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Primer group, probe and kit for detecting porcine reproductive and respiratory syndrome NADC30-like strain

A detection kit and a technology for porcine PRRS, applied in the direction of recombinant DNA technology, DNA/RNA fragments, microorganisms, etc., to achieve the effects of improving efficiency, rapid detection, and saving detection costs

Pending Publication Date: 2021-04-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of detecting porcine PRRS encountered in clinics, farms and experimental institutions, and to provide a detection method for accurately and independently detecting NADC30-like strains from mixed porcine PRRS strains Method, through the designed specific primers and probes, it can accurately detect whether the samples infected with porcine PRRS are infected with NADC-30-like strains, so as to accurately distinguish them from other popular PRRS viruses

Method used

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  • Primer group, probe and kit for detecting porcine reproductive and respiratory syndrome NADC30-like strain
  • Primer group, probe and kit for detecting porcine reproductive and respiratory syndrome NADC30-like strain
  • Primer group, probe and kit for detecting porcine reproductive and respiratory syndrome NADC30-like strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Design and synthesis of NADC30-like strain-specific primers and probes

[0042] 1. Design and synthesis of qPCR primers and probes

[0043] The flow chart of the synthesis design of primers and probes is as follows: figure 1 shown. According to a NADC30-like strain isolated and preserved in our laboratory and three classic PRRS strains Jxa-1, GM2, and XH-GD popular in South China, the shared missing sequence and NADC30-like strain Unique partial sequence design qPCR specific primers and probes.

[0044] First, use SnapGene software to analyze the whole genome sequence obtained by sequencing, and obtain a unique sequence of NADC30-like strain by comparing four PRRS strains. The sequence is about 420bp. For this sequence, a pair of The positive primers of NADC30-like strains can be run out, and then design probes for this 420bp sequence, and then redesign the primers. The probe sequence is between the upstream and downstream primers, and then use ordinary PCR...

Embodiment 2

[0056] Example 2 The kit for detecting the NADC30-like strain of porcine PRRS

[0057] A detection kit for accurately and independently detecting NADC30-like strains from mixed strains of porcine PRRS, consisting of the following components:

[0058] Specific primers and probes whose sequences are shown in SEQ ID NO.1-3 in Example 1; Premix Ex Tap, cDNA template and ddH 2 O, specifically as shown in Table 2.

[0059] Table 2 Components and contents of the kit

[0060]

[0061] Wherein, the reaction condition of qPCR is: 95° C. for 30 seconds of pre-denaturation, followed by 40 cycles of qPCR reaction. The reaction conditions are 95° C. for 5 seconds and 60° C. for 30 seconds.

Embodiment 3

[0062] Example 3 TapMan fluorescent quantitative PCR method for detecting the NADC30-like strain of PRRS

[0063] 1. Experimental method

[0064] 1. Preparation of strain samples

[0065] Take the samples of pig farms infected with PRRS provided by the swine disease testing room of our laboratory, including lung tissue blocks and serum (note: the pig farm collects blood from the anterior vena cava of pigs with diseased pigs, and the serum separates out after placement. , centrifuged and frozen after extraction), all samples are guaranteed to be transported in the cold chain. The samples were delivered and kept in a safe place. For the lung tissue, cut it and put it into a 2mL EP tube, add 3 special steel balls for grinding, grind it with a liquid nitrogen cryogenic grinder, and centrifuge to collect the supernatant.

[0066] All serum and tissue fluids were extracted with the RNAfast200 Total RNA Rapid Extraction Kit from Shanghai Feijie Biotechnology Co., Ltd. After confirm...

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Abstract

The invention discloses a primer group, a probe and a kit for detecting a porcine reproductive and respiratory syndrome NADC30-like strain. The invention provides a specific primer group and probe for detecting a porcine reproductive and respiratory syndrome NADC30-like strain, wherein the nucleotide sequences of the specific primer group and the probe are shown as SEQ ID NO.1-3; the invention provides a detection kit for detecting the NADC30-like strain and a TapMan fluorescent quantitative PCR detection method based on the specific primer group and the probe; the kit and the detection method only need 3 hours from sample nucleic acid extraction to result, the detection time is shortened, the detection result can be quickly detected and fed back, the efficiency of a detection mechanism is improved, and a pig farm is helped to know the infection condition of pigs more quickly. Meanwhile, technologies such as enzyme digestion, sequencing and the like are not needed, a kit is not used, the detection cost is saved, and a wide application prospect is achieved.

Description

technical field [0001] The invention relates to the technical field of virus molecular biology detection, and more specifically, relates to a primer set, a probe and a kit for detecting the NADC30-like strain of PRRS. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome), also known as porcine blue ear disease, is a severe infectious disease of pigs bred by porcine reproductive and respiratory syndrome virus (PRRSV), and its clinical features are mainly severe reproductive disorders in sows , Respiratory diseases of weaned piglets. The disease spreads quickly and is highly contagious. It is one of the 93 statutory reportable animal diseases listed by the World Organization for Animal Health (Office international des épizooties, OIE). PRRSV is a single-stranded positive-sense RNA virus, which belongs to the order Necroviridae, Arteriviridae, and Arterivirus genus. An important feature of this virus family ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2561/101
Inventor 张桂红梁冠
Owner SOUTH CHINA AGRI UNIV
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