 or other provided sequence or variant thereof. The form of labeling of a probe used in an embodiment of the invention may be any that is appropriate, such as the use of radioisotopes, for example, 32P and 35S, etc. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of suitably labeled bases using methods well known in the art. For use in some aspects of the invention, probes may be detectably labeled with a fluorescent (also referred to herein as a chemiluminescent or luminescent) molecule, examples of which include, but are not limited to: Hexachloro-Fluorescein (HEX), VIC fluorescent dye, 4-5-Dichloro carboxy fluorescein (JOE), Cy3 fluorescent dye, and TexasRed (TxRed). Additional types of detectable labels, known in the art and/or disclosed herein may also be used in conjunction with polynucleotide probes of the invention. In some embodiments of the invention a V. parahaemolyticus molecule may be detected as part of a complex with one or more additional molecules such as polynucleotides or labeling agents.
Pathogenic V. parahaemolyticus RNA Detection Techniques
Detection of RNA transcripts may be achieved by Northern blotting, for example, wherein a preparation of RNA is run on a denaturing agarose gel, and transferred to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Radiolabeled cDNA or RNA is then hybridized to the preparation, washed and analyzed by autoradiography.
Detection of RNA transcripts can further be accomplished using known amplification methods. For example, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by real-time polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, or reverse transcribe mRNA into cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR). Each of these methods is well known and routinely used in the art. Other known amplification methods can also be utilized in methods of the invention. In situ hybridization visualization may also be employed, wherein a radioactively labeled antisense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography. Biological samples may be stained with haematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion. Non-radioactive labels such as digoxigenin, or others referred to herein and/or known in the art may also be used.
Pathogenic V. parahaemolyticus Antibodies and Additional Binding Agents
In addition to using PCR and RNA detection methods for detection of a pathogenic V. parahaemolyticus molecule in diagnostic methods of the invention, it may also be possible to use other molecules or compounds that bind to a pathogenic V. parahaemolyticus molecule identified as being expressed in pathogenic but not non-pathogenic V. parahaemolyticus bacteria. For example, it may be possible to identify compounds, and/or molecules such as polypeptides that specifically bind to a pathogenic V. parahaemolyticus molecule. In addition, it may also be possible to use an antibody or other compound or molecule that binds to, and permits detection of a pathogenic V. parahaemolyticus molecule in a sample. In some embodiments a pathogenic V. parahaemolyticus molecule may be detected as part of a complex with one or more additional polypeptides.
An isolated pathogenic V. parahaemolyticus polypeptide, or fragment thereof, can be used as an immunogen to generate antibodies that bind pathogenic V. parahaemolyticus polypeptides using standard techniques for polyclonal and monoclonal antibody preparation. The full-length pathogenic V. parahaemolyticus polypeptide can be used or, alternatively, the invention provides antigenic peptide fragments of a pathogenic V. parahaemolyticus for use as immunogens. The antigenic peptide of pathogenic V. parahaemolyticus may comprise at least 8 amino acid residues of the amino acid sequence set forth herein and encompasses an epitope of a polypeptide from a pathogenic V. parahaemolyticus that is not present in a non-pathogenic V. parahaemolyticus polypeptide.
Pathogenic V. parahaemolyticus Protein Detection Techniques
In certain embodiments of the invention, methods include the use of diagnostic molecules (e.g., antibodies, antibody equivalents, binding molecules, etc.) to detect pathogenic V. parahaemolyticus polypeptides. Amino acid sequences of pathogenic V. parahaemolyticus polypeptides prp, cps, and flp are provided herein as SEQ ID NOs: 41, 42, and 46, respectively. Methods for the detection of polypeptides are well known to those skilled in the art, and include ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), Western blotting, and immunohistochemistry. Methods for immunoassays are routinely used and are well known in the art.
ELISA and RIA procedures may be conducted such that a pathogenic V. parahaemolyticus standard is labeled (with a radioisotope such as 125I or 35S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, pathogenic V. parahaemolyticus in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-pathogenic V. parahaemolyticus antibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable. Enzymatic and radiolabeling of a detection agent (e.g., antibodies, binding molecules, etc.) may be carried out by conventional means.
Other techniques may be used to detect pathogenic V. parahaemolyticus molecules according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting (Towbin et al., 1979 Proc. Nat. Acad. Sci. 76:4350), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti-pathogenic V. parahaemolyticus antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including but not limited to 125I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
Immunohistochemistry may be used to detect expression of pathogenic V. parahaemolyticus in a biological sample. A suitable antibody is brought into contact with, for example, cells, washed, and then contacted with a second, labeled antibody. Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabelling. The assay may be scored visually, using microscopy, or using any other suitable methods.
Pathogenic V. parahaemolyticus Detection Kit
The invention also encompasses kits for detecting the presence of pathogenic V. parahaemolyticus in a sample (e.g., a cell sample, tissue sample, water sample, seafood sample, fish sample, wound sample, etc.). For example, a kit can comprise a labeled or labelable agent capable of detecting a pathogenic V. parahaemolyticus polypeptide or polynucleotide (e.g., RNA, DNA, etc.) in a sample and a means for determining a status (e.g., at least one of the presence and level/amount) of a pathogenic V. parahaemolyticus molecule in the sample. The agent can be packaged in a suitable container. The kit can further comprise a means for comparing the level of the pathogenic V. parahaemolyticus molecule in the sample with a standard or control and/or can further comprise instructions for using the kit to detect the pathogenic V. parahaemolyticus polynucleotide or polypeptide. A kit may also include a detectably labeled probe.
This invention in some aspects also provides a kit for measuring pathogenic V. parahaemolyticus molecules. Such a kit may include a diagnostic agent (e.g., an antibody or antibody fragments, or binding molecule, etc.) that selectively bind a pathogenic V. parahaemolyticus molecule or a set of DNA oligonucleotide primers that allows synthesis of cDNA encoding the polypeptide or a DNA probe that detects expression of a pathogenic V. parahaemolyticus mRNA, etc. In some embodiments of the invention, the primers and probes may comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more nucleotides and hybridize under stringent conditions to a DNA fragment having the nucleic acid sequence set forth herein, for example, the nucleic acid sequence of an ST36prp polynucleotide, a ST36cps polynucleotide, a tlh polynucleotide, a tdh polynucleotide, a trh polynucleotide, an ST36flp polynucleotide, an ST631end polynucleotide, ST631-ENV polynucleotide, ST34lys polynucleotide, an ST34reg polynucleotide, an ST1127hyp polynucleotide, an ST36Phage or other polynucleotide set forth in Table 2, Table 3, or elsewhere herein, or a variant thereof. As herein used, the term “stringent conditions” means hybridization will occur only if there is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, identity between the sequences. Table 1 provides additional information in reference to the prp, cps, flp, tdh, trh, and tlh sequences and
Methods of Detection
The invention in some aspects provides methods for detecting the presence of a pathogenic V. parahaemolyticus molecule in a sample. The method may comprise contacting the sample with an agent capable of detecting pathogenic V. parahaemolyticus polypeptide or polynucleotide molecules (e.g., pathogenic V. parahaemolyticus RNA, cDNA, mRNA, or DNA, etc.) such that the presence of pathogenic V. parahaemolyticus is detected in the sample. An agent for detecting pathogenic V. parahaemolyticus polynucleotide using methods of the invention may be a labeled or labelable nucleic acid probe capable of hybridizing to V. parahaemolyticus mRNA. The nucleic acid probe may be, for example, the full-length V. parahaemolyticus sequences set forth herein, including but not limited to: SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus) and SEQ ID NO:38 (flp locus), or a portion thereof, such as an oligonucleotide of at least 3, 4, 5, 10, 15, 30, 50, 100 or more nucleotides in length and sufficient to specifically hybridize under stringent conditions to pathogenic V. parahaemolyticus mRNA. Polypeptides that may be detected using methods of the invention, and whose presence may indicate the presence of a pathogenic V. parahaemolyticus bacteria include, but are not limited to the polypeptides set forth herein as SEQ ID NO:41, 42, and 46. Polynucleotides that may be used as probes in certain embodiments of methods and kits of the invention include, but are not limited to primers and probes set forth in Tables 2 and 3.
The term “labeled or labelable”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the diagnostic molecule (e.g., probe, antibody, binding molecule, etc.), as well as indirect labeling of the molecule by reactivity with another reagent that is directly labeled. Some non-limiting examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with a fluorescent molecule, with biotin such that it can be detected with fluorescently labeled streptavidin, or other detectable label.
Detection methods useful in certain embodiments of methods of the invention, including but not limited to those described above herein, can be used as the basis for a method of detecting the presence or absence of a pathogenic V. parahaemolyticus molecule, can be used as the basis for a method of monitoring the progression of an infection or contamination by the pathogenic V. parahaemolyticus bacterium in a subject or substrate, or can be used as the basis for a method of determining a prognosis for a subject at risk for infection with or a subject or substrate at risk for contamination by a pathogenic V. parahaemolyticus bacterium.
Some aspects of the invention provide a prognostic method for determining whether a subject is at risk for developing an infection by pathogenic V. parahaemolyticus bacteria, or whether a substrate of subject is at risk for contamination by a pathogenic V. parahaemolyticus bacterium. Such methods may include contacting a sample obtained from the subject or substrate (or isolate of the sample) with an agent capable of detecting a pathogenic V. parahaemolyticus polypeptide or polynucleotide such that the presence and/or level of the pathogenic V. parahaemolyticus polypeptide or polynucleotide is detected in the sample or isolate, thereby determining whether the subject or substrate has an infection and/or contamination and/or is at risk for developing an infection or contamination by the pathogenic V. parahaemolyticus bacterium.
In some aspects and embodiments, diagnostic methods of the present invention may include determining the level of a pathogenic V. parahaemolyticus polynucleotide in a sample or isolate from a sample. In certain embodiments, methods of the present invention may include comparing the level of a pathogenic V. parahaemolyticus polypeptide or polynucleotide in a sample or isolate with the level of the pathogenic V. parahaemolyticus polypeptide or polynucleotide in a control sample. In yet another embodiment, a diagnostic or prognostic method of the invention may also include a step of forming a prognosis or forming a diagnosis, and may also include a step of determining an appropriate treatment for a subject or substrate by a health-care provider, environmental scientist, scientist, public health personnel, or other appropriate party, based at least in part on the determination of the presence, absence, and/or amount of the pathogenic V. parahaemolyticus molecule in a sample from the subject or substrate.
In certain aspects of the invention, determining the presence and/or amount of a pathogenic V. parahaemolyticus molecule can be carried out in conjunction with the determination of one or more other biomarkers of the pathogenic V. parahaemolyticus bacteria. For example, the level and/or presence of a pathogenic V. parahaemolyticus molecule (e.g., a polynucleotide that encodes a pathogenic V. parahaemolyticus polypeptide, a pathogenic V. parahaemolyticus polypeptide or signature fragment thereof), may be detected using a panel that also permits detection at least one additional molecule whose expression and/or level is may be useful to characterize an infection or contamination with the pathogenic V. parahaemolyticus bacteria. For example, a panel of the invention may comprise a means to determine at least one of the level or presence one of more of a prp locus, a cps locus, and an ftp locus, etc. to generate an aggregate biomarker panel. In certain embodiments, a panel of the invention may comprise a means to determine at least one of the level or presence one of more of a prp locus, a cps locus, a tlh locus, a tdh locus, a trh locus, and an ftp locus, etc. to generate a biomarker panel that may be used in certain embodiments of the invention. A biomarker panel of the invention may include detection means for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 50, 100 or more biomarkers (including each integer in between the listed integers) in addition to a detection means for a pathogenic V. parahaemolyticus bacteria as set forth herein. As used herein, in certain aspects of the invention the term biomarker means a polynucleotide or polypeptide that is specific to a pathogenic V. parahaemolyticus bacteria
Pathogenic V. parahaemolyticus Sequences and Variants
One aspect of the invention involves isolated polynucleotide molecules that encode polypeptides of pathogenic V. parahaemolyticus bacteria or biologically active portions thereof, as well as polynucleotide fragments sufficient for use as hybridization probes to identify a pathogenic V. parahaemolyticus bacteria polypeptide-encoding polynucleotide. A polynucleotide or polypeptide of the invention may be a “signature” polynucleotide or polypeptide, which, as used herein means a polynucleotide or polypeptide that is present in a pathogenic V. parahaemolyticus bacteria but not present in a non-pathogenic bacterium, including but not limited to a non-pathogenic parahaemolyticus bacterium. Thus, the presence of a signature nucleic acid or polypeptide of a pathogenic V. parahaemolyticus bacterium in a biological sample indicates the presence of the pathogenic V. parahaemolyticus bacterium in the sample. Examples of signature polynucleotide sequences provided herein, though not intended to be limiting are V. parahaemolyticus sequences set forth as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), and SEQ ID NO:38 (flp locus), or fragments thereof that are sufficiently unique so as to identify the presence of the prp locus, cps locus, or flp locus, respectively. Examples of some of the signature polypeptide sequences provided herein, though not intended to be limiting are V. parahaemolyticus sequences set forth as SEQ ID NO:41 (prp locus), SEQ ID NO:42 (cps locus), and SEQ ID NO:46 (flp locus), or fragments thereof that are sufficiently unique so as to identify the presence of the prp locus, cps locus, or flp locus, respectively.
As used herein, the term “polynucleotide” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., RNA, mRNA). A polynucleotide may be single-stranded or double-stranded or may be a double-stranded DNA molecule. An “isolated” polynucleotide molecule is free of sequences that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. The terms: “polynucleotide”, “oligonucleotide”, may be used interchangeably. For example an oligonucleotide primer may also be referred to as a polynucleotide primer, etc. Moreover, an “isolated” polynucleotide such as a cDNA molecule may be free of other cellular material.
In some aspects of the invention, an isolated polynucleotide of the invention comprises the nucleic acid sequence set forth herein as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID NO. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus), or another sequence set forth in Tables 2 and 3 or elsewhere herein, or a variant and/or fragment thereof. Some nucleic acid sequences provided herein are identified as sequences of polynucleotides found in pathogenic V. parahaemolyticus bacteria, for example SEQ ID NOs: 1, 8, and 38. Some nucleic acid sequences provided herein are identified as sequences of polynucleotides found in V. parahaemolyticus bacteria that need not be pathogenic, for example SEQ ID NOs:11, 15, and 18. Table 1 provides additional information in reference to the prp, cps, flp, tdh, trh, and tlh sequences.
The invention further encompasses polynucleotide molecules that differ from the sequences for the V. parahaemolyticus sequences set forth as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus) or another sequence set forth in Tables 2 and 3 or elsewhere herein, or a portion thereof, due to degeneracy of the genetic code and thus encode the same V. parahaemolyticus polypeptide as that encoded by the sequences set forth as set forth as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus), or another sequence set forth in Tables 2 and 3 or elsewhere herein, respectively. Encoded V. parahaemolyticus polypeptides include, but are not limited to sequences set forth herein as SEQ ID NOs:41, 42, 43, 44, 45, and 46, and additional amino acid sequences disclosed herein.
A polynucleotide having the nucleic acid sequence as set forth herein as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus), or another sequence set forth in Tables 2 and 3 or elsewhere herein, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a cDNA library can be probed using all or portion of a sequence set forth as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus) as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd. edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
Moreover, a polynucleotide encompassing all or a portion of a sequence set forth as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus) or other polynucleotide sequence disclosed herein including but not limited to a ST36end locus, a ST34reg locus, a ST674hyp locus, a ST1127hyp locus, etc. can be isolated using any suitable method, including as a non-limiting example, use of the polymerase chain reaction using oligonucleotide primers set forth herein and/or designed based upon the sequence set forth herein as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus), ST36end locus, ST34reg locus, ST674hyp locus, ST1127hyp locus, etc. can be isolated. For example, V. parahaemolyticus mRNA can be isolated from samples using standard, art-known methods and cDNA can be prepared using reverse transcriptase and art-known methods. Synthetic oligonucleotide primers for PCR amplification, such as those provided herein, can be designed based upon one or more nucleic acid sequences set forth herein as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus), a ST36end locus, a ST34reg locus, a ST674hyp locus, a ST1127hyp locus, etc. and polynucleotides of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The polynucleotide so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to pathogenic and non-pathogenic V. parahaemolyticus polypeptide-encoding nucleic acid sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In addition to the pathogenic and non-pathogenic V. parahaemolyticus bacterial nucleic acid sequences set forth herein as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), and SEQ ID NO:38 (flp locus), ST36end locus, ST34reg locus, ST674hyp locus, ST1127hyp locus, etc., it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of pathogenic or non-pathogenic V. parahaemolyticus may exist within a population (e.g., the bacterial population). Such genetic polymorphism in the V. parahaemolyticus gene may exist among bacteria within a population due to natural allelic variation. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in V. parahaemolyticus that are the result of natural allelic variation and that do not alter the pathogenicity of the V. parahaemolyticus bacteria are intended to be within the scope of the invention. Polynucleotide molecules corresponding to natural allelic variants of a V. parahaemolyticus cDNA of the invention can be isolated based on their homology to the polynucleotides disclosed herein using the cDNA, or a portion thereof, as a hybridization probe—according to standard hybridization techniques under stringent hybridization conditions, which are recognized in the art.
In some aspects of the invention, an isolated polynucleotide of the invention that hybridizes under stringent conditions to a nucleic acid sequence set forth as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus), ST36end locus, ST34reg locus, ST674hyp locus, a ST1127hyp locus, or other sequence provided herein, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleic acid sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural V. parahaemolyticus polypeptide.
In addition to naturally occurring allelic variants of the V. parahaemolyticus sequence that may exist in the bacterial population, the skilled artisan will further appreciate that changes may be introduced by mutation into a nucleic acid sequence set forth as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus), ST36end locus, ST34reg locus, ST674hyp locus, ST1127hyp locus, or other sequence provided herein thereby leading to changes in the amino acid sequence of the encoded V. parahaemolyticus protein without altering the functional ability of the V. parahaemolyticus protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made in the sequences. A “non-essential” amino acid residue is a residue that can be altered from a V. parahaemolyticus polypeptide without altering the activity of the V. parahaemolyticus polypeptide, whereas an “essential” amino acid residue is required for the V. parahaemolyticus polypeptide activity.
The invention, in some embodiments, includes use of full-length sequences of fragments of sequences of polynucleotides that encode pathogenic and non-pathogenic V. parahaemolyticus polypeptides, but that differ in sequence from the nucleic acid sequences set forth as SEQ ID NO:1 (prp locus), SEQ ID NO:8 (cps locus), SEQ ID No. 11 (tlh locus), SEQ ID NO:15, (tdh locus), SEQ ID NO:18 (trh locus), SEQ ID NO:38 (flp locus), ST36end locus, ST34reg locus, ST674hyp locus, ST1127hyp locus, or other sequence provided herein and yet can still be used in method of the invention to detect one or more of the presence and level of a pathogenic V. parahaemolyticus bacteria in a sample. In certain embodiments of the invention, a variant of a prp polynucleotide, useful in a method of the invention, comprises a nucleic acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:1 or fragment thereof; a variant of a cps polynucleotide useful in a method of the invention comprises a nucleic acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:8 or fragment thereof; a variant of a tlh polynucleotide useful in a method of the invention, comprises a nucleic acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:11 or fragment thereof; a variant of a tdh polynucleotide useful in a method of the invention, comprises a nucleic acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:15 or fragment thereof; a variant of a trh polynucleotide useful in a method of the invention, comprises a nucleic acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:18 or fragment thereof; a variant of a flp polynucleotide useful in a method of the invention, comprises a nucleic acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:38 or fragment thereof; and a variant; or a ST36end locus, ST34reg locus, ST674hyp locus, ST1127hyp locus, or other polynucleotide sequence provided herein that is useful in a method of the invention, comprises a nucleic acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to its corresponding polynucleotide sequence of the ST36end locus, ST34reg locus, ST674hyp locus, ST1127hyp locus, or other polynucleotide sequence provided herein, respectively.
It will be understood that a fragment of one or more polynucleotides comprising a nucleic acid sequence set forth herein as SEQ ID NO:1, SEQ ID NO:8, SEQ ID No. 11, SEQ ID NO:15, SEQ ID NO:18, or SEQ ID NO:38 or a polynucleotide sequence of the ST36end locus, ST34reg locus, ST674hyp locus, ST1127hyp locus, or other polynucleotide sequence provided herein can be used in methods of the invention, for example, though not intended to be limiting, as primers or probes. A fragment as used herein may be a portion of a sequence set forth herein, or variant thereof. As used herein, a portion of a polynucleotide having a length of “n” means a polynucleotide having a length of “n” minus at least one, two, three, four, five, or more nucleotides. For example if “n” equals the number of nucleotides in a sequence, a fragment may have a length of “n” minus at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides (including all intervening integers). It will be understood by those in the art that the number of nucleotides that can be subtracted from a sequence to result in a fragment will depend on the number of nucleotides in the sequence, for example, more fragment sizes can be obtained from a polynucleotide having nucleic acid sequence that is 2000 nucleotides in length than can be obtained from a polynucleotide having a nucleic acid sequence that is 100 nucleotides in length. In certain embodiments of the invention, a polynucleotide fragment includes between 4 and “n” minus one nucleotides of the nucleic acid sequence having “n” nucleotides set forth herein, or a variant thereof.
To determine the percent similarity/identity of two polynucleotides the nucleic acid sequences of the polynucleotides can be aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one polynucleotide for optimal alignment with the other polynucleotide). The nucleotide residues at corresponding positions are then compared. When a position in one sequence is occupied by the same nucleotide residue as the corresponding position in the other sequence (e.g., a variant form of the V. parahaemolyticus sequence), then the molecules have identity at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % similarity=number of identical positions/total number of positions×100). Such an alignment can be performed using any one of a number of well-known computer algorithms designed and used in the art for such a purpose. In some aspects of the invention, a variant of a polynucleotide comprises a fragment of the polynucleotide. In certain aspects of the invention, a variant of a polynucleotide is or comprises a portion of the polynucleotide's nucleic acid sequence that has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the corresponding region of the polynucleotide's nucleic acid sequence when the sequences are aligned. For example, a polynucleotide fragment that is 20 nucleotides in length may have 100% identity with the nucleic acid sequence of a polynucleotide that is 200 nucleotides in length if all 20 nucleotides have identical positions shared by the sequences (e.g., are identical across the corresponding region) when the two polynucleotide sequences are aligned. Similarly, the sequence of a polynucleotide fragment that is 20 nucleotides long has identity of at least 90% to a nucleic acid sequence of a polynucleotide that is 200 nucleotides long if 18, 19, or 20 of the 20 nucleotides of the fragment are the same in the corresponding region of the nucleic acid sequence of the 200 nucleotide polynucleotide when the nucleic acid sequences of the fragment and the 200 nucleotide polynucleotide are aligned.
Polynucleotide fragments that may be useful in some embodiments of the invention may be primers or probes. Non-limiting examples of primer or probe sequences that may be useful in some embodiments of the invention are set forth herein as SEQ ID NOs: 2-7, 9, 10, 12-14, 16, 17, 19, 20, 36, 37, and other sequences set for in Tables 2 and 3, or complements thereof, some of which are set forth herein as SEQ ID Nos: 49, 50, 55, 56, 61, 62, etc. Those skilled in the art will understand how to determine a complement sequence using standard methods. In certain embodiments a primer sequence may also include additional elements, for example, though not intended to be limiting: a tag, detectable label, identifying sequence for purification, visualization, additional nucleotides, etc.
An isolated polynucleotide useful in methods of the invention can be created by introducing one or more nucleotide substitutions, additions, or deletions into a nucleic acid sequence such as, but not limited to, SEQ ID NO: 1, 8, 11, 15, 18, and 38, sequences set forth herein of the ST36end locus, ST34reg locus, ST674hyp locus, ST1127hyp locus, other polynucleotide sequence provided herein; or fragments thereof. Mutations can be introduced into the sequences by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. The following examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.
Identification of strains as ST36 currently requires costly and time-consuming DNA isolation, PCR amplification and sequencing of 7 housekeeping loci; however, a subset of the loci informs whether isolates are likely to be or are related to ST36. From among 94 clinical isolates from infections reported in Massachusetts, N.H., and Maine between 2010-2013, during which time infections from the ST36 strain were first reported from Atlantic sources [Martinez-Urtaza J, et al., (2013) N Engl J Med 369:1573-1574], 43 isolates were identical to ST36 at four housekeeping loci (dnaE, dtdS, pntA and tnaA) [González-Escalona N, et al., (2008) J Bacteriol 190:2831-2840]. The relationships of these probable ST36 isolates to 47 of the remaining clinical isolates from the region was determined by constructing a neighbor-joining tree of the concatenated and aligned sequences [Tamura K, et al. (2013) Mol Biol Evol 30:2725-2729] (
To identify genomic differences for use in a ST36-diagnostic assay, whole genome comparisons were performed between the published draft genome for serotype 04:K12 ST36 strain 10329 (the 33 contigs including NZ_AFBW01000001.1-33.1) [Gonzalez-Escalona N, et al., (2011) J Bacteriol 193:3405-3406] and the genomes of two other pathogenic strains, including the pandemic strain RIMD 2210633 (NC 004605.1, NC 004603.1) [Makino K, et al, (2003) The Lancet 361:743-749] and pre-pandemic strain BB22OP (NC 019955.1, NC 019971.1) [Jensen R V, et al., (2013). Genome Announce 1:e00002-00012]. The unique genome content was visualized [Alikhan N-F, et al., (2011) BMC Genomics 12:402 DOI:410.1186/1471-2164-1112-1402; Darling A C, et al., (2004) Genome Res 14:1394-1403] and re-annotated using a Vibrio sp. specific database in NCBI with Prokka 1.8 [Seemann T. (2014) Bioinformatics: DOI:10.1093/bioinformatics/btu1153]. Few coding regions (˜six) in three different contigs appeared unique to strain 10329. It was then determined the extent the ORFs within these regions were present in and potentially exclusive to all strains in the 10329 genome group based on comparisons with draft genomes of V. parahaemolyticus available at the time of this analysis (289 total) (www.ncbi.nlm.nih.gov/genome/691), accessed Oct. 12, 2014) [Altschul S F, et al., (1997). Nucl Acids Res 25:3389-3402].
ORFs that were absent from more than one high-quality draft genome of the 10329 genome group (not all of which are ST36), or that were present in many other genome groups were eliminated from further consideration thereby focusing further analysis on two different regions of contig 10329_28 that were also present in the four Northeast regional ST36 isolates. ST36 diagnostic loci were identified. Genomic were identified, including regions that were present in 10329_28 of strain 10329 and in Northeastern ST36 isolates, including MAVP-V, MAVP-26, MAVP-36 and MAVP-45, but that were absent in pandemic and pre-pandemic strains including the prp, cps and O-antigen flippase (flp) genes. From the first region in this contig, an ORF identified as a pathogenesis-related protein (locus prp) (EGF42613) based on its similarity to a single ORF in Vibrio sp. Ex25 (YP_003285914.1) was selected as a potential diagnostic locus due to its uniqueness, even outside the species.
The designation of prp was proposed for this locus even though the gene more likely encodes an endonuclease or DNA helicase based on similarity searches in NCBI [Altschul S F, et al., (1997) Nucl Acids Res 25:3389-3402] in the absence of compelling evidence for function and a different name. Two additional ORFs including one identified as encoding a capsular polysaccharide (locus cps) (EGF42671) and another as 0-antigen flippase (locus flp) (EGF42675) in a second region of the same contig were also identified as diagnostic targets due to their potential role in conferring the 04 antigenic property of the strain, which is a useful diagnostic phenotype.
To determine the extent that one or a combination of these loci are present only in strains closely related to ST36, multiple reference whole genome phylogenies were constructed with REALPHY v. 1.09 for a few high quality genomes from each NCBI genome group lineage that harbored at least one of the three loci under evaluation, and for strains branching relatively closely with the 10329 genome group (www.ncbi.nlm.nih.gov/genome/?term=vibrio %20parahaemolyticus) [Bertels F, et al., (2014) Mol Biolo Evol 31:1077-1088] (see
Only one high quality genome (MDVP13, ST678) in the 10329 genome group apparently lacks prp (see
Oligonucleotide primers that produce prp-, cps- and flp-specific amplicons were developed for simultaneous detection along with both hemolysin-encoding genes (tdh and trh) and the species specific locus (tlh) to improve an existing multiplex PCR assay [see for example: Panicker G, et al., (2004) Appl Environ Microbiol 70:7436-7444 and J. L. Nordstrom, et al., App Environ Microbiol (2007) 73:5840-5847]. Table 2 shows non-limiting examples of primers utilized in PCR amplifications.
Alignments of 29 available prp sequences [Tamura K, et al., (2013) Mol Biol Evol 30:2725-2729] allowed identification of regions of high sequence identity, which informed primer design to produce optimal amplicon size separation with the existing multiplex PCR amplicons. Secondary structure and primer cross-dimerization were minimized by design with the aid of the NetPrimer program (PREMIER Biosoft, CA, US). A similar strategy was used to design the cps and flp-specific PCR. Amplification of the prp locus was evaluated in individual and multiplex assays using genomic DNA from positive (F11-3A), and negative (G61) [Mahoney J C, et al., (2010) Appl Environ Microbiol 76:7459-7465; Ellis, C N, et al., (2012) Appl Environ Microbiol 78: 3778-3782] control strains using AccuStart PCR Supermix (Quanta, Md., US) with an initial denaturation at 94° C. for 3 minutes, followed by 30 cycles with a denaturation at 94° C. for 1 minute, primer annealing at 55° C. for 1 minute, and extension at 72° C. for 1 minute, and with a final extension at 72° C. at the completion of the cycling for 5 minutes.
Amplicons from the amplifications were evaluated using standard procedures, for example: by electrophoresis of 1.5 μl of sample on 1.2% SeaKem LE agarose (Lonza, Rockland, Me., USA) gel with 1× GelRed (Phenix Research Products, Candler, N.C., USA) in Tris-acetate-EDTA (TAE) buffer compared against a 1-kb Plus DNA ladder (Invitrogen, Grand Island, N.Y., USA).
The developed assay was also applied to the collection of 94 clinical strains and several closely related environmental strains (i.e. G1350) from the Northeast. The prp amplicon was detected in all 43 strains that grouped within the ST36 clade and no other clinical or environmental isolates from northern New England, even the three strains identified as most closely related to the ST36 clade (
These data indicate that the presence of the prp amplicon is diagnostic for ST36 complex strains, and that the cps (or flp) amplicon could further enhance accuracy of identification. Not only was the assay useful for clinical identification, but it should also aid in surveillance. It will help determine the extent of this strain's geographic expansion, establishment of stable local populations and the seasonal dynamics of these strains, thereby aiding in management of shellfish harvesting and reducing public health risk.
Specific Identification of Strains by Sequence Type Using PCR
In addition to ST36, several other sequence types have contributed to the rise in infections, and have been recovered from shellfish harvest areas suggesting they are not transient but resident pathogens in harvest areas. ST631 has caused infections from Atlantic harvest areas from FL to PEI Canada, and currently accounts for ˜10% of infections from Gulf of Maine sources. ST34 (and closely related ST324), ST674, and ST1127 are more rarely detected in harvest areas and cause fewer infections but may be emergent types. All five of these strain types carry the same pathogenicity island harboring both tdh and trh.
A similar approach to that described for identifying sequence-type (ST)-specific genetic loci for the invasive ST36 strain, (See Example 1; and Whistler et al, 2015), was used to develop a detection process for additional pathogenic lineages. Whole genome sequence comparisons were conducted, and using BLAST Ring Image Generator (BRIG) and Multiple Alignment of Conserved Genomic Sequence With Rearrangements (MAUVE), highly unique regions were visualized, located, and compared to various reference genomes. To ensure identified DNA was uniquely present only in specific lineages (or Sequence Types), BLAST and alignment comparisons were carried out using the NCBI database of all complete and draft genomes. Primers were designed (See Table 2 for invented strain identification primers) and evaluated using PCR amplification methods for specificity and accuracy on a collection of several hundred unique isolates, and the expected high degree of specificity/accuracy was confirmed.
Methods
Amplification
PCR amplifications were performed with AccuStart PCR Supermix (Quanta, Gaithersburg, Md., US) with an initial denaturation at 94° C. for 3 minutes, followed by 30 cycles with a denaturation at 94° C. for 1 minute, primer annealing at 55° C. for 1 minute, and extension at 72° C. for 1 minute, and with a final extension at 72° C. at the completion of the cycling for 5 minutes. Altered extension times were also used as appropriate. For example, Table 2 provides PCR annealing temps and indicates certain primer pairs for which a 1.5 min extension time was used. For example, in multiplex assays, a 1.5 min extension time was used when multiple amplifications were performed in the same cycler. As noted in Table 2, the extension time used for the TdhUreG primer pair was 3 minutes. Standard procedures to optimize PCR amplifications were used.
For the locus PCR amplifications, the amplicon length determined the length in time of the PCR extension reaction, which was 1 Kb per minute. All of the reactions were 3 minutes or less and most were 1 minute. The annealing temperatures used were one degree below the melt temperature. If two primer pairs were used in multiplex, the lower annealing temperature of the primers was used. For example, if the annealing for one was 55° and the other 57°, the 55° temperature was used.
Amplicon Detection
Amplicons from the amplifications were evaluated using standard procedures, for example: by electrophoresis of 1.5 μl of sample on 1.2% SeaKem LE agarose (Lonza, Rockland, Me., USA) gel with 1× GelRed (Phenix Research Products, Candler, N.C., USA) in Tris-acetate-EDTA (TAE) buffer compared against a 1-kb Plus DNA ladder (Invitrogen, Grand Island, N.Y., USA).
Additional studies have been performed using multiplex amplification methods including a tiered approach for strain identification in which the first round was the 5-amplicon (tlh/tdh/trh/prp3/cps) amplification, which was then double checked with prp2/flp amplification from the sample (see
Enumeration of ST36 and ST631 by Quantitative PCR-Based Most Probable Number (MPN) Methods
In order to use these specific loci for quantification of pathogenic Vibrio parahaemolyticus, qPCR assays were designed that are compatible with the currently employed FDA protocol designed by J. Jones (Nordstrom et al. 2007) and based on the Bacteriological Analytical Manual (see fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm). Table 3 herein provides a list of oligonucleotide primers used for pathogen quantification.
Methods
Amplification Conditions
Amplification methods were designed and optimized as needed using routine optimization procedures. For example, cycling was based on the length of the amplicon and annealing was based on temperature of melting. Table 3 provides locus information, primer sequences, and melting temperatures for the primers used in qPCR procedures. Table 3 also provides sequences of probes and some examples of detectable labels that were used. The Quanta ToughMix (Quanta Biosciences Inc, Gaithersburg, Md.) was used (according to manufacturer's instructions) for multiplex amplifications with primers for prp, flp, and end. The cycling parameters were as follows:
Cycle 1: 95° C. hold for 60 s (this was increased as necessary depending on enzyme used) Cycle 2 (45 times):
The MPN protocol of Nordstrom uses a two-step enumeration: In step one, enriched oyster homogenate lysates are evaluated for the presence of V. parahaemolyticus by detection of tlh with an internal amplification control IAC and in step two, lysates that are positive for V. parahemoltyicus are subsequently evaluated for the presence of two virulence associated markers, including tdh and trh (with the IAC control) allowing the determination of the most probable number of these bacteria per gram of oyster.
Studies that were performed identified that a major limitation of the Nordstrom test was that these markers are present in environmental strains that are likely not pathogenic, and the markers are absent in some clinical strains so they are associated with virulence, but are not very useful for predicting the abundance of specific pathogen type strains.
Experiments were performed to develop a more specific detection strategy that can be added to this widely used platform and/or with other testing described herein, to improve risk assessment by specific quantification of the two strains that cause a predicted 85% of the cases from Atlantic harvest areas in the US. ST36 also causes more than 50% of the cases from the Pacific harvest areas of the US.
Experiments were carried out and a third assay step was developed. Studies were performed to evaluate lysates (samples) that were positive for either tdh or trh, and if found to be positive for one or both, the samples were further tested for the presence of ST36 by targeted amplification of both prp and flp, and for ST631 by the targeted amplification of end using probes and primers designed to these loci (Table 3), using qPCR amplification methods.
The probes were fluorescently labeled (see label information in Table 3) and they attached to the loci and when the primers amplified the sequence it released the quencher from the fluorofor, which was detected. These assays can also be performed using the detectable label: SYBR green or other detectable labels incorporated in the amplicon or linked to the probes, as indicated in Table 3 and elsewhere herein. Evaluation and quantitation of amplicons was performed using standard methods and manufacturer's instructions.
The developed PCR reactions were compatible with the FDA-required IAC internal control. Presence of these pathogenic bacteria in the lysates was further confirmed by use of the strain specific PCR primers by end-point binary PCR (Table 2) including the cps locus, and the prp2 primer pair for validation, and ST631-ENV or end primers.
Other types of qPCR assays that were carried out included a combined qPCR detection of prp, flp, and end polynucleotides. In the assays, an initial step included one cycle of tlh/IAC amplification/detection, and if results were positive a next step was to amplify and detect tdh/trh/IAC, which was followed by a third step of amplifying and detecting prp/flp/end/IAC.
Identification of Sub Populations of ST36 that are Localized to the Gulf of Maine (Vipa-26 Marker) or Katama Bay of Martha's Vineyard (Vipa-36 Marker) and Invention of a Population Marker Based PCR
Whole genome phylogenies revealed that there were distinctive lineages with diagnostic characteristics of the populations in the Gulf of Maine, for Katama Bay of Martha's Vineyard that would allow traceback of isolates. The unique attributes were the presence of phage Pathogenicity Islands: Vipa-26 in the Gulf of Maine, and Vipa-36 only in Katama Bay. Experiments were performed and a detection assay that included PCR amplification processes for specific identification of strains by population/harvest areas was developed. Following application of the multiplex PCR (see Example 1 and Whistler et al., 2015), isolates that were positive for the ST36 marker were subsequently analyzed for the presence of f237-like or Vipa-like phage, followed by a PCR reaction that distinguished the Gulf of Maine, and Katama Bay population by size difference of the amplicon (see
Identification of a Shared Pathogenicity Island Architecture Among Tdh/Trh Positive Pathogens of Several Sequence Types (ST36, ST631, ST34/324, ST674, ST1127, ST749, and ST110), and Invention of PI-Hybrid Marker Based PCR
Through the application of PacBio genome sequencing and highly accurate genome assembly, it was identified that the major emergent and invasive pathogenic strains of V. parahaemolyticus harboring both tdh and trh share a previously undiscovered hybrid pathogenicity island architecture. The unique feature of the island is the presence of a smaller tdh island flanked by transposases that have integrated within the major pathogenicity island containing the urease cluster, trh, and type three secretion beta genes that are implicated in defining virulence. It was identified that it is not simply the presence of the tdh and trh genes in the genomes that is characteristic, but the synteny or architecture of these elements within the same island. Therefore a strategy was developed whereby all strains with this specific island architecture could be detected by PCR amplification through the juncture of these two islands from tdh to ureG and detection, for example, by electrophoresis.
The primers used in the PCR amplification are the primers listed in Table 2 as TdhUreG. The forward primer, SEQ ID NO:100, anneals to tdh gene, the reverse primer, SEQ ID NO: 101, anneals to ureG, and this unique juncture is the pathogenicity island. The region amplified is the region is this unique island. The reaction conditions are also provided in Example 1 and 2 herein with additional detail in Table 2. PCR amplifications were performed with AccuStart PCR Supermix (Quanta, Gaithersburg, Md., US) with an initial denaturation at 94° C. for 3 minutes, followed by 30 cycles with a denaturation at 94° C. for 1 minute, primer annealing at 55° C. for 1 minute, and extension at 72° C. for 3 minutes, and with a final extension at 72° C. at the completion of the cycling for 5 minutes.
Amplicons from the amplifications were evaluated using standard procedures, for example: by electrophoresis of 1.5 μl of sample on 1.2% SeaKem LE agarose (Lonza, Rockland, Me., USA) gel with 1× GelRed (Phenix Research Products, Candler, N.C., USA) in Tris-acetate-EDTA (TAE) buffer compared against a 1-kb Plus DNA ladder (Invitrogen, Grand Island, N.Y., USA).
In the gel shown in
Although several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified, unless clearly indicated to the contrary.
All references, patents and patent applications and publications that are cited or referred to in this application are incorporated in their entirety herein by reference.
This application is a National Stage filing under U.S.C. § 371 of PCT International Application PCT/US2016/021089, filed Mar. 5, 2016, which designates the U.S., and claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/128,764, filed Mar. 5, 2015, the content of each of which is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/021089 | 3/5/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/141370 | 9/9/2016 | WO | A |
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| 20030176687 | Ishizuka | Sep 2003 | A1 |
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| Number | Date | Country | |
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| 20180044719 A1 | Feb 2018 | US |
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| 62128764 | Mar 2015 | US |
