A kind of primer combination, probe combination and its application in detecting porcine virus, detection reagent, kit and detection method

A detection reagent and primer combination technology, applied in the field of probe combination and its application in the detection of porcine virus, and primer combination, can solve the problems of difficult clinical sample detection, and achieve improved detection efficiency, cost saving, and simple operation Effect

Active Publication Date: 2021-04-27
北京市动物疫病预防控制中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some scholars have established five or more target fluorescent quantitative PCR methods, they are all based on dye-based fluorescent PCR. Due to the problem of false positives, it is difficult to apply to clinical sample detection.
[0010] At present, there are many fluorescent quantitative PCR methods that can detect ASFV, FMDV, CSFV, HP-PRRSV and PRV wild virus alone, but there are no reports on the TaqMan probe five-fold fluorescent quantitative PCR method that can detect the above five porcine viruses simultaneously.

Method used

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  • A kind of primer combination, probe combination and its application in detecting porcine virus, detection reagent, kit and detection method
  • A kind of primer combination, probe combination and its application in detecting porcine virus, detection reagent, kit and detection method
  • A kind of primer combination, probe combination and its application in detecting porcine virus, detection reagent, kit and detection method

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Experimental program
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Effect test

Embodiment 1

[0068] Embodiment 1: This embodiment provides a method for joint detection of five porcine viruses. Specifically, a five-fold fluorescent quantitative PCR detection method was established to establish a joint detection method for five porcine viruses.

[0069] 1. Design of primers and TaqMan probes

[0070] The B646L gene sequences of different African swine fever virus (ASFV) strains published by GenBank (accession numbers: MN393476.1, MN715134.1, MN172368.1, LR743116.1, MK333180.1, MH910495.1, MN336500.2); 3D gene sequences of foot-and-mouth disease virus (FMDV) strains (accession numbers: FJ175661.1, GU384683.1, DQ989323.1, DQ989308.1, FJ175662.1, FJ175665.1, MF372126.1, DQ989320. 1); Nsp2 gene sequence of porcine reproductive and respiratory syndrome virus (PRRSV) strain (accession numbers: NC_043487.1, EF635006.1, EF112445.1, EF641008.1, AY150564.1, MH500776.1, MN119307.1 , KP771777.1); 5´UTR gene sequence of classical swine fever virus (CSFV) strain (accession numbers:...

Embodiment 2

[0127] Example 2: Sensitivity Verification of Five-fold Fluorescent Quantitative PCR Detection Method

[0128] ASFV (5.8 × 10 4 copies / μL), FMDV (8.1×10 4 copies / μL), CSFV (5.1×10 4 copies / μL), HP-PRRSV (9.7×10 4 copies / μL), PRV wild virus (7.6×10 4 copies / μL) Take 10 μL of each nucleic acid and mix evenly (concentrations: 1.16×10 4 copies / μL, 1.62×10 4 copies / μL, 1.02×10 4 copies / μL, 1.94×10 4 copies / μL, 1.52×10 4 copies / μL) after serial dilution of 10 times, 100 times, 1000 times and 10000 times, then used as a template for five-fold fluorescent quantitative PCR detection, each gradient was repeated 3 times, and the minimum detection limit and linearity of the five-fold fluorescent quantitative PCR method were calculated. relation. Amplification results such as Image 6 As shown in Table 11, ASFV is the standard curve of the Texas Red channel (No. 1-Texas Red) regression equation is Y= -3.251X+40.547, and the correlation coefficient (R 2 ) was 0.990, the a...

Embodiment 3

[0131] Embodiment 3: Five-fold fluorescent quantitative PCR detection method specific verification

[0132] FMDV (A type, O type, Asian type I), PRRSV (LV strain, JXA1 strain, VR2332 strain), CSFV (C strain), PRV standard strain, PRV vaccine strain (Bartha -K61 strain), TGEV-PEDV - PoRV was extracted according to step 3 of Example 1, and then the nucleic acid of the ASFV positive sample was used as a template for five-fold fluorescent quantitative PCR detection.

[0133] Test results such as Figure 7 As shown, except ASFV (No. 1), FMDV-A (No. 2), FMDV-O (No. 3), FMDV-Asia Ⅰ (No. 4), PRRSV-JXA1 (No. 5), CSFV (No. 6), PRV Except for wild virus (No. 7), other viruses (No. 8-10) were not detected, indicating that this method has no cross-reaction with other viruses, and can specifically detect ASFV, FMDV, HP-PRRSV, CSFV and PRV wild poison.

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Abstract

The invention discloses a combination of primers, a combination of probes and their application in detecting porcine viruses, a detection reagent, a kit and a detection method; the invention provides a combination of primers, a combination of probes and their application in detecting porcine viruses Application, detection reagents, kits and detection methods, establish a joint detection method for five kinds of porcine viruses, which can quickly detect five major swine diseases of ASFV, FMDV, CSFV, HP‑PRRSV and PRV wild virus, and can achieve high detection of five kinds of viruses Throughput screening improves detection efficiency and reduces reagent consumption, thereby saving costs, shortening detection time, and reducing workload; providing detection reagents that can realize joint detection of five porcine viruses, using optimized primer combinations and probe combinations, so that reagents and The kit using this reagent can specifically and sensitively detect five porcine viruses of ASFV, FMDV, CSFV, HP-PRRSV and PRV wild virus; it can simply and quickly detect five porcine virus infections with different types of nucleic acids in parallel in a single reaction tube, Realized single tube PCR to detect DNA virus and RNA virus at the same time.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer combination, a probe combination and their application in detecting porcine viruses, a detection reagent, a kit and a detection method. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contagious zoonotic disease of pigs caused by African swine fever virus (ASFV), characterized clinically by high fever and reticuloendothelial bleeding Characterized by high mortality and high mortality, the case fatality rate of susceptible pigs is as high as 100%. The World Organization for Animal Health (OIE) has listed African swine fever as a legally notifiable animal disease, and my country has listed it as a first-class animal disease. In August 2018, my country's first case of African swine fever was diagnosed, and then African swine fever broke out in many places, which brought a heavy blow to my country's pig breeding industry. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q1/705C12Q2600/16C12Q2531/113C12Q2537/143C12Q2545/113C12Q2563/107
Inventor 高晓龙梅力王英超冯小宇赵浩军程汝佳张启龙王培吴迪陈会玲程敏姮
Owner 北京市动物疫病预防控制中心
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