Primer pair, kit and detection method for rt-raa fluorescence detection of porcine epidemic diarrhea virus n gene

A porcine epidemic diarrhea and kit technology, applied in the field of molecular biology, can solve the problems of false positive PCR, complicated operation, low sensitivity, etc., and achieve the effect of strong specificity

Active Publication Date: 2022-07-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PEDV is relatively difficult to isolate, and it is possible to isolate the virus only by adding an appropriate amount of trypsin. The sensitivity is not high, and the operation is cumbersome, and the cycle is long; although the conventional RT-PCR detection method is simple, convenient, and fast, there are still problems with long detection cycle. Problems with false positives and PCR contamination

Method used

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  • Primer pair, kit and detection method for rt-raa fluorescence detection of porcine epidemic diarrhea virus n gene
  • Primer pair, kit and detection method for rt-raa fluorescence detection of porcine epidemic diarrhea virus n gene
  • Primer pair, kit and detection method for rt-raa fluorescence detection of porcine epidemic diarrhea virus n gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Initial primer design

[0052] 以NCBI数据库中PEDV N基因序列(GenBank登录号:JX188454、KJ020932、KJ646613、KC243782、JF690780、JF700126、JQ743654、JX406135、MG373547、MK458324、MK458327、KT799997、KY619826、KY619826、KY619825)为参考,根据基因比对 Conserved regions were analyzed, and after homology analysis, primers and probes (P1) were designed as follows:

[0053] P1: CTAGCGGACTCTTACGAGATTACATACAATTATAAAATGACTGTGCCAAAG (SEQ ID NO.5); the modified probe is as follows: CTAGCGGACTCTTACGAGATTACATACAA / i6FAMdT / / THF / / iBHQ1dT / AAAATGACTGTGCCAAAG[C3-spacer];

[0054] F-1: CTCTTTGGTGGTAATGTGGCTGTTCGTGAG (shown in SEQ ID NO. 6);

[0055] F-2: CCAGTTTAGCACCAAATGTTGCAGCATTG (shown in SEQ ID NO. 7);

[0056] R-1: CAGTTTTTAAATGCATCCACCTGTGAAACAAGAAG (shown in SEQ ID NO. 8);

[0057] R-2: CATTCCCAGTTTTAAATGCATCCACCTGTG (shown in SEQ ID NO. 9);

[0058] R-3: TTCCCAGTTTTAAATGCATCCACCTGTG (shown in SEQ ID NO. 10);

[0059] Divided into 6 groups: F-1 / R-1, F-1 / R-2, F-1 / R-3, F-2 / R-1, F-2 / R-2 and F-2 / R -3.

[0060] Primer Sc...

Embodiment 2

[0080] Validation of primer pair for detection of porcine epidemic diarrhea virus N gene RT-RAA

[0081]The primers screened in Example 1 were verified to the F5R3 amplified fragment, the template was PEDVN gene plasmid (GenBank accession number JX188454: 26376-27701), and the reaction system was: forward primer F5 (10 μM) 1.0 μL, reverse primer R3 ( 10μM) 1.0μL, ddH 2 O 6.0 μL, 2x Es Taq MasterMix 10.0 μL, Template 2 μL. The reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30s, renaturation at 58°C for 30s, extension at 72°C for 30s, 35 cycles; extension at 72°C for 2 min. The resulting product was analyzed by 1% agarose gel electrophoresis.

[0082] The result is as Figure 4 As shown, the amplified fragment pair of the screened primer pair is consistent with the expected result, and the size is 171bp. The lanes are Marker, PEDV N gene plasmid, ddH from left to right. 2 O negative control.

Embodiment 3

[0084] A kit for detecting porcine epidemic diarrhea virus, the kit is composed of the following components: RT-RAA reaction dry powder, forward primer F5 screened in Example 1, reverse primer R3 screened in Example 1, and Example 1 Modified probes P, A, B, positive control and negative control after screening; the molar ratio of forward primer, reverse primer and probe is 1:1:1; the positive control is a pig containing pig Epidemic diarrhea virus AJ1102 nucleic acid (shown in SEQ ID No. 4), negative control substance is ddH 2 O.

[0085] The components of RT-RAA reaction dry powder are: MLV reverse transcriptase, recombinase, single-chain binding protein, polymerase, ATP, dNTP Mix, magnesium chloride (MgCl 2 ); A liquid composition is: polyethylene glycol (PEG); B liquid composition is: magnesium acetate (MgAc 2 ); the RT-RAA reaction dry powder, liquid A and liquid B are the components in the RT-RAA nucleic acid amplification reagent with item number ZBA22001 purchased fro...

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Abstract

The invention relates to the technical field of molecular biology, in particular to a primer pair, a kit and a detection method for RT-RAA fluorescence detection of the N gene of porcine epidemic diarrhea virus. The primer pair of the present invention includes a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2 . The primer pair provided by the invention can specifically amplify the porcine epidemic diarrhea virus N gene, and the porcine epidemic diarrhea virus can be detected by using the primer pair, and the detection period is shorter and the specificity is strong.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer pair, a kit and a detection method for RT-RAA fluorescence detection of the N gene of porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea virus (PEDV) is currently a widespread coronavirus worldwide. PEDV is an enveloped virus belonging to the family Coronaviridae and alphacoronavirus. Day-old pigs, especially, can infect the small intestine of piglets, and quickly lead to watery diarrhea in piglets, accompanied by clinical symptoms such as vomiting and dehydration, and the fatality rate is as high as 100%. [0003] The latest research data show that the S1 gene in the PEDV epidemic strains in my country is mutating, and the infection of the mutant strains leads to the severity of porcine epidemic diarrhea. With the prevalence of PEDV mutant strains, vaccine strains also need to be replaced urgently. Since detection is one ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2521/107C12Q2563/107
Inventor 张新珩谢青梅巫秀红刘远佳严专强
Owner SOUTH CHINA AGRI UNIV
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