Patent Application
20250092470
A sequence listing is electronically submitted in XML format in compliance with 37 C.F.R. § 1.831 (a) and is incorporated by reference herein. The XML file is named D7962SEQ, was created on Sep. 18, 2024 and is 267 KB in size.
The present invention relates to the field of identification of pathogenic bacteria and pathogenic fungal. More particularly, the present invention relates to methods to detect and to discriminate among a set of bacteria and fungi via microarray analysis.
Bacteria and fungi are a major cause of disease in humans and animals especially infections of urine, blood, or wounds. Such infection can manifest symptomatically as a “syndrome” which may be caused by any one of or combinations among a set of bacteria or fungal pathogens. The early detection of the disease-causing pathogen or pathogens in such syndromic presentation requires the ability to detect and discriminate multiple candidate microbial species quickly and simultaneously among a much larger set of microbes that can produce an infection in the blood or urine or a wound. Such testing is often referred to as “Syndromic Testing for Infectious Disease” that use multiplex methods for testing a large set of pathogens in parallel, which alone or in combination could be linked to a syndrome such as urinary tract infection or blood infection or wound infection. Generally, such a syndromic pathogen set may comprise bacteria, fungi or viruses and the syndromic testing could be based on nucleic acid or protein analytes. The present invention is focused on nucleic acid testing only and to detect and to discriminate causative bacteria and fungal pathogens.
Historically, such syndromic infections have been interrogated by culture methods, often requiring multiple days of culture testing on multiple growth media to detect and discriminate the causative pathogens. In some cases, such pathogens of concern culture slowly or are hard to differentiate from other possible members of a syndromic test panel. Thus, rapid screening of a sample to detect and discriminate one or combinations of microbial contamination, without culture, in hours rather than days could save lives by enabling early identification of the specific combination of microbes presenting in a subject, to guide diagnosis and treatment.
Thus, the prior art is deficient in means and methods of fast, culture-free detection and differentiation of multiple bacterial and fungal species in a single assay. Specifically, the prior art is deficient in methods that enable discrimination among elements of a syndromic test panel comprising multiple candidate bacteria and fungi as a high throughput lab-based molecular test.
The present invention is directed to a method for method for detecting and discriminating among multiple bacterial species and/or fungal species in a sample. In the method a sample is obtained from a subject. Bacteria and fungi are harvested from the sample and nucleic acids are isolated from the harvested bacteria and fungi. The bacterial and/or fungal ribosomal DNA or ribosomal RNA are amplified using at least two fluorescently-labeled primer pairs selective for bacterial and fungal hypervariable regions to generate at least two fluorescently labeled ribosomal nucleic acid amplicons. The fluorescently-labeled ribosomal nucleic acid amplicons are hybridized to a plurality of nucleic acid probes, where each probe in the plurality has a sequence complementary to a sequence determinant in one of the amplified ribosomal nucleic acid hypervariable regions and each probe is attached to a microarray. The microarray is washed at least once and imaged to detect a fluorescent signal from each of the two or more fluorescently-labeled ribosomal gene amplicons hybridized to the complementary probe. At least two hypervariable regions are analyzed concurrently, thereby detecting the bacterial species and/or fungal species in the sample.
The present invention also is directed to a related method that further comprises amplifying a region of interest in at least one additional gene using at least one fluorescently-labeled primer pair selective for the region of interest to generate at least one additional fluorescently labeled amplicon. The at least one additional fluorescently labeled amplicon is hybridized where the plurality of nucleic acid probes comprising the at least one additional probe has a sequence complementary to the region of interest. The microarray is imaged to detect a fluorescent signal from each of the at least one additional fluorescently-labeled amplicons hybridized to the complementary probe.
The present invention is directed further to a related method that further comprises quantifying an abundance of each species in the sample. In the method a known number of copies of a quantitative reference standard is added to the amplifying step where the quantitative reference standard has a nucleotide sequence similar to the nucleotide sequences of the hypervariable regions that are amplified. A probe with a sequence complementary to the nucleotide sequence of the quantitative reference standard is added to the microarray and the quantitative reference standard is hybridized to the complementary probe. A ratio of the hybridization signal in relative fluorescence units from the fluorescently-labeled amplicons hybridized to their complementary probes to a hybridization signal in relative fluorescence units from the quantitative reference standard hybridized to its complementary probe is measured. The ratio is uniquely correlated to a gene copy number of bacterial species or fungal species in the sample relative to the number of copies of the quantitative reference standard.
These and other features, aspects, and advantages of the embodiments of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings.
So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.
The articles “a” and “an” when used in conjunction with the term “comprising” in the claims and/or the specification, may refer to “one”, but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”. Some embodiments of the invention may consist of or consist essentially of one or more elements, components, method steps, and/or methods of the invention. It is contemplated that any composition, component or method described herein can be implemented with respect to any other composition, component or method described herein.
The term “or” in the claims refers to “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or”.
The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included.
The terms “consist of” and “consisting of” are used in the exclusive, closed sense, meaning that additional elements may not be included.
The term “including” is used herein to mean “including, but not limited to”. “Including” and “including but not limited to” are used interchangeably.
As used herein, the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., +/−5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.
As used herein, the terms “microarray” and “microarray support” are interchangeable.
As used herein, the term “subject” refers to a human subject or other mammal.
As used herein, the term “ESKAPE++pathogens” refers to the six pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., for example Enterobacter cloacae, plus the two additional pathogens Escherichia coli and Klebsiella aerogenes that are characterized by having increased levels of resistance to multiple classes of antibiotics.
In one embodiment of this invention, there is provided a method for detecting and discriminating among multiple bacterial species and fungal species in a sample, comprising obtaining a sample from a subject; harvesting bacteria and fungi from the sample; isolating nucleic acids from the harvested bacteria and fungi; amplifying bacterial and/or fungal ribosomal DNA or ribosomal RNA using at least two fluorescently-labeled primer pairs selective for bacterial and fungal hypervariable regions to generate at least two fluorescently labeled ribosomal nucleic acid amplicons; hybridizing the fluorescently-labeled ribosomal nucleic acid amplicons to a plurality of nucleic acid probes, each probe in the plurality having a sequence complementary to a sequence determinant in one of the amplified ribosomal nucleic acid hypervariable regions and each probe attached to a microarray; washing the microarray at least once; and imaging the microarray to detect a fluorescent signal from each of the two or more fluorescently-labeled ribosomal gene amplicons hybridized to the complementary probe; and analyzing concurrently at least two hypervariable regions, thereby detecting the bacterial species and/or fungal species in the sample. In this embodiment the amplifying step may comprise a PCR amplification, an RT-PCR amplification or an isothermal Transcription Mediated NASBA Amplification of rRNA.
Further to this embodiment the method comprises amplifying a region of interest in at least one additional gene using at least one fluorescently-labeled primer pair selective for the region of interest to generate at least one additional fluorescently labeled amplicon; hybridizing the at least one additional fluorescently labeled amplicon, where the plurality of nucleic acid probes comprises at least one additional probe having a sequence complementary to the region of interest; and imaging the microarray to detect a fluorescent signal from each of the at least one additional fluorescently-labeled amplicons hybridized to the complementary probe. In one aspect of both embodiments one of the at least one additional genes may comprise human RNaseP as an internal standard. In this aspect the fluorescently-labeled primer pair for the human RNaseP gene may have a nucleotide sequence of SEQ ID NOS: 10-11. In this aspect, the human RNaseP probe may have a nucleic acid of SEQ ID NO: 171. In another aspect of both embodiments, the at least one additional genes may comprise genes that confer antibiotic resistance to the bacterial species. In this aspect the antibiotic resistant bacterial species may be Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli and Klebsiella aerogenes.
In another further embodiment, the method comprises a step of quantifying an abundance of each species in the sample. In this further embodiment, the method may comprise adding a known number of copies of a quantitative reference standard to the amplifying step wherein said quantitative reference standard has a nucleotide sequence similar to the nucleotide sequences of the hypervariable regions that are amplified; adding to the microarray a probe with a sequence complementary to the nucleotide sequence of the quantitative reference standard; hybridizing the quantitative reference standard to the complementary probe; measuring a ratio of the hybridization signal in relative fluorescence units from the fluorescently-labeled amplicons hybridized to their complementary probes to a hybridization signal in relative fluorescence units from the quantitative reference standard hybridized to its complementary probe; and correlating uniquely the ratio to a gene copy number of bacterial species or fungal species in the sample relative to the number of copies of the quantitative reference standard. In this further embodiment, the quantitative reference standard may have a probe nucleotide sequence of SEQ ID NO: 173.
In all embodiments and aspects thereof, the fluorescently-labeled primer pair comprises a pair of nucleotide sequences that targets a bacterial 16S rDNA hypervariable region or a 28S D2 region, said fluorescently-labeled primer pair selected from the group consisting of SEQ ID NOS: 1 and 2, SEQ ID NOS: 1 and 3, SEQ ID NOS: 4 and 5, SEQ ID NOS: 6 and 7, SEQ ID NOS: 8 and 9 and a combination thereof.
In all embodiments and aspects thereof, the plurality of hybridization probes may comprise a set of bacterial probes each with a nucleotide sequence corresponding to the sequence determinant in a 16S hypervariable region 1 selected from the group consisting of SEQ ID NOS: 12-31, SEQ ID NOS: 32-50 and a combination thereof. Further to this, the plurality of hybridization probes may comprise a set of bacterial probes each with a nucleotide sequence corresponding to the sequence determinant in a 16S hypervariable region 3 selected from the group consisting of SEQ ID NOS: 51-75, SEQ ID NOS: 76-100, SEQ ID NOS: 101-111, SEQ ID NOS: 112-113 and a combination thereof. Further yet the plurality of hybridization probes may comprise a set of bacterial probes each with a nucleotide sequence corresponding to the sequence determinant in a 16S hypervariable region 6 selected from the group consisting of SEQ ID NOS: 114-122, SEQ ID NOS: 123-140, SEQ ID NOS: 141-152, SEQ ID NOS: 153-166 and a combination thereof. Further yet the plurality of hybridization probes may comprise a set of bacterial probes each with a nucleotide sequence corresponding to the sequence determinant in a 28S D2 region selected from the group consisting of SEQ ID NO: 167, SEQ ID NOS: 168-169 and a combination thereof.
In all embodiments and aspects thereof, the bacteria and the fungi may be associated with detecting and treating a urinary tract infection, a blood infection or a wound infection. Particularly, the bacteria associated with the urinary tract infection may be Acinetobacter baumannii, Aerococcus urinae, Citrobacter freundii, Citrobacter koseri, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Mycoplasma hominis, Mycoplasma genitalium, Proteus mirabilis, Proteus vulgaris, Providencia stuartii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus agalactiae, Ureaplasma parvum, or Ureaplasma urealyticum and the fungi associated with the urinary tract infection are Candida albicans or Candida glabrata. In addition, the subject may be a human or other mammal or a plant. Furthermore, the sample is a blood sample, a urine sample, a wound swab, a urogenital swab or other equivalent sample type, an air sample, a water sample, or a surface swab.
Provided herein are methods for detecting and discriminating among multiple bacterial species and fungal species in a sample. In the method, a sample is obtained from a subject, Bacteria and fungi are harvested from a sample obtained from a subject and nucleic acids are isolated therefrom. An amplification reaction is performed on the Bacterial and/or fungal ribosomal DNA or ribosomal RNA are amplified using at least two fluorescently-labeled primer pairs selective for two or more hypervariable regions of ribosomal genes or the RNA product of such genes. Two or more fluorescently-labeled ribosomal DNA or ribosomal RNA amplicons are generated thereby.
Such hypervariable (HV) regions of the ribosomal genes (rDNA) and the rRNA transcribed from them are well known. There are 9 such regions known in the bacterial 16S gene and the rRNA transcribed from it (
The sequence-specific diversity resident in the hypervariable regions of 16S and 28S ribosomal genes is well known and is widely used, especially in the context of sequencing, to detect and differentiate microbes in environmental and clinical samples. Such studies of sequence variation among the hypervariable regions have shown that in many cases, a single hypervariable region does not display enough sequence diversity to differentiate a species of bacteria from all others. In those cases, multiple hypervariable regions are sequenced concurrently. The present invention is based on a similar conceptual approach. However, rather than using next generation sequencing to measure sequence diversity among multiple hypervariable regions, a combination of multiplex amplification and highly multiplex sequence analysis by microarray hybridization provides the ability to detect and discriminate complex mixtures in a way that is much simpler, faster and less expensive than a sequencing-based alternative.
Specifically, fluorescently-labeled ribosomal hypervariable region amplicons are hybridized to a plurality of nucleic acid probes (see Tables 3A-3N), each probe in the plurality having a sequence corresponding to a sequence determinant resident in an amplicon and each designed to bind to a specific microbial species, for example, bacterial and fungal species, and each probe attached to a microarray. The microarray is washed at least once and the microarray is imaged to detect at least one fluorescent signal each from the two or more fluorescently-labeled bacterial or fungal amplicons with concurrent analysis of hybridization of two or more hypervariable regions in the same assay, thereby detecting the bacterial and/or fungal species in the sample.
Thus, the amplification with hybridization methods described herein are useful for resolving specific bacterial and fungal species in a sample. Up to four hypervariable regions may be amplified as a single multiplex amplification assay, that is, 16S-HV1, 16S-HV3, 16S-HV6 and 28S-D2. Simultaneous hybridization-based analysis of sequence determinants in multiple Hypervariable regions, is used to discriminate among species in a syndromic panel of microbial contaminants known to be causative for clinical presentation, such as a urinary tract infection or a blood infection or wound infection, where each of the nucleic acid probes is attached at a specific position on a microarray support.
Also provided is hybridizing the fluorescently-labeled amplicons to a sub-set of several nucleic acid probes in parallel, resident on the same microarray. Each of the probes comprising such a sub-set is specific to at least one sequence determinant within one of the ribosomal Hypervariable regions. For example, four hypervariable regions may be amplified, such as 16S-HV1, 16S-HV3, 16S-HV6 for bacteria and 28S-D2 or, alternatively, ITS2 for fungi. There may be, correspondingly, at least four probes provided on the microarray as a set, at least one such probe per amplified region. Each probe in that sub-set is designed to be specific for the same species.
The rational for such concurrent analysis of multiple hypervariable sites is shown in in
In the representative example of
The methods provided herein utilize both rDNA and rRNA target analytes. It is well known in the art that individual hypervariable regions (HV) of the ribosomal gene or its RNA product contain sequences which detect the presence of a species, i.e. have good inclusivity, but in many instances cannot unambiguously resolve the species of interest from other contaminating microbes or host factors, i.e. have poor exclusivity. In particular, many bacterial or fungal sequences presenting in a single HV region might display 90% inclusivity, that is, fail to detect the species of interest 10% of the time, but only deliver 50% exclusivity, that is, inadvertently misidentify as a false positive, a contaminating species as the species of interest 50% of the time.
The several hypervariable regions of the rRNA gene are known to have evolved semi-independently and thus are only weakly correlated. Thus, if (m) such weakly correlated HV sites are analyzed in parallel, as provided herein, if the statistical likelihood of a false positive were 0.5 for each, the likelihood of a false positive among (m) such sites measured as multiplex hybridization could approach 0.5m. For instance, for m=3 quasi-independent HV sites, the false positive rate would thus decrease from 50% to 12%, while the inclusivity would increase from 90% (1-10−1) to >99% (1-10−3). Such multiplex amplification of the multiple hypervariable regions presented in rRNA or rDNA, followed by hybridization of the resulting amplicons to a species-specific probe sub-set generates a substantial improvement of inclusivity and exclusivity and is central to the present invention.
The methods provided herein are useful for testing a biological specimen for the presence of bacteria and/or fungi. A sample is obtained from a human subject or other mammal, or from matter such as a swab in previous contact with the subject from which total nucleic acids are isolated. At least one amplification reaction is performed on the total nucleic acid using at least one fluorescently-labeled primer pair. Preferably, two or more fluorescently-labeled primer pairs, which are selective for two or more ribosomal hypervariable regions, are used to generate two or more fluorescently-labeled hypervariable rDNA or rRNA amplicons as described herein.
The fluorescently-labeled rDNA or rRNA amplicons are hybridized to nucleic acid probes each of which has a sequence complementary to a sequence determinant in at least one hypervariable region among the syndromic panel of microbes relevant to a specific clinical indication, such as urinary tract infection or blood infection or wound infection. The specific pattern of the fluorescent signal on the microarray support, among a panel of species-specific probes, differentiates the bacteria and/or fungal species present in the human or animal specimen and identifies the specific clinical indication.
An internal control may be incorporated into the methods provided herein. Particularly, one or more additional genes may be amplified and hybridized with the bacterial and/or fungal ribosomal DNA (rDNA) or ribosomal RNA (rRNA). For example, but not limited to, the human RNAseP gene (Table 3M) may be used as an internal control. Alternatively, the methods provided herein are useful for obtaining information about these additional genes within the bacterial and/or fungal species in the sample, for example, but not limited to, which, if any, of the genes or their RNA products confer antibiotic resistance to the identified bacteria and/or fungi.
Additionally, a quantitative reference standard (Table 3N) may be incorporated into the methods provided herein to quantify the abundance of each species in the sample. A known number of copies of the quantitative reference standard is added to the amplification step and is similar in sequence to the pathogen DNA regions being amplified, for example, but not limited to, the probe sequences for 16S-HV3-6 (Tables 3C-3J). The abundance of each species detected in the multiplex amplification assay is obtained from the measured ratio of the hybridization signal from a species specific probe to that of a corresponding probe which binds only to the Quantitative Reference Standard. A hybridization signal ratio [Pathogen/Standard RFU] is obtained from those data and may be uniquely linked to the ratio of pathogen gene copies present in a sample relative to the (known) number of standard copies per reaction (
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
PCR is performed on the rDNA Hypervariable regions of purified bacteria and fungi at the cycling conditions shown in Table 1. The PCR is performed as an asymmetric endpoint PCR reaction. Primer pairs for each of the N=4 elements of the multiplex are shown in Table 2.
The multiplex PCR is performed using one fluorescently labeled primer pair for each HV region (16S-HV1, -HV3, -HV6, 28S-D2). Table 2 lists non-limiting examples of primer pairs suitable for the asymmetric endpoint reaction for the entire species set of
Bacterial Hypervariable region amplicons, as in Example 1, are hybridized to a microarray or a microarray support, such as, but not limited to, a microarray with a functionalized solid surface, to which a plurality of bacterial and fungal nucleic acid probes are directly or indirectly covalently attached. The attachment site correlates to a specific nucleic acid sequence. The nucleic acid probes may be indirectly covalently attached via linker, for example, a bifunctional oligonucleotide linker, such as, but not limited to, the oligothymidine linker OLIGO-T, which is covalently attached at one terminal nucleotide to the functionalized and covalently crosslinked to at least one nucleic acid probe at the other terminus.
The approach is as follows. Define a specific set (N) of bacteria or fungi to be analyzed, typically 10 to 100 species such as the syndromic panel suitable for urinary tract infection testing in
Generally, each HV region is amplified by a single primer pair, found to be common to the “Universal” ribosomal gene sequence flanking each HV region. Specific examples are shown in Table 2, that is, one “Universal” primer pair each for the several HV regions to be used in the assay. At each of the HV regions, a gene sequence within each HV region is picked which is unique or nearly unique to each species in the set. Such “highly discriminating” sequences are used to design hybridization probes which are used to detect the presence of the unique sequence for that bacterium of fungi among the set of (N) species. Representative examples of such “highly discriminating” probes are shown in Tables 3A-3M. Updated probe sequence content is presented in a bold italic.
It is generally possible to find a “highly discriminating” sequence for a species in each 16S Hypervariable region (HV1, HV3, HV6) and fungal 28S D2. As a result, discrimination of a bacterial species is not based on hybridization to a single probe, but is instead based on analysis of hybridization to a probe sub-set, which for any species of interest comprises at least one “highly discriminating” probe in HV1 and HV3 and HV6 for bacterial species and at least one “highly discriminating” probe in D2 for fungal species, where species discrimination is obtained by concurrent analysis of hybridization to probes from all three HV regions.
Having found amplification primers and associated highly discriminating probe sequences in each of the 4 amplicons and having designed hybridization probes for them in all (N) species in the set, a multiplex assay is then performed comprising three steps. Firstly, multiplex PCR or RT-PCR or isothermal amplification of all Hypervariable regions under study. Secondly, hybridization of the resulting set of amplified nucleic acid products to a microarray. Thirdly, detect and discriminate all members of the set of N species, based on simultaneous sequence analysis by hybridization to the amplicon products from Hypervariable regions in parallel.
Tables 3A-3B list non-limiting examples of nucleic acid probes selective for sequence determinants complementary to specific bacterial species, as detected per the present invention, in the 16S hypervariable regions 1a and 1c. Tables 3C-3F list non-limiting examples of nucleic acid probes selective for sequence determinants complementary to specific bacterial species as detected per the present invention, in 16S hypervariable regions 3a, 3c, 3e, and 3g. Tables 3G-3J list non-limiting examples of nucleic acid probes selective for sequence determinants complementary to specific bacterial species as detected per the present invention in 16S hypervariable regions 6a, 6c, 6e, and 6g. Tables 3K-3L lists non-limiting examples of nucleic acid probes selective for sequence determinants complementary to specific fungal species as detected per the present invention in the fungal 28S-D2 hypervariable regions 1a and 1b. Table 3M lists non-limiting examples of nucleic acid probes selective for sequence determinants complementary to human RNAseP gene controls. Table 3M is an example of a quantitation reference standard for the 16S gene.
16s.HV1c.013
16s.HV3a.030
16s.HV3a.101
16s.HV3c.013
16s.HV3c.101
16s.HV6c.026
16s.HV6c.101
16s.HV6g.012
Multiplex PCR primer design (Example 1) and microarray hybridization probe design (Example 2) were used to design a PCR+Microarray assay for detection and discrimination of the set of pathogens known to be causative for urinary tract infection (
Hybridization was then followed by 15 min of washing at room temp, then imaging of the entire 96-well plate on a Sensospot Imager, using automated image analysis (Augury™). The resulting data comprise the CY-3 fluorescent signal obtained at each probe of the array measured in relative fluorescence units (RFU).
In
Per a standard naming used throughout, probes designed for species numbered (n) are given the same number (n). Thus in
The experimentally-derived hybridization data (in RFU) are presented in
Inspection of the data in
Analytical Limit of Detection Among Microbial Species that are Causative for Uti
The microarray workflow of
As more is learned about sequence heterogeneity within species or as more is learned about Species of Concern that may cause disease, it may be necessary to add or modify the PCR primers and/or the hybridization probes of the present invention, especially those probes which can interrogate such sequence variation within the hypervariable regions of the ribosomal 16S or 28S regions provided as examples in the present invention (see
In
Analytical LOD and Specificity Obtained with Updated Probe and Primer Design Using the Information Content of HV3 and HV6 Hypervariable Region for Bacteria and 28s Region for Fungi
Analytical Specificity was obtained with the update Primer and Probe set (HV3 and HV6) following the same protocol described in Example 3 and is presented in a similar format (
Clinical Concordance Obtained Vs qPCR on 377 Urine Samples Previously Analyzed by qPCR
377 clinical urine isolates have been obtained as anonymous samples from a CAP-CLIA lab. These samples has been previously subjected to DNA isolation via a combination of MagMax magnetic beads executed on a Kingfisher robot, per the workflow suggested by Thermo Fisher. The resulting purified DNA has been previously analyzed by multiplex qPCR (Thermo Fisher Open Array) for which species detection and discrimination had been obtained for the full set of Species of Concern described in
Acinetobacter baumannii
Aerococcus urinae
Candida albicans
Candida glabrata
Citrobacter freundii
Citrobacter koseri
Enterobacter cloacae
Enterococcus faecalis
Enterococcus faecium
Escherichia coli
Klebsiella oxytoca
Klebsiella pneumoniae
Morganella morganii
Proteus mirabilis
Proteus vulgaris
Providencia stuartii
Pseudomonas aeruginosa
Serratia marcescens
Staphylococcus aureus
Staphylococcus saprophyticus
Streptococcus agalactiae
Acinetobacter baumannii
Aerococcus urinae
Candida albicans
Candida glabrata
Citrobacter freundii
Citrobacter koseri
Enterobacter cloacae
Enterococcus faecalis
Enterococcus faecium
Escherichia coli
Klebsiella oxytoca
Klebsiella pneumoniae
Morganella morganii
Proteus mirabilis
Proteus vulgaris
Providencia stuartii
Pseudomonas aeruginosa
Serratia marcescens
Staphylococcus aureus
Staphylococcus
saprophyticus
Streptococcus agalactiae
Introduction of Quantitation into Multiplex Analysis Via the Use of Internal Quantitative Reference Standards for Hypervariable Regions
Quantitation of bacterial pathogens is performed internally within each array of the present invention by hybridization via simultaneous analysis of a synthetic internal ribosomal gene standard which contains the consensus sequence of its cognate HV regions (bacterial or fungal). Thus, if added to a PCR or RT-PCR master mix, the standard is amplified in parallel with the corresponding pathogen nucleic acid present in the same reaction (
Upon completion of PCR or RT-PCR amplification the two amplicon variants “Pathogen” vs “Standard” can be resolved by quantitation of hybridization signals obtained for the two types of probe, pathogen and standard as described in Table 5 and Table 6 (
Details of the process are as follows. The standard undergoes PCR amplification with the same efficiency as matched bacterial or fungal pathogens in the same sample via identical PCR priming sites in the conserved 16S rDNA or 28s rDNA flanking regions. In the present example, the standard's PCR product hybridizes to engineered HV3a, HV3c and HV6c probes complementary to its slightly altered 16sDNA sequence (Tables 3A, 3B, 3H), At least 3 independent measurements of these Pathogen/Standard hybridization rations are measured for each Species of Concern resident in such a test. Thus, a quantitative measure is obtained, in parallel, for all species detected in the assay.
The seminal data quantity is the Hyb ratio [Pathogen/Standard] which is related to the number of DNA molecules (and in turn cell equivalents) for each pathogen in a sample relative to the known number of standard molecules introduced per reaction, which is 5000 in Table 6 and
Ribosomal RNA (rRNA) Analysis by RT-PCR Amplification Followed by Array Hybridization Analysis
Example 9 provides a reduction to practice showing that rRNA, followed by Rt-PCR amplification, can be used as the Pathogen nucleic acid target in the present invention. For this first RT-PCR deployment, a pair of highly standardized E. faecalis bacterial cell lines ATCC 29212: Vancomycin Susceptible were chosen because it lacks van A/B antibiotic resistance genes) and ATCC 51299: Vancomycin Resistant because it contains a vanB gene). These two lines have been validated by CLSI (the Clinical Lab Safety Institute) and are used widely as standards for antibiotic susceptibility testing in CLIA labs. For the present example, they have both been expanded by fluid culture to mid log phase (Brain Heart Infusion Broth) harvested and enumerated by analytical plate culture using Brain Heart Infusion Agar Plates. The OD600 was also measured post overnight culture to estimate a cell/mL value for each. Total RNA was then extracted using a standard Zymo Bacterial RNA kit, including DNA removal with DNAse1 per manufacturer instructions: Zymo Quick-DNA/RNA MagBead kit catalog number R2130. For the two E. faecalis cell lines, the resulting total RNA preparation was then quantified by UV/VIS (nanodrop) with the resulting concentration converted to cell equivalents per unit volume assuming @100% recovery of RNA from the original enumerated cell preparation. Based on that quantitation, Serial dilutions were prepared to yield a total RNA input per RT-PCR RXN (5 uL/50 uL RXN) equivalent to the following 10-step dilution series [4.66×10+6, 4.66×10+5, 4.66×10+4, 4.66×10+3, 466, 46, 4.66, 0.466, 0.046 and 0] cell equivalents per RT-PCR reaction. An endpoint RT-PCR reaction was performed on each with cycling conditions (Table 5) very similar to the analogous endpoint PCR reaction used for rDNA (Table 1) with RT-PCR primers also identical to those used for PCR (Table 6). Subsequent to endpoint PCR, array hybridization was performed via the same microarray workflow used for PCR (
The raw hybridization results of those titrations are displayed in
In order to confirm that approximate aLOD and to interrogate the specificity of the analysis, all data on the 21×21 1319 array were submitted to concurrent hybridization analysis via Augury software (Table 7 shows representative Augury data output), over the entire titration range, for both E. faecalis lines see (Table 8).
Enterococcus faecalis
Candida glabrata
Enterococcus faecalis
Detection and Discrimination of these two well characterized E. faecalis (CLSI) standards was obtained over the entire 7-log range of input rRNA density (Table 8). Data obtained from this initial autonomous (Augury™) range finding analysis (Table 8) suggest an aLOD of <0.05 cell equivalent of rRNA for both lines, in good agreement with the visual inspection of the raw RFU data (Tables 7-8;
E faecalis
E faecalis
Enterococcus
Enterococcus
faecalis &
faecalis &
Candida
Candida
glabrata
glabrata
Enterococcus
Enterococcus
faecalis &
faecalis &
Candida
Candida
glabrata
glabrata
Enterococcus
Enterococcus
faecalis
faecalis
Enterococcus
Enterococcus
faecalis
faecalis
Enterococcus
Enterococcus
faecalis
faecalis
Enterococcus
Enterococcus
faecalis
faecalis
Enterococcus
Enterococcus
faecalis
faecalis
Enterococcus
Enterococcus
faecalis
faecalis
Enterococcus
Enterococcus
faecalis
faecalis
Autonomous Augury™ software analysis shows that the only bacterial species consistent with the observed pattern of hybridization was that of E. faecalis, even up to a 10+6 excess of input RNA above that of the apparent aLOD, suggesting excellent Discrimination. Interestingly at the two highest inputs (4.66×10+6, 4.66×10+5 cells/RXN) C. glabrata (a fungi) was also detected, selectively as distinct from C. albicans, which is also among the Species of Concern. It is likely that the presence of C. glabrata detected at very high sample input (>10+5 cells/RXN) may be real and due to small, but measurable trace contamination in the RNA preparation.
rRNA was extracted from cells (Table 9) purchased from Zeptometrix using the PDx OCTA automated system per manufacturers IFU. NASBA amplification was done using NASBA kit NWK-1 from Life Sciences Advanced Technologies Inc. following manufacturer's instruction. NASBA primers were customized for multiplex NASBA amplification to produce short HV3 and HV6 amplicons (Table 10). The principles used for such NASBA primer design have been previously described in a companion patent awarded U.S. Pat. No. 12,054,794 B2 issued Aug. 6, 2024 which is incorporated herein by reference.
Enterococcus faecium Z347, vanA
Staphylococcus aureus Z482, tittered
Klebsiella pneumoniae Z138,
Acinetobacter baumannii 307-0294
Pseudomonas aeruginosa Z189, VIM-1
Escherichia coli Z136, CTX-M-15
Klebsiella aerogenes Z052
Enterobacter cloacae Z101
Hybridization and Detection conditions were taken directly from the PCR based UTI array assay (
TTTCTACCGTACTCTAGCT.CC
ATAA CTCAACAG TGTATTA
TTTCTACCGTACTCTAGCT.CA
TTTCTACCGTACTCTAGCT
E. faecium
S. aureus
K. pneumoniae 1
A. baumannii
P. aeruginosa
Enterobacter
(cloacae)
Result: Data shown in
bacterial species and/or fungal species in the sample.
This non-provisional patent application claims benefit under 35 U.S.C. § 119 (e) of provisional application U.S. Ser. No. 63/584,008, filed Sep. 20, 2023, the entirety of which is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63584008 | Sep 2023 | US |
