CN116287443A - Fluorescent PCR method for detecting PEDV with quality control system and specific primer probe combination thereof - Google Patents
Fluorescent PCR method for detecting PEDV with quality control system and specific primer probe combination thereof Download PDFInfo
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Abstract
The invention relates to the technical field of virus detection, in particular to a fluorescent PCR method for detecting PEDV with a quality control system and a specific primer probe combination thereof, wherein the specific primer comprises an upstream primer and a downstream primer of the PEDV and a PEDV probe, and the sequences of the upstream primer and the downstream primer of the IPC and the IPC probe are respectively shown as SEQ ID NO: 1-6. Based on the primer and the probe, the fluorescence PCR method comprises the following steps: 1) Extracting template RNA; 2) Preparing a double fluorescence quantitative reaction system; 3) Performing PCR reaction; 4) Quality control; 5) And judging the conditions and experimental results of the experiment. The method can monitor the false negative in the sample due to the existence of the PCR reaction inhibitor and has a complete quality control system, so that the PCR detection flow is monitored, and the epidemic diarrhea virus infection condition can be diagnosed in a short time; the sensitivity was high, and at the lowest, 1 copy/. Mu.l was detectable.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to a fluorescent PCR method for detecting PEDV.
Background
In recent years, outbreaks and epidemics of porcine epidemic diarrhea (Porcine epidemicdiarrhea, PED) caused by porcine epidemic diarrhea virus (Porcine epidemic diarrheavirus, PEDV) have caused devastating attacks to the pig industry in China and even worldwide. Outbreaks of porcine epidemic diarrhea in 2013-2015 cause the United states pig industry to lose 9-18 billions of dollars each year, with about 40% of pig farms affected and tragic. The novel porcine epidemic diarrhea virus strain has become one of the most important factors in the occurrence of the group diarrhea disease in the swinery.
In 2021, the animal husbandry production of the inner Mongolia autonomous region presents the following characteristics: firstly, the production of live pigs is quickened and recovered, the number of live pigs in stock and in stock is increased rapidly by 565.2 ten thousand for the inner Mongolian autonomous region at the end of 2021, the number of live pigs in stock and in stock is increased by 5.8% compared with the last year, and the number of live pigs in stock and in stock is increased by 812.9 ten thousand for the last year, and the number of live pigs in stock and in stock is increased by 9.5% compared with the last year. In this situation, pigs in the inner Mongolia autonomous region have good vigor of increasing year by year, and the diarrhea of the pigs is taken as the most common disease in pig breeding and is included in the technical guidelines for prevention and control of common epidemic disease of pigs by the Chinese animal epidemic disease prevention and control center.
At present, the detection method of the porcine epidemic diarrhea virus by the national standard mainly comprises a common RT-PCR detection method and a fluorescent quantitative diagnosis method of single pathogen, lacks a complete quality control system, and lacks monitoring of the whole PCR process, including monitoring of nucleic acid extraction reagents, personnel operation, equipment, whether inhibitors exist in samples and the like.
Therefore, developing a method for detecting PEDV with a quality control system is one of the problems in the art.
Disclosure of Invention
The invention aims to provide a fluorescence PCR method for detecting PEDV with a quality control system and a specific primer probe combination thereof.
According to the invention, according to the published Porcine Epidemic Diarrhea Virus (PEDV) (accession number: MH 726401.1) and human HBB gene (accession number: MK 476504.1) in GenBank, which mainly represent virus strain sequences, the PEDV conserved N gene and human HBB gene are respectively used as target genes, 1 pair of PEDV specific Primer probes and internal positive quality control (IPC) specific Primer probes are designed by using Primer Express 3.0.1 software, and 2 groups of specific probes marked by different fluorescent groups (PEDV is modified by FAM groups and IPC is modified by VIC groups). The method is established according to a double fluorescence PCR method by designing and synthesizing two groups of primer probes, and simultaneously, technologies such as antipollution enzyme and the like are introduced, so that the fluorescent detection method of the epidemic diarrhea virus (PEDV) with a complete quality control system is established.
Specifically, the invention provides the following technical scheme:
a specific primer probe combination for a fluorescent PCR method for detecting PEDV, comprising:
PEDV upstream primer: 5'-AAGAACAAATCCAGGGCCA-3';
PEDV downstream primer: 5'-TAAACTGGCGATCTGAGCATA-3';
PEDV probe: FAM-AAAGACATCCCAGAGTGGAGGAGAATTC-BHQ1;
IPC upstream primer: 5'-TGCTCGGTGCCTTTAGTGAT-3';
IPC downstream primer: 5'-AGTCCCATAGACTCACCCTGAA-3';
IPC probe: VIC-TGGCTCACCTGGACAACCTCAAGG-TAMRA; the sequences are respectively shown as SEQ ID NO: 1-6.
The fluorescence PCR method for detecting PEDV with the quality control system by adopting the specific primer probe combination comprises the following steps:
(1) Extracting template RNA: extracting RNA of a sample to be detected, simultaneously adding 5 μl of internal positive quality control (IPC) into an extraction plate to participate in the whole nucleic acid extraction process, monitoring nucleic acid extraction and subsequent PCR reaction, and using the extracted nucleic acid in the subsequent PCR amplification reaction; the genome of a clinical sample was extracted using a KingFisher Flex nucleic acid extraction apparatus using a Thermo Fisher MagMAX CORE nucleic acid extraction method, and the specific procedure is as follows:
a. full vortex MagMAX TM CORE beads ensure complete resuspension of the beads.
b. Depending on the number of samples to be extracted, the following components were mixed and 10% margin was increased. Required volume MagMAX of each sample TM CORE magnetic bead 20. Mu.L, magMAX TM 10. Mu.L of CORE proteinase K and 30. Mu.L of the total volume of the magnetic bead/proteinase K mixture;
c. depending on the number of samples to be extracted, the following components were mixed and 10% margin was increased. Component name required volume MagMAX per sample TM 350 mu L of CORE lysate and MagMAX TM A volume of 350. Mu.L of CORE binding solution and 700. Mu.L of total lysis/binding solution;
d. separately packing 500 μl of Wash1 and Wash2 reagents into 96 deep well plates;
e. split charging 90 μl of the solution reagent into 96 deep well plates;
f. 200 mu L of the sample to be tested is added into a sample plate containing magnetic beads, proteinase K, lysate and binding solution according to MagMAX TM The CORE nucleic acid extraction procedure performs nucleic acid extraction.
(2) The configuration of a double fluorescence quantitative reaction system: the reaction system was prepared in 200. Mu.l PCR tubes: 10 μl of one-step fluorescence PCR reaction solution containing UNG enzyme, 0.8 μl of each of PEDV upstream primer, PEDV downstream primer and PEDV probe, 0.4 μl of each of IPC upstream primer, IPC downstream primer and IPC probe, and ddH 2 O1 μl was prepared; premixing the reaction liquid, split charging 15 μl, adding 5 μl of template RNA, and performing machine loading operation;
(3) Premixing the reaction liquid, split charging 15 μl each tube, adding 5 μl of template RNA, and performing on-machine PCR reaction, wherein the reaction procedure is as follows:
UNG enzyme anti-pollution: 25 ℃ for 10min;1Cycle.
Reverse transcription: 5min at 52 ℃;1Cycle.
Pre-denaturation: 95 ℃ for 10s;1Cycle.
And (3) PCR reaction: 95 ℃ for 5sec; 30s at 60 ℃;40Cycles.
(4) Quality control
(1) Control set up:
positive control: DNA templates prepared from known pathogen-positive samples.
Negative extraction control: pure sterile, water (added during extraction) was used to monitor contamination throughout.
Negative no template control: pure sterile, water was used.
(2) IPC addition
Before sample addition, 5 μl IPC was added to each extraction well and the experimental procedure was monitored throughout from nucleic acid extraction to fluorescent PCR.
(5) The experiment establishment condition is as follows:
negative control: FAM channels have no Ct value or no typical S-type amplification curve;
negative extraction control: the FAM channel has no Ct value or typical S-shaped amplification curve, and the CT value of the IPC is within a preset value (which is an actual measured value after the IPC manufacturing process is finished) +/-3 CT value ranges;
positive control: the Ct value of the FAM channel is less than or equal to 35, and the amplification curve is a typical S-shaped amplification curve; meanwhile, the three points are met, the experiment is established, otherwise, the result is not established, and the experimental result is invalid.
(6) And (3) judging an experimental result:
negative results: the IPC value of the sample is within +/-3 CT values of negative extraction control IPC value, no Ct value and no typical S-type amplification curve, which indicates that the detected pathogenic nucleic acid is not present in the sample;
positive results: ct value is less than or equal to 35, and a typical S-shaped amplification curve appears, which indicates that the detected pathogenic nucleic acid exists in the sample;
suspicious results: samples with Ct values greater than 35 need repeated experiments; and if no Ct value exists in the repeated experiment results, the result is negative, otherwise, the result is positive.
The detection result needs to be repeated: the IPC value of the sample is outside the negative extraction control IPC value ± 3 CT value ranges, no CT value and no typical S-type amplification curve, indicating that there may be reaction inhibition or extraction failure in the sample.
Compared with the prior art, the invention has the beneficial effects that:
(1) The method can monitor the false negative in the sample due to the existence of the PCR reaction inhibitor, has a complete quality control system (negative extraction control, no template control, IPC, positive control and UNG enzyme use), comprehensively monitors the human, machine, material, method and ring of the PCR detection flow, and can diagnose the infection condition of the epidemic diarrhea virus in a shorter time; the sensitivity of the method of the invention is high, and 1 copy/. Mu.l can be detected at the lowest.
(2) The specificity of the method is good, and the invention has higher specificity as proved by the experiment result by carrying out the specificity experiment on common pig pathogens such as blue-ear, swine fever, pseudorabies and other diarrhea viruses such as rotavirus, transmissible gastroenteritis virus, delta coronavirus and the like.
(3) For the fluorescent quantitative PCR method, the pollution is easy to cause due to the high sensitivity, especially the pollution of amplified products in a laboratory is one of the most common pollution sources, and the false positive result caused by the amplified products can be greatly reduced by adopting the UNG enzyme amplification product pollution technology.
(4) The existing commercialized kit usually has only positive and negative controls, and the invention adds IPC to monitor the whole process, simultaneously sets up two negative controls, monitors the whole process, monitors the reagent, introduces UNG enzyme on the basis, can prevent or reduce the pollution of the product, thus forming an integral quality control system.
Therefore, the method can greatly improve the accuracy and reliability of the detection of the epidemic diarrhea virus, is convenient and quick, has low cost, has higher reference value for diagnosing, preventing and controlling the porcine epidemic diarrhea virus of the farm, and is suitable for popularization and application.
Drawings
FIG. 1 is a flow chart of a fluorescent PCR method for detecting PEDV with a quality control system according to the present invention.
FIG. 2 is an optimal amplification plot for dual fluorescent quantitative PCR system optimization.
FIG. 3 is an amplification curve of a specificity experiment.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to FIG. 1, a fluorescent PCR method for detecting PEDV with a quality control system comprises the following steps:
(1) And (3) primer probe synthesis:
according to the PEDV conserved N gene and human HBB gene in GenBank as target genes, primer Express 3.0.1 software is used for designing 1 a Primer probe specific to PED and a Primer probe specific to internal positive quality control (IPC), and different fluorescent group modifications (FAM group modification for PEDV and VIC group modification for IPC) are carried out on the two probes.
The following primers and probes were submitted to Gene company for synthesis (sequences shown in SEQ ID NOS: 1-6, respectively).
PEDV upstream primer: 5'-AAGAACAAATCCAGGGCCA-3';
PEDV downstream primer: 5'-TAAACTGGCGATCTGAGCATA-3';
PEDV probe: FAM-AAAGACATCCCAGAGTGGAGGAGAATTC-BHQ1;
IPC upstream primer: 5'-TGCTCGGTGCCTTTAGTGAT-3';
IPC downstream primer: 5'-AGTCCCATAGACTCACCCTGAA-3';
IPC probe: VIC-TGGCTCACCTGGACAACCTCAAGG-TAMRA.
(2) Positive control preparation
The RT-qPCR amplification was performed on "porcine epidemic diarrhea virus" (PEDV) according to the above-synthesized primer probe using a TAKARA (cat# RR 600A) kit, and the amplification conditions and reaction procedure were performed according to the kit instructions. And (3) extracting nucleic acid from the PED attenuated vaccine by using a Thermo Fisher MagMAX CORE nucleic acid extraction method, wherein the extracted pure nucleic acid is used as a template for PCR amplification reaction. The amplified product was subjected to cloning and sequencing by Shanghai Biotechnology Co., ltd, and plasmid and bacterial liquid were returned, and the returned bacterial liquid was subjected to expansion culture using LB liquid medium (50. Mu.l bacterial liquid+3 mL LB ampicillin resistant medium), and cultured overnight at 37℃at 180rpm, and turbidity of the medium was observed.
Extracting 50 microliters of the bacterial liquid after the amplification culture to obtain pure nucleic acid, respectively extracting 5 microliters of the nucleic acid and the returned plasmid to perform RT-qPCR verification, performing RT-qPCR amplification by using a TAKARA (product number: RR 600A) kit, performing amplification conditions and a reaction program according to a kit instruction, and preserving the bacterial liquid according to 20% glycerol for later use after verification. And diluting part of returned plasmids to the working concentration (CT value is 25+/-1), and preserving at 20 ℃ for standby, thereby completing the positive control preparation.
Manufacturing IPC:
the partial sequence of human HBB gene (accession number: MK 476504.1) is biosynthesized by a hybrid company, and the specific synthetic gene sequence is shown in SEQ ID NO: shown at 7.
The synthesized and returned plasmid was diluted at working concentration (IPC amplification CT value 29±1) according to the positive control preparation procedure.
(3) Optimization of a double fluorescent quantitative PCR system:
under the condition of establishing a single fluorescent quantitative RT-PCR detection method to verify that the detection method is error-free, an experiment is utilized to optimize a dual reaction system of the Porcine Epidemic Diarrhea Virus (PEDV) and an internal positive quality control, the volume of a primer probe is optimized according to the range of 0.1 mu L-1 mu L, the optimized concentration is specifically shown in the following table, and the optimal PED dual RT-qPCR detection method is established.
The primers were tested by selecting the 3 concentrations of 0.4. Mu.L/0.8. Mu.L// 1. Mu.L, and the positive control quality and positive quality control (IPC) plasmids prepared as described above were used as amplification templates. The system is optimized, and the specific test results are as follows:
| system (mu L) | 20 | 20 (optimal) | 20 | 20 | 25 |
| |
10 | 10 | 10 | 10 | 12.5 |
| IPC upstream | 0.4 | 0.4 | 0.4 | 0.8 | 0.5 |
| IPC downstream | 0.4 | 0.4 | 0.4 | 0.8 | 0.5 |
| IPC probe | 0.4 | 0.4 | 0.4 | 0.8 | 0.5 |
| PED upstream | 0.4 | 0.8 | 1 | 0.8 | 1 |
| Downstream of PED | 0.4 | 0.8 | 1 | 0.8 | 1 |
| PED probe | 0.4 | 0.8 | 1 | 0.8 | 1 |
| ROX | 0.4 | 0.4 | 0.4 | 0.4 | 0.5 |
| Water and its preparation method | 1.8 | 1 | 0.4 | 0 | 2.5 |
| Template | 5 | 5 | 5 | 5 | 5 |
| IPC mean value | 29.78 | 29.08 | 29.92 | 30.44 | 29.93 |
| Mean value of positive control | 25.77 | 24.89 | 25.78 | 25.3 | 25.01 |
Experimental results show that the optimal reaction system of the method is as follows: 10 μl of one-step fluorescence PCR reaction solution, 0.8 μl of each of PEDV upstream primer, PEDV downstream primer and PEDV probe, 0.4 μl of each of IPC upstream primer, IPC downstream primer and IPC probe, and ddH 2 O1. Mu.l, 5. Mu.l of template and the optimal amplification effect are shown in FIG. 2.
(4) Specificity experiments
The invention relates to a double RT-qPCR detection method for detecting PEDV, which takes foot-and-mouth disease virus (FMDV), swine fever virus (CSFV), blue ear virus (PRRSV), pseudorabies virus (PRV), parvovirus (PPV) and transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PoRV) vaccines as research objects to respectively extract nucleic acid, and the extracted nucleic acid is subjected to double RT-qPCR detection of PEDV, so that positive amplification curves do not appear, and the amplification curves are shown in figure 3, thus proving the specificity of the method.
(5) Sensitivity test
The positive clone plasmid prepared in the step (2) is used for measuring the OD260nm absorbance value and converting the value into a nucleic acid concentration value. According to plasmid copy number/. Mu.L= [ total content (. Mu.g/. Mu.L)]/(plasmid molecular base number X10) -15 μg), the purified plasmid DNA was converted to gene copy number. The recombinant plasmid with known copy number is serially diluted 10 times, 5 dilutions with the copy number of 10000 copies/. Mu.L to 1 copy/. Mu.L are selected, and then 1 copy/. Mu.L is diluted 2 times for 2 times, so that 7 dilutions are obtained in total. The sensitivity of the method is determined by detecting by using the established double RT-qPCR method, and experiments prove that the minimum detection limit of the method is 1 copy/. Mu.L. The specific results are shown in the following table:
note that: neg stands for negative.
(6) Repeatability test
Experiments were repeated within groups: in the same experiment, 3 concentrations of PED of recombinant plasmid 10 were selected -5 、10 -6 、10 -7 After dilution, mixing each concentration uniformly as a template, carrying out 10 repeated detection on three concentrations of recombinant plasmid according to the established PED double RT-qPCR detection method, calculating the standard deviation and variation coefficient of Ct value, and carrying out in-group repeatability test, wherein specific experimental results are shown inThe following table:
experiments were repeated between groups: the fluorescence PCR reaction kit with the same production lot number is adopted for detection under the same reaction condition every 1 week, the detection is carried out continuously for 3 weeks, the standard deviation and the variation coefficient of the Ct value are calculated, the inter-group repeatability test is carried out, and the specific results are shown in the following table:
| number of weeks | |
|
|
| 1 | 24.33 | 28.01 | 31.32 |
| 2 | 24.56 | 28.24 | 30.98 |
| 3 | 24.49 | 28.12 | 31.09 |
| Average of three weeks | 24.46 | 28.12 | 31.13 |
| Standard deviation of | 0.12 | 0.12 | 0.17 |
| Coefficient of variation | 0.48％ | 0.41％ | 0.56％ |
From the repeated test data in the group and among the groups, the variation coefficient is smaller than 1, and the stability and the repeatability of the detection method are good.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A specific primer probe combination for a fluorescent PCR method for detecting PEDV, comprising:
PEDV upstream primer: 5'-AAGAACAAATCCAGGGCCA-3';
PEDV downstream primer: 5'-TAAACTGGCGATCTGAGCATA-3';
PEDV probe: FAM-AAAGACATCCCAGAGTGGAGGAGAATTC-BHQ1;
IPC upstream primer: 5'-TGCTCGGTGCCTTTAGTGAT-3';
IPC downstream primer: 5'-AGTCCCATAGACTCACCCTGAA-3';
IPC probe: VIC-TGGCTCACCTGGACAACCTCAAGG-TAMRA; the sequences are respectively shown as SEQ ID NO: 1-6.
2. A fluorescent PCR method for detecting PEDV with a quality control system using the specific primer probe combination of claim 1, comprising the steps of:
(1) Extracting template RNA;
(2) The configuration of a double fluorescence quantitative reaction system: the reaction system was prepared in 200. Mu.l PCR tubes: 10 μl of one-step fluorescence PCR reaction solution containing UNG enzyme, 0.8 μl of each of PEDV upstream primer, PEDV downstream primer and PEDV probe, 0.4 μl of each of IPC upstream primer, IPC downstream primer and IPC probe, and ddH 2 O1 μl was prepared; premixing the reaction solution, sub-packaging 15 μl, adding template RNA5 μl, and performing machine-on operation
(3) Performing PCR reaction;
(4) Quality control
(1) Control set up:
positive control: DNA templates prepared from known pathogen-positive samples;
negative extraction control: using pure sterile and aseptic water;
negative no template control: using pure sterile and aseptic water;
(2) IPC addition
Before the sample is added, adding 5 mu L IPC into each extraction hole, and monitoring the experimental process from nucleic acid extraction to fluorescence PCR;
(5) The experiment establishment condition is as follows:
negative control: FAM channels have no Ct value or no typical S-type amplification curve;
negative extraction control: the FAM channel has no Ct value or typical S-shaped amplification curve, and the CT value of the IPC is within the range of preset value +/-3 CT values; the preset value is an actual measured value after the IPC manufacturing process is completed;
positive control: the Ct value of the FAM channel is less than or equal to 35, and the amplification curve is a typical S-shaped amplification curve;
meanwhile, the three points are met, the experiment is established, otherwise, the result is not established, and the experimental result is invalid;
(6) And (3) judging an experimental result:
negative results: the IPC value of the sample is within +/-3 CT values of negative extraction control IPC value, no Ct value and no typical S-type amplification curve, which indicates that the detected pathogenic nucleic acid is not present in the sample;
positive results: ct value is less than or equal to 35, and a typical S-shaped amplification curve appears, which indicates that the detected pathogenic nucleic acid exists in the sample;
suspicious results: samples with Ct values greater than 35 need repeated experiments; and if no Ct value exists in the repeated experiment results, the result is negative, otherwise, the result is positive.
The detection result needs to be repeated: the IPC value of the sample is outside the negative extraction control IPC value ± 3 CT value ranges, no CT value and no typical S-type amplification curve, indicating that there may be reaction inhibition or extraction failure in the sample.
3. The fluorescent PCR method for detecting PEDV with quality control system of claim 2, wherein: the step (1) is specifically that RNA of a sample to be detected is extracted, meanwhile, 5 μl of internal positive quality control IPC is added into an extraction plate to participate in the whole nucleic acid extraction process, nucleic acid extraction and subsequent PCR reaction are monitored, and the extracted nucleic acid is used for the subsequent PCR amplification reaction.
4. The fluorescent PCR method for detecting PEDV with quality control system of claim 3, wherein: the PCR reaction program in the step (3) is that,
UNG enzyme anti-pollution: 25 ℃ for 10min;1Cycle;
reverse transcription: 5min at 52 ℃;1Cycle.
Pre-denaturation: 95 ℃ for 10s;1Cycle;
and (3) PCR reaction: 95 ℃ for 5sec; 30s at 60 ℃;40Cycles.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506186A (en) * | 2016-02-17 | 2016-04-20 | 南阳师范学院 | Primers for detecting porcine epidemic diarrhea viruses and fluorescent quantitative RT-PCR detecting method |
| CN109097499A (en) * | 2018-09-19 | 2018-12-28 | 浙江农林大学 | It is a kind of for detecting the dual fluorescence quantification RT-PCR detection kit of PEDV-TGEV nucleic acid |
| CN109487014A (en) * | 2019-01-09 | 2019-03-19 | 中生方政生物技术股份有限公司 | Herpes simplex virus type II detects marker, primed probe to, kit and detection method |
| CN112391497A (en) * | 2020-11-30 | 2021-02-23 | 山东绿都生物科技有限公司 | Primer probe set, application thereof and kit for detecting African swine fever virus and porcine epidemic diarrhea virus |
| CN112760320A (en) * | 2020-12-02 | 2021-05-07 | 扬州大学 | Exogenous artificial miRNA for effectively inhibiting porcine epidemic diarrhea virus replication and application thereof |
| CN113462820A (en) * | 2021-07-21 | 2021-10-01 | 河北工程大学 | Multiplex RT-PCR primer probe set for real-time fluorescent quantitative detection of four porcine diarrhea viruses, kit and detection method thereof |
-
2022
- 2022-12-26 CN CN202211675470.9A patent/CN116287443A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506186A (en) * | 2016-02-17 | 2016-04-20 | 南阳师范学院 | Primers for detecting porcine epidemic diarrhea viruses and fluorescent quantitative RT-PCR detecting method |
| CN109097499A (en) * | 2018-09-19 | 2018-12-28 | 浙江农林大学 | It is a kind of for detecting the dual fluorescence quantification RT-PCR detection kit of PEDV-TGEV nucleic acid |
| CN109487014A (en) * | 2019-01-09 | 2019-03-19 | 中生方政生物技术股份有限公司 | Herpes simplex virus type II detects marker, primed probe to, kit and detection method |
| CN112391497A (en) * | 2020-11-30 | 2021-02-23 | 山东绿都生物科技有限公司 | Primer probe set, application thereof and kit for detecting African swine fever virus and porcine epidemic diarrhea virus |
| CN112760320A (en) * | 2020-12-02 | 2021-05-07 | 扬州大学 | Exogenous artificial miRNA for effectively inhibiting porcine epidemic diarrhea virus replication and application thereof |
| CN113462820A (en) * | 2021-07-21 | 2021-10-01 | 河北工程大学 | Multiplex RT-PCR primer probe set for real-time fluorescent quantitative detection of four porcine diarrhea viruses, kit and detection method thereof |
Non-Patent Citations (1)
| Title |
|---|
| FANFAN ZHANG ET AL.: ""A simple and rapid identifcation method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplifcation"", 《BIOLOGICAL RESEARCH》, vol. 50, no. 30, pages 1 - 7 * |
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