Patent Application
20160244849
The present invention relates generally to a method for the detection of salmonid pathogens in a sample. More specifically, the present invention relates to a molecular diagnostic assay for detecting more than one salmonid pathogen in a sample simultaneously, for example using fluidic bead-based technology and a multiplexed PCR platform.
Viral and bacterial diseases are a major problem in the aquaculture industry. Outbreaks of infectious disease are a challenge facing fish farming operations, including those involving dense populations of fish in the open sea (Robertson, B. “Can we get the upper hand on viral diseases in aquaculture of Atlantic salmon?” Aquaculture Research, 42, 125-131, 2011). Viral and bacterial infections can have severe effects on the fish farming industry, and the importance of prevention, detection, and treatment of outbreaks is well-recognized.
Infectious Hematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Salmon Alphaviruses (SAV) and Viral Hemorrhagic Septicemia Virus (VHSV) are 5 of the most globally detrimental and economically damaging salmonid viruses. IHNV, IPNV, ISAV, SAV, and VHSV are RNA viruses. RNA viruses such as these cause the highest ecological and socio-economical impacts due to disease in European farmed finfish (Gomez-Casado, E.; Estepa, A.; Coll, J. M. “A comparative review of European-farmed finfish RNA viruses and their vaccines” Vaccine, 29(15), 2657-2671, 2011). Clinical and/or postmortem disease diagnosis of these viruses in fish is highly important, especially during disease outbreaks. Some countries have mandated inspections of artificially propagated fish for the presence of these types of fish pathogens as part of programs to limit fish exposure (Williams, K.; Blake, S.; Sweeny, A.; Singer, J. T.; Nicholson, B. L. “Multiplex Reverse Transcriptase PCR Assay for Simultaneous Detection of Three Fish Viruses” Journal of Clinical Microbiology, 37(12), 4139-4141, 1999).
Renibacterium salmoninarum is a gram-positive bacteria that causes bacterial kidney disease (BKD) in salmon. Bacterial kidney disease is a major cause of morbidity and mortality in salmon, and so R. salmoninarum is another economically and environmentally important salmonid pathogen. The detection of R. salmoninarum through the traditional methods (i.e. media culture and immunogical testing) requires a long time, and these methods are not sensitive enough to detect carrier fish. PCR and nested PCR has demonstrated a better sensitivity (Toranzo, A. E.; Magariños, B.; Romalde J. L. “A review of the main bacterial fish diseases in mariculture systems”. Aquaculture 246; 37-61, 2005). Quantitative PCR (qPCR) has also been developed, however, there is no significant difference in sensitivity between qPCR and nPCR (Elliot D. G; Applegate, L. J.; Murray, A. L.; Purcell, M. K.; McKibben, C. L. “Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture”. Journal of Fish Disease 36(9): 779-809, 2013).
Detection and monitoring of IHNV, IPNV, ISAV, SAV, and VHSV is currently done using PCR-based assays and/or Virus Isolation. PCR and bacterial isolation are currently used for detection of R. salmoninarum. Traditional polymerase chain reaction (PCR) and real time PCR both detect a single target at a time in one sample. This can be time consuming and costly, and thus there is a need for more efficient detection methods.
Multiplex PCR techniques have been developed for detection of various pathogens, including agricultural (US Patent Application No. 2011/0070586) and fish (Williams, K. et al. (1999) Journal of Clinical Microbiology. 37(12), 4139-4141) pathogens. However, continued improvement and development is still needed in order to develop a more robust multiplexing assay for salmonid pathogen detection.
It is an object of the invention to provide a method for detecting salmonid pathogens in a test sample.
It is also an object of the invention to provide reagents to use in the detection assay, including PCR primers and TSPE primers specific for pathogens such as Infectious Hematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Salmon Alphaviruses (SAV), Viral Hemorrhagic Septicemia Virus (VHSV), or Renibacterium salmoninarum.
Accordingly, there is provided herein a method for detecting the presence or absence of at least one pathogen including: IHNV, IPNV, ISAV, SAV VHSV, or Renibacterium salmoninarum in a sample. The method comprises:
There is also herein provided a method for detecting the presence or absence of at least one pathogen including: IHNV, IPNV, ISAV, SAV, VHSV, or Renibacterium salmoninarum in a sample. The method comprises:
In addition, there is provided a method for detecting the presence or absence of at least one pathogen including: IHNV, IPNV, ISAV, SAV VHSV, or Renibacterium salmoninarum in a sample. The method comprises:
The above-described methods may be provided in the form of an assay, including a PCR assay, a RT-PCR assay or a multiplexing RT-PCR assay. In addition, a system may also be provided for performing the aforementioned method or assay.
In certain non-limiting embodiments of the above-described methods, two or more of the pathogens may be detected in the sample simultaneously. Moreover, in other embodiments it may be preferred for three, four, five, or all six of the pathogens to be detected in the sample simultaneously. It is also envisioned that additional pathogens may be added to the methods or assays in panels where more than the six mentioned pathogens are tested.
Also provided herein are polynucleotide primers, including primers comprising a nucleic acid sequence having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 16, or 17, or a fragment thereof of at least 15 contiguous nucleotides.
Also provided herein is a primer pair for the detection of Infectious Hematopoietic Necrosis Virus (IHNV), comprising isolated nucleic acid primers having at least 80% identity to the sequence of SEQ ID NO: 4 and 9, or fragments thereof of at least 15 contiguous nucleotides.
Also provided herein is a primer pair for the detection of Infectious Pancreatic Virus (IPNV), comprising isolated nucleic acid primers having at least 80% identity to the sequence of SEQ ID NO: 2 and 7, or fragments thereof of at least 15 contiguous nucleotides.
Also provided herein is a primer pair for the detection of Infectious Salmon Anemia Virus (ISAV), comprising isolated nucleic acid primers having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ ID NO: 1 and 6, or fragments thereof of at least 15 contiguous nucleotides.
Also provided is a primer pair for the detection of Salmon Alphaviruses (SAV) comprising isolated nucleic acid primers having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ ID NO: 5 and 10, or fragments thereof of at least 15 contiguous nucleotides.
Also provided is a primer pair for the detection of Viral Hemorrhagic Septicemia Virus (VHSV), comprising isolated nucleic acid primers having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ ID NO: 3 and 8, or fragments thereof of at least 15 contiguous nucleotides.
Also provided is a primer pair for the detection of Renibacterium salmoninarum, comprising isolated nucleic acid primers having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ 11) NO: 16 and 17, or fragments thereof of at least 15 contiguous nucleotides.
Also provided is a use of a polynucleotide primer as described above in the detection of one or more pathogen including: Infectious Hematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Salmon Alphaviruses (SAV), Viral Hemorrhagic Septicemia Virus (VHSV), or Renibacterium salmoninarum.
Also provided is a target specific primer extension (TSPE) primer for the detection of Infectious Hematopoietic Necrosis Virus (IHNV), comprising a nucleic acid sequence having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ ID) NO: 14, or a fragment thereof of at least 15 contiguous nucleotides.
Also provided is a target specific primer extension (TSPE) primer for the detection of Infectious Pancreatic Virus (IPNV), comprising a nucleic acid sequence having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ ID NO: 12, or a fragment thereof of at least 15 contiguous nucleotides.
Also provided is a target specific primer extension (TSPE) primer for the detection of Infectious Salmon Anemia Virus (ISAV), comprising a nucleic acid sequence having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ ID NO: 11, or a fragment thereof of at least 15 contiguous nucleotides.
Also provided is a target specific primer extension (TSPE) primer for the detection of Salmon Alphaviruses (SAV), comprising a nucleic acid sequence having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ ID NO: 15, or a fragment thereof of at least 15 contiguous nucleotides.
Also provided is a target specific primer extension (TSPE) primer for the detection of Viral Hemorrhagic Septicemia Virus (VHSV), comprising a nucleic acid sequence having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ ID NO: 13, or a ligament thereof of at least 15 contiguous nucleotides.
Also provided is a target specific primer extension (TSPE) primer for the detection of Renibacterium salmoninarum, comprising a nucleic acid sequence having at least 80%, 85%, 95%, or 99% identity to the sequence of SEQ ID NO: 18, or a fragment thereof of at least 15 contiguous nucleotides.
Optionally, the ligaments noted above may all comprise 16, 17, 18, 19 or more contiguous nucleotides of the noted sequences. The primers may be synthetically prepared according to known methods.
Herein, there is also provided the use of a TSPE primer as described above in a method for the detection of one or more pathogen including: Infectious Hematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Salmon Alphaviruses (SAV), Viral Hemorrhagic Septicemia Virus (VHSV), or Renibacterium salmoninarum.
In addition, there is further provided a kit for detecting at least one pathogen including: Infectious Hematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Salmon Alphaviruses (SAV), Viral Hemorrhagic Septicemia Virus (VHSV), or Renibacterium salmoninarum in a sample, the kit including:
The assay kit may also comprise instructions for carrying out the methods as described herein.
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
Described herein is a diagnostic assay, as well as various PCR primers and TSPE primers which can be used for the detection of salmonid pathogens. In certain embodiments, these primers and probes can be used in a method capable of simultaneous detection of more than one pathogen in a sample using multiplexing technology.
In certain non-limiting embodiments, the assay is capable of simultaneous detection of two or more salmonid pathogens in a sample, including IHNV, IPNV, ISAV, SAV, VHSV, and Renibacterium salmoninarum. In further non-limiting embodiments, the assay can be customized for specific detection of 1, 2, 3, 4, 5, or 6 pathogens as a singleplex (e.g. ISAV, IHNV, IPNV, SAV, VHSV, or Renibacterium salmoninarum only), duplex (e.g. ISAV and IPNV, or other duplex combinations of IHNV, IPNV, ISAV, SAV, VHSV, and Renibacterium salmoninarum), triplex (e.g. IHNV, ISAV, and VHSV, or other triplex combinations of IHNV, IPNV, ISAV, SAV, VHSV, and Renibacterium salmoninarum), quadruplex (IHNV, ISAV, SHSV, and IPNV, or other quadruplex combinations of IHNV, IPNV, ISAV, SAV, VHSV, and Renibacterium salmoninarum), pentaplex (e.g. ISAV, IPNV, IHNV, SAV, and VHSV, or other pentaplex combinations of IHNV, IPNV, ISAV. SAV, VHSV, and Renibacterium salmoninarum), or hexaplex (e.g. IHNV, IPNV, ISAV, SAV, VHSV, and Renibacterium salmoninarum), depending on the needs of the user.
The assay may be especially useful for disease detection in salmonid stocks during disease outbreaks, and for surveillance and regulatory testing.
Without wishing to be limiting in any way, it is envisioned that some or all of the following steps may be performed when carrying out an assay according to the present invention:
[1] Amplification of viral RNA and/or bacterial genetic material in a sample using multiplex RT-PCR with primer pairs that specifically target each of the salmonid pathogens to be detected (i.e. one or more of ISAV, IPNV, IHNV, SAV, VHSV, Renibacterium salmoninarum, and any others that may be included in the detection panel). The amplified region of each pathogen to be detected is preferably unique to that pathogen, and conserved such that pathogen mutations will not prevent primer annealing.
[2] Carrying out target specific primer extension (TSPE) reactions to prepare labeled (e.g. with biotin or other label) oligonucleotides from the PCR products of the previous step, which may be accomplished using specifically designed tagged TSPE primers (TAG-TSPEs). TAG-TSPEs may have a pathogen-specific primer sequence, and a specifically assigned TAG sequence.
[3] Hybridizing the TSPE reaction products via the TAG-TSPE TAG sequences to fluorescent microbeads containing a corresponding anti-tag sequence (for example, microbeads commercially available from Luminex Corporation). The anti-tag sequence of each fluorescent microbead may be matched to a specific microbead label. For example, a microbead with an anti-TAG sequence specific for an ISAV tag sequence may be labeled with one fluorophore, and a microbead with an anti-TAG sequence for an IPNV tag sequence may be labeled with a different and distinguishable fluorophore. In this way the microbead fluorophore may be used to identify the pathogen specificity of the anti-TAG sequence carried on the microbead.
[4] Reacting the microbead-bound TSPE reaction products, which carry a label (e.g. biotin or other label) as described above, with a reporter (e.g. streptavidin and phycoerythrin, SAPE, or other reporter) which causes fluorescent emission from the biotinylated product.
[5] Detecting the reporter in a detection step. For example, fluorescence from the biotin-SAPE reactions, and the co-localized fluorescence from the labeled microbeads to which the biotinylated TSPE reaction products are bound, may be detected and transmitted into a computer that transforms the emission into numerical values for detecting, interpreting, and evaluating the presence or absence of one or more salmonid pathogens in the sample. By way of example, the Luminex Mag-Pix system may be used for these purposes, in which fluorescence from the SAPE reaction is matched to microbead fluorescence. As described above, microbead fluorescence may be used to determine the pathogen specificity of the microbead anti-TAG sequence, and thus the presence and identity of pathogen in a sample may be identified.
An example of a multiplex diagnostic assay for detection of salmonid pathogens according to an embodiment of the present invention is described in further detail below with reference to
As illustrated, multiplexed real time-PCR is carried out in a first step (Multiplex RT-PCR, shown in
For this initial multiplex PCR step of the present example, target regions within the genomes of each of the 6 pathogens detected by the assay (ISAV, IPNV, IHNV, SAV, VHSV, and Renibacterium salmoninarum) have been selected, and conserved segments within each target region have been identified. In this example, PCR primer pairs with specificity and sensitivity for detecting each of the conserved segments from each pathogen to be detected have been optimized and verified as described below in Example 2. The selection of conserved regions in this example is such that the regions are unique to each pathogen, and remain mostly resistant to pathogen mutation which might otherwise affect detection using the specified PCR primer pair. Table 1 contains PCR primer pairs utilized in this non-limiting example, which have been optimized for the multiplex RT-PCR-based simultaneous detection of one or more pathogens from ISAV, IPNV, IHNV, SAV, VHSV, and Renibacterium salmoninarum.
In a next step of this example, PCR amplified products produced in the multiplex PCR step (in this example, multiplex PCR amplified an ISAV product, as shown in
The TAG-TSPE primer extension step is used to produce labeled (in the present example biotinylated) TAG-TSPE primer extension products. As shown in the example of
As discussed above, TAG-TSPEs may carry a TAG region, which may be retained in the TAG-TSPE primer extension products produced in the step described above, as shown in
In a further step of the current example, as shown in
In a subsequent step of the present example, as shown in
In the detection step shown in the example in
A non-limiting example of an experimental protocol for performing a multiplex diagnostic assay to detect salmonid pathogens may include the following reagents, supplies, equipment, and steps:
List of Reagents and Laboratory Supplies:
List of Equipment:
A. Multiplex RT-PCR
1. Using QIAGEN® One Step RT-PCR kit, prepare a master mix in a total volume of 50 μL per reaction. The master mix also contains a primer pool (0.4 μM) with a primer set for each of the six pathogens.
2. Transfer aliquots of 5 μL of RNA/genomic material extracted from each pathogen in tissue culture to each reaction microtube.
3. Put the microtubes in a thermo cycler under the following conditions for the RT-PCR reaction: reverse transcription 30 min/50° C., initial PCR activation 15 min/95° C., 35 cycles of denaturation 1 min/94° C., annealing 45 sec/58° C., and extension 1 min/72° C., followed by a step of final extension 10 min/72° C.
4. Visualize the PCR products in a 1.5% agarose gel through a UV transilluminator (optional).
B. Exo-SAP-IT Treatment
5. Treat the PCR products by mixing 7.5 μL of them with 3 μL of Exo/SAP-IT (Biolynx®).
6. Incubate at 37° C. for 30 minutes and inactivate Exo/SAP-IT by heating to 80° C. for 15 minutes. Hold the treated reactions at 4° C. (steps can be performed in a thermal cycler).
C. Multiplex TSPE Reaction
7. Prepare a Master mix containing 2 μL of 10×TSPE reaction buffer, 0.5 μL of 50 mM MgCl2, 1 μL of 20× (500 nM each) Tag-TSPE primer mix, 0.15 μL of 5 U/μL Taq DNA polymerase, 1 μL of 20× (100 μM each) dNTP mix (except dCTP), 0.25 μL of 400 μM biotin-dCTP, and 10.1 μL of dH2O, per sample.
Note: All these reagents are from Invitrogen®.
8. Transfer aliquots of 15 μL of Master mix to 200 μL microtubes, and add 5 μL of treated PCR reaction (sample).
9. Put the microtubes in a thermocycler under the following cycling conditions: 96° C./2 min; 30 cycles of 94° C./30 sec, 55° C./1 min and 74° C./2 min.
D. Hybridization to MagPlex-TAG Microspheres
10. Select the 5 microsphere sets (Luminex®) from storage at 4° C. to room temperature. Resuspend them by vortexing (30 sec) and sonication (1 min).
11. Transfer 41.6 μL from each set into a 1.5 mL microtube. This tube will have a mixture of 520000 beads in 208 μL.
12. Centrifuge at ≧8000×g for 1-2 min, and remove the supernatant (being careful not to disturb the pellet).
13. Resuspend the pellet with 1 mL of 1.1×Tm hybridization buffer. Vortex (10 sec) and sonicate (10 sec) twice. This stock solution will contain 520000 beads mix/mL or 104000 bead set/mL (104 bead set/μL).
14. Transfer 24 μL of this solution to each well of a microplate.
15. Add 1 μL of dH2O to each background well or 1 μL of TSPE reactions to appropriate wells. Cover the plate to prevent evaporation.
16. Denature at 96° C. for 90 sec and hybridize at 37° C. for 30 min (thermocycler).
17. Prepare reporter mix by diluting streptavidin-R-phycoerythrin (Invitrogen®) to 5 μg/mL in 1×Tm hybridization buffer with 0.1% BSA.
18. Add 100 μL of reporter mix to each well. Mixing gently.
19. Incubate at 37° C. for 15 min.
20. Analyze 50 μL at 37° C. on the Luminex Mag Pix analyzer.
Note: The block should be pre-heated and the protocol created in the analyzer should include a wash step before reading.
The non-limiting example provided above is for demonstrative purposes only, and is in no way intended to limit the scope of the present invention.
As described above, the multiplex diagnostic assay for salmonid pathogens described herein may employ multiple primer sets and TAG-TSPE primers in multiplex PCR steps. The assay may benefit from high specificity (a primer set for PCR amplification of a target region in one pathogen genome should not PCR amplify sequence from any other pathogen in the assay), high sensitivity (low concentrations of pathogen in the sample should be detectable), and compatibility between primer sets (and TAG-TSPEs) during multiplex PCR, which may simultaneously amplify more than one sequence. Optimized pathogen-specific primer sets utilized in the previously described example are shown in Table 1, and optimized TAG-TSPE primers in Table 2. Genomic segments/genes targeted by the primers shown in Tables 1 and 2 are outlined in Table 3.
Renibacterium salmoninarum.
CAT AAT CAA TTT CAA CTT TCT ACT
CAA ATA CAT AAT CTT ACA TTC ACT
TAC TTC TTT ACT ACA ATT TAC AAC
CAC TTA ATT CAT TCT AAA TCT ATC
indicates data missing or illegible when filed
The performance of a multiplex diagnostic assay for salmonid pathogens employing the primer sets and TAG-TSPEs shown in Tables 1 and 2 (and discussed in Example 1) has been verified experimentally. Table 4 shows the results of experimentation designed to determine the specificity of the exemplified assay. In these experiments, the extracted nucleic acid of each of the ISAV, IPNV, VHSV, IHNV, SAV, and Renibacterium salmoninarum pathogens was subjected to the exemplified assay for detecting one or more of the 6 pathogens. As shown in Table 4, in each case the assay correctly detected only the pathogen present in the sample, and indicated an absence of any of the other 5 pathogens. These results demonstrate the specificity of the assay.
R.
salmoninarum
R.
salmoninarum
Table 5 provides experimental results related to the sensitivity of the multiplex assay for salmonid pathogens provided herein. Several 10-fold dilutions of specific virus preparations from the Regional Diagnostic Virology Services (RDVS) at AVC-UPEI were used in the comparative sensitivity experiments, which compare the sensitivity of the present example to assays employing the PCR design and methodologies currently being utilized in the field. As shown in the results presented in Table 5, the present example has remarkable sensitivity compared to the existing molecular diagnostic assays being utilized. For the 5 viruses being detected in the comparative sensitivity experiments shown in Table 5, the multiplex assay of the present example was consistently able to detect virus at lower concentrations than the other assays in the comparison study. The sensitivity of the core multiplex diagnostic assay for salmonid viruses of the present example is similar in sensitivity to the gold standard, which is Virus Isolation.
Renibacterium salmoninarum
1OIE “Manual of Diagnostic Tests for Aquatic Animals”, Bacterial Kidney Disease (Renibacterium salmoninarum), 2000.
2OIE “Manual of Diagnostic Tests for Aquatic Animals”, Bacterial Kidney Disease (Renibacterium salmoninarum), 2003.
3Köningsson, M. H.; Ballagi, A.; Jansson, E.: Johansson, K-E. “Detection of Renibacterium salmoninarum in tissue samples by sequence capture and fluorescent PCR based on the 16S rRNA gene”. Veterinary Microbiology 105: 235-243, 2005.
In summary, the example of the multiplex assay described in the examples is useful for the simultaneous detection of salmonid pathogens in a sample. The examples illustrated herein provide 6 primer pairs that are especially useful for simultaneous detection of up to 6 pathogens (ISAV, IPNV, VHSV, IHNV, SAV, and Renibacterium salmoninarum), and have been optimized for pathogen specificity and sensitivity in a multiplex PCR-based assay. These 6 primer pairs were designed to amplify specific regions within the genomes of each of the 6 pathogens. The amplification regions of the described examples have been selected on the basis of resistance to mutation, uniqueness to each pathogen, compatibility with multiplex PCR amplification, and proportion of the 4 nucleotides in the sequence. The proportion of the 4 nucleotides, which is related to the quantity of labeled (e.g. biotinylated) nucleotide that may be produced during the TSPE reaction, may be related to the sensitivity of the assay. The description illustrated herein also provides TAG-TSPE primer sequences and tag sequences for simultaneous detection of one or more of the 6 pathogens (ISAV, IPNV, VHSV, IHNV, SAV, and Renibacterium salmoninarum), and which are compatible with the amplification regions and multiplex PCR steps of the assay described in the examples above.
One or more currently preferred embodiments have been described by way of example. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2014/050972 | 10/8/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61888336 | Oct 2013 | US |
