CN116024387A - Quadruple fluorescence PCR detection kit for porcine viral diarrhea pathogen and application thereof - Google Patents
Quadruple fluorescence PCR detection kit for porcine viral diarrhea pathogen and application thereof Download PDFInfo
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Abstract
The invention relates to a quadruple fluorescence PCR detection kit for porcine viral diarrhea pathogen and application thereof, which comprises a PCR premix 2 multiplied by Taq PCR Master Mix and a detection system consisting of four pairs of specific primers and probes for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis, porcine rotavirus A and porcine delta coronavirus. The invention can rapidly and accurately detect porcine epidemic diarrhea virus, transmissible gastroenteritis, porcine rotavirus A and porcine delta coronavirus at the same time, solves the problem of time and labor waste in differential diagnosis of mixed infection in pig farm detection, and effectively reduces economic loss of pig farms.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a quadruple fluorescence PCR detection kit for porcine viral diarrhea pathogen and application thereof.
Background
The diarrhea related diseases of live pigs cause great loss to live pig cultivation. In particular to the suckling piglet and the piglet in the early period of conservation. For piglets, diarrhea disease is one of the biggest effects affecting the mortality of primary piglets. Therefore, it is particularly important to find diarrhea virus, especially diarrhea virus of suckling piglet and early stage piglet.
Porcine epidemic diarrhea is caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), a highly contagious intestinal infectious disease characterized by diarrhea, vomiting, dehydration and high mortality for young piglets. Porcine pathological changes in PEDV-infected pigs are mainly manifested by atrophy and shedding of intestinal villi in the jejunum and ileum portions of the pigs.
The pig infectious gastroenteritis causes great variability in the harm to pigs of different ages, usually causes 100% death of piglets, especially suckling piglets, and seriously jeopardizes the safety of pig raising industry.
Pig groups have a high susceptibility to rotavirus, especially for piglets of low age, and once infected, can cause a huge economic loss to the pig farm.
The delta coronavirus infection of piglets is easy to occur in a large-scale pig farm and can clinically infect pigs at each stage, and mainly causes watery diarrhea and vomiting of the suckling piglets, if preventive and effective treatment measures are not actively taken, serious patients can cause dehydration and failure of the sick piglets to die, and certain economic loss is caused to the pig farm.
In recent years, diarrhea of piglets mainly occurs by mixed infection of diarrhea viruses, and the infection rate is sequentially from large to small: porcine epidemic diarrhea, porcine rotavirus a, transmissible gastroenteritis of swine, porcine delta coronavirus. The detection of diarrhea viruses of piglets in the market at present mainly uses single fluorescence detection, so that single diseases are easily detected aiming at diarrhea samples, pathogens of other same diseases are easily ignored, and the four viruses can be diagnosed respectively only by carrying out detection for 4 times, thereby being time-consuming and labor-consuming. Not only greatly prolongs the diagnosis time, but also easily misses the optimal treatment time.
Disclosure of Invention
The invention aims to provide a quadruple fluorescence PCR detection kit for porcine viral diarrhea pathogens, which is used for detecting four viruses, namely porcine epidemic diarrhea virus, transmissible gastroenteritis of swine, porcine rotavirus A and porcine delta coronavirus, and can rapidly and accurately detect the four viruses at the same time, solve the problem of time and labor waste in differential diagnosis of mixed infection in pig farm detection, and effectively reduce economic loss of pig farms.
The invention adopts the following technical scheme: a quadruple fluorescence PCR detection kit for porcine viral diarrhea pathogen comprises PCR premix 2 xTaqPCR Master mix and a detection system consisting of four pairs of specific primers and probes for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis of swine, porcine rotavirus A and porcine delta coronavirus, wherein:
the nucleotide sequences of the primers PEDV-F and PEDV-R and the probe PEDV-P for detecting porcine epidemic diarrhea virus are as follows:
PEDV-F：ctttcagcatccttatggctt；
PEDV-R：ccacaaccgratgctattraca；
PEDV-P：atgctgtggataatgta；
the nucleotide sequences of the primers TGEV-F and TGEV-R and the probe TGEV-P for detecting transmissible gastroenteritis of swine are as follows:
TGEV-F：aggtgatgtgacaagattyta；
TGEV-R：araatgctrgacacagatgga；
TGEV-P：actgctcccattggcaa；
the nucleotide sequences of the primers PDCov-F and PDCov-R and the probe PDCov-P for detecting the porcine rotavirus A type are as follows:
PDCov-F：agtagactccttgcagggayt；
PDCov-R：cttgccatgyttaacgactg；
PDCov-P：atgcacctccatgtacc；
the nucleotide sequences of primers PoRV-F and PoRV-R and probe PoRV-P for detecting porcine delta coronavirus are as follows:
PoRV-F：agargatattggaccwtctgatt；
PoRV-R：agcrtctgcatttgtcttaact；
PoRV-P：tggtgagtggatcgtt。
further, each 25 mu L of detection system comprises 12.5 mu L of PCR premix liquid 2 xTaqPCR Master mix, and the concentrations of four pairs of specific primers for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis, porcine rotavirus A and porcine delta coronavirus are all 0.2 mu M, and the concentrations of four probes are all 0.05 mu M
Further, the fluorescent group marked at the 5' end of the sequence in the four pairs of specific primers and probes is FAM, VIC, CY or ROX.
Further, the group marked at the 3' end of the sequence in the four pairs of specific primers and probes is MGB.
The invention adopts another technical scheme that: the quadruple fluorescence PCR detection kit disclosed by the technical scheme is applied to detection of porcine epidemic diarrhea virus, transmissible gastroenteritis of swine, porcine rotavirus A and porcine delta coronavirus.
Further, the detection method comprises the following steps:
s100: obtaining a nucleic acid sample to be detected;
s200: and adding the accounting sample to be detected into the quadruple fluorescence PCR detection kit for detection, wherein each 25 mu L of detection system contains 5 mu L of nucleic acid sample during detection.
Further, the reaction procedure of the quadruple fluorescent PCR detection kit in step S200 is as follows: the quadruple fluorescent PCR detection kit was sequentially subjected to pre-denaturation at 95℃for 1min, reverse transcription at 60℃for 10min, pre-denaturation at 94℃for 2min, denaturation at 94℃for 15s and annealing and extension at 60℃for 45s, 45 cycles were performed, and FAM, VIC, rox and Cy5 fluorescent signals were collected when annealing and extension at 60℃for 45 s.
The invention has the beneficial effects that:
(1) The invention can detect porcine epidemic diarrhea virus, transmissible gastroenteritis of swine, porcine rotavirus A and porcine delta coronavirus simultaneously in one reaction, realizes rapid differential diagnosis of diarrhea samples infected by multiple pathogens clinically, has better simplicity compared with single pathogen detection reagent, avoids the risk of missed detection of mixed infection, shortens the time cost in the detection process, has advantages for large-scale sample detection or differential diagnosis of mixed infection samples, and saves time and labor cost;
(2) The invention has the advantages that the detection sensitivity and the specificity are not inferior to those of single fluorescence PCR, so that the false detection and the omission detection can be effectively avoided; the method can be repeatedly used, and under the condition of repeated detection for 5 times, the variation coefficient is not more than 2%, so that the method has good economic benefit;
(3) In the porcine rotavirus type A detection reagent, in consideration of rotavirus type A is double-stranded RNA virus, template pre-denaturation treatment is needed before RT-PCR is carried out, however, reverse transcriptase (MMLV) is inactivated by adding the step in the reaction of the same program, so that the invention utilizes Tth ferment contained in a premix 2 xTaqPCR Master mix to carry out the template pre-denaturation treatment under the same reaction program, and the pre-treatment time is saved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the gradient dilution detection result of a PEDV sensitive quality control obtained in a sensitivity test experiment according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a gradient dilution test result of a TGEV sensitive property control product obtained in a sensitivity test experiment according to an embodiment of the invention;
FIG. 3 is a schematic diagram of a gradient dilution detection result of a PDCoV sensitive property control product obtained in a sensitivity test experiment according to an embodiment of the invention;
FIG. 4 is a schematic diagram of a gradient dilution detection result of a PoRV sensitive property control product obtained in a sensitivity test experiment according to the embodiment of the present invention; FIG. 5 is a schematic diagram showing the detection results of specific detection of PEDV in 32 positive samples obtained in a specific test experiment according to an embodiment of the present invention;
FIG. 6 is a schematic diagram showing the detection results of TGEV specificity detection in 32 positive samples obtained in a specificity test experiment according to the embodiment of the invention;
FIG. 7 is a schematic diagram showing the detection results of PDCoV specificity detection in 32 positive samples obtained in a specificity test experiment according to the embodiment of the invention;
FIG. 8 is a schematic diagram of detection results of PoRV specific detection in 32 positive samples obtained in a specific test experiment according to the embodiment of the present invention;
FIG. 9 shows the detection of PEDV in a simulated clinical positive nucleic acid sample according to an embodiment of the present invention; FIG. 10 shows the detection results of TGEV in a simulated clinically positive nucleic acid sample according to an embodiment of the present invention;
FIG. 11 shows the result of detecting PDCoV in a simulated clinical positive nucleic acid sample according to an embodiment of the invention;
FIG. 12 shows the detection results of PoRV in a simulated clinical positive nucleic acid sample according to the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those described herein, and therefore the present invention is not limited to the specific embodiments disclosed below.
The invention adopts the following technical scheme: the kit comprises PCR premix 2 xTaqPCR Master mix and a detection system consisting of four pairs of specific primers and probes for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis, porcine rotavirus A and porcine delta coronavirus, wherein:
the nucleotide sequences of the primers PEDV-F and PEDV-R and the probe PEDV-P for detecting porcine epidemic diarrhea virus are as follows:
PEDV-F：ctttcagcatccttatggctt；
PEDV-R：ccacaaccgratgctattraca；
PEDV-P：atgctgtggataatgta；
the nucleotide sequences of the primers TGEV-F and TGEV-R and the probe TGEV-P for detecting transmissible gastroenteritis of swine are as follows:
TGEV-F：aggtgatgtgacaagattyta；
TGEV-R：araatgctrgacacagatgga；
TGEV-P：actgctcccattggcaa；
the nucleotide sequences of the primers PDCov-F and PDCov-R and the probe PDCov-P for detecting the porcine rotavirus A type are as follows:
PDCov-F：agtagactccttgcagggayt；
PDCov-R：cttgccatgyttaacgactg；
PDCov-P：atgcacctccatgtacc；
the nucleotide sequences of primers PoRV-F and PoRV-R and probe PoRV-P for detecting porcine delta coronavirus are as follows:
PoRV-F：agargatattggaccwtctgatt；
PoRV-R：agcrtctgcatttgtcttaact；
PoRV-P：tggtgagtggatcgtt。
the fluorescent group marked at the 5 'end of the sequence in the four pairs of specific primers and probes is FAM, VIC, CY or ROX, and the group marked at the 3' end is MGB. Every 25 mu L of detection system comprises 12.5 mu L of PCR premix liquid 2 xTaqPCR Master mix, and the concentration of four pairs of specific primers for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis of swine, porcine rotavirus A and porcine delta coronavirus is 0.2 mu M, and the concentration of four probes is 0.05 mu M.
The detection effect of the quadruple fluorescence PCR detection kit disclosed by the invention is described below by combining a specific embodiment:
in the embodiment of the invention, the 5 'end and 3' end marked groups of PEDV-F, PEDV-R and PEDV-P are 6-FAM and MGB respectively; the 5 'and 3' end marked groups of TGEV-F, TGEV-R and TGEV-P are VIC and MGB respectively; the 5 'end and 3' end marked groups of PDCov-F, PDCov-R and PDCov-P are CY5 and MGB respectively; the 5 'and 3' end-labeled groups of PoRV-F, poRV-R and PoRV-P are ROX and MGB, respectively.
The quadruple fluorescence PCR detection kit provided by the embodiment of the invention is used for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis, porcine rotavirus A and porcine delta coronavirus, and the specific detection method comprises the following steps:
s100: obtaining a nucleic acid sample to be detected from a clinical pig farm;
s200: and adding the accounting sample to be detected into the quadruple fluorescence PCR detection kit for detection, wherein each 25 mu L of detection system contains 5 mu L of nucleic acid sample, the concentration of the PCR premix liquid 2 xTaqPCR Mastermix is 12.5 mu L, the concentration of PEDV-F, PEDV-R, TGEV-F, TGEV-R, PDCov-F, PDCov-R, poRV-F and the concentration of PoRV-P are both 0.2 mu M, and the concentration of the PEDV-P, TGEV-P, PDCov-P and the concentration of the PoRV-P are both 0.05 mu M. The reaction program of the quadruple fluorescence PCR detection kit is as follows: the quadruple fluorescent PCR detection kit was sequentially subjected to pre-denaturation at 95℃for 1min, reverse transcription at 60℃for 10min, pre-denaturation at 94℃for 2min, denaturation at 94℃for 15s and annealing and extension at 60℃for 45s, 45 cycles were performed, and FAM, VIC, rox and Cy5 fluorescent signals were collected when annealing and extension at 60℃for 45 s.
(1) Kit sensitivity test
The porcine epidemic diarrhea virus, transmissible gastroenteritis of swine, porcine rotavirus type A and porcine delta coronavirus gene plasmids were diluted with nuclease-free water, respectively, each virus was diluted to a standard substance concentration of 100 copies/. Mu.L and 10 copies/. Mu.L, each virus was divided into five parts at each concentration, and the study of the minimum detection limit of the fluorescent quantitative PCR detection kit was performed, respectively, and the detection results shown in Table 1 and FIGS. 1 to 4 were obtained.
As can be seen from Table 1 and FIGS. 1 to 4, the examples of the present invention can stably detect the corresponding virus species when the concentrations of PEDV, TGEV, PDCoV and PoRV are 10/uL copies, and demonstrate that the examples of the present invention have good sensitivity and can effectively avoid missed detection.
TABLE 1 detection results (Ct values) of gradient dilution of sensitive quality control
(2) Kit specificity verification
The nucleic acid extracted from 32 positive disease materials of PCV2 and PRV, CSFV, PRRSV collected in clinic is taken as a template, wherein sample numbers 1-8 are PCV2 positive samples, sample numbers 9-16 are PRV positive samples, sample numbers 17-24 are CSFV positive samples, and sample numbers 25-32 are PRRSV positive samples. The quadruple fluorescence PCR detection kit disclosed by the embodiment of the invention is used for detection, so that the specificity of the detection kit is good when no detection is performed. Specific detection results are shown in Table 2 and FIGS. 5 to 8.
As can be seen from Table 2 and FIGS. 5 to 8, in the nucleic acid detection of 32 total positive disease samples of PCV2 and PRV, CSFV, PRRSV, none of the examples of the present invention was detected, which indicates that the specificity of the examples of the present invention is good, the occurrence of false detection and false positive is effectively avoided, and the time cost of secondary confirmation by the experimenter is saved.
TABLE 2 specificity test results (Ct value) for 32 negative samples
(3) Kit stability verification
The nucleic acid extracted from PEDV, TGEV, PORV and PDCOV positive disease materials is mixed as a template, three different templates are taken, the quadruple fluorescence PCR detection kit disclosed by the embodiment of the invention is used for detection, the detection is repeated for 5 times, and the variation coefficient is not more than 5%, so that the method is good in stability. Specific detection results are shown in Table 3 and FIGS. 9 to 12.
TABLE 3 repeatability test results (Ct value)
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. The quadruple fluorescence PCR detection kit for the porcine viral diarrhea pathogen is characterized by comprising a PCR premix 2×TaqPCR Master mix and a detection system consisting of four pairs of specific primers and probes for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis, porcine rotavirus A and porcine delta coronavirus, wherein:
the nucleotide sequences of the primers PEDV-F and PEDV-R and the probe PEDV-P for detecting porcine epidemic diarrhea virus are as follows:
PEDV-F：ctttcagcatccttatggctt；
PEDV-R：ccacaaccgratgctattraca；
PEDV-P：atgctgtggataatgta；
the nucleotide sequences of the primers TGEV-F and TGEV-R and the probe TGEV-P for detecting transmissible gastroenteritis of swine are as follows:
TGEV-F：aggtgatgtgacaagattyta；
TGEV-R：araatgctrgacacagatgga；
TGEV-P：actgctcccattggcaa；
the nucleotide sequences of the primers PDCov-F and PDCov-R and the probe PDCov-P for detecting the porcine rotavirus A type are as follows:
PDCov-F：agtagactccttgcagggayt；
PDCov-R：cttgccatgyttaacgactg；
PDCov-P：atgcacctccatgtacc；
the nucleotide sequences of primers PoRV-F and PoRV-R and probe PoRV-P for detecting porcine delta coronavirus are as follows:
PoRV-F：agargatattggaccwtctgatt；
PoRV-R：agcrtctgcatttgtcttaact；
PoRV-P：tggtgagtggatcgtt。
2. the kit for detecting the porcine viral diarrhea pathogen according to claim 1, wherein each 25 mu L of the detection system comprises 12.5 mu L of PCR premix liquid 2 xTaqPCR Master mix, the concentration of the four pairs of specific primers for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis, porcine rotavirus A and porcine delta coronavirus is 0.2 mu M, and the concentration of the four probes is 0.05 mu M.
3. The kit for detecting the swine viral diarrhea pathogen through quadruple fluorescence PCR according to claim 1, wherein the fluorescent groups marked at the 5' ends of the sequences in the four pairs of specific primers and probes are FAM, VIC, CY or ROX.
4. The kit for detecting swine viral diarrhea pathogen according to claim 1, wherein the group marked at the 3' -end of the sequences in the four pairs of specific primers and probes is MGB.
5. Use of the quadruple fluorescence PCR detection kit according to any one of claims 1-4 for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis, porcine rotavirus type A and porcine delta coronavirus.
6. The use according to claim 5, wherein the detection method comprises the steps of:
s100: obtaining a nucleic acid sample to be detected;
s200: and adding the accounting sample to be detected into the quadruple fluorescence PCR detection kit for detection, wherein each 25 mu L of detection system contains 5 mu L of nucleic acid sample during detection.
7. The use according to claim 6, wherein the reaction procedure of the quadruple fluorescent PCR detection kit in step S200 is: the quadruple fluorescent PCR detection kit was sequentially subjected to pre-denaturation at 95℃for 1min, reverse transcription at 60℃for 10min, pre-denaturation at 94℃for 2min, denaturation at 94℃for 15s and annealing and extension at 60℃for 45s, 45 cycles were performed, and FAM, VIC, rox and Cy5 fluorescent signals were collected when annealing and extension at 60℃for 45 s.
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| CN117512225A (en) * | 2024-01-04 | 2024-02-06 | 南京农业大学三亚研究院 | Primer probe combination capable of detecting porcine epidemic diarrhea and porcine delta coronavirus, freeze-dried pellet and application thereof |
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| CN117512225A (en) * | 2024-01-04 | 2024-02-06 | 南京农业大学三亚研究院 | Primer probe combination capable of detecting porcine epidemic diarrhea and porcine delta coronavirus, freeze-dried pellet and application thereof |
| CN117512225B (en) * | 2024-01-04 | 2024-04-02 | 南京农业大学三亚研究院 | Primer probe combination capable of detecting porcine epidemic diarrhea and porcine delta coronavirus, freeze-dried pellet and application thereof |
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