Liquid chip detection method for four types of pathogens of porcine virus diarrhea

A liquid-phase chip detection technology for porcine viral diarrhea, which is applied in the field of virology, can solve the problems of cumbersome operation, long experiment cycle, and no simultaneous detection, and achieve the effect of good repeatability and specificity

Active Publication Date: 2016-06-15
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In recent years, the incidence and mortality of porcine viral diarrhea diseases have gradually increased, and the infection of a single pathogen has developed into a mixed infection of multiple pathogens, which brings difficulties to clinical diagnosis, delays the timing of diagnosis, and brings economic losses to the pig industry.
At present, routine laboratory detection methods for diagnosing such pathogens include virus isolation and identification, ELISA, real-time fluorescent quantitative PCR technology, etc., but these methods have long experimental cycles, cumbersome operations, or are limited to a single virus, or 2-3 viruses Detection, there is no technology to detect the above four viruses at the same time

Method used

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  • Liquid chip detection method for four types of pathogens of porcine virus diarrhea
  • Liquid chip detection method for four types of pathogens of porcine virus diarrhea
  • Liquid chip detection method for four types of pathogens of porcine virus diarrhea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Design and synthesis of embodiment 1 primers and probes

[0063] Select the M gene of conservative PEDV on GENBANK (GenBank accession number is AF353511.1), the N gene of TGEV (GenBanK accession number is EU074218), the VP6 gene of PRoV (GenBank accession number is FJ617209.1) and pig Source BVDV's N pro After the gene sequence (GenBanK accession number is HQ258810.1) was analyzed online, DNASTAR software was used to analyze and design specific primers, probes and complementary probe chains for these genes. Amino (NH 2 (CH 2 ) 12 ) modification, all complementary probe strands and the 5' end of the downstream primers are labeled with biotin (Biotin), and the test screening is carried out after synthesis. The sequences of each viral primer and probe after screening are shown in Table 2. The probe and primer design of the present invention mainly pay attention to the following points:

[0064] Primer design principles: 1) The length of the general primer is 18-25 bas...

Embodiment 2

[0069] The preparation of embodiment 2 positive plasmid

[0070] Viral RNA extraction: Trizol lysis method was used to extract the RNA of the sample to be tested, and the operation steps were as follows: (1) Take 250 μl of each cytotoxicity and add 3 times the volume of Trizol lysate, mix with a vortex shaker, and stand on ice for 10 minutes; ( 2) Add 200 μl of chloroform, mix well with a vortex shaker, and let stand on ice for 10 minutes; (3) Centrifuge at 12000×g at 4°C for 15 minutes; (4) Take the supernatant, add an equal volume of isopropanol, and mix well. Stand on ice for at least 10 minutes, repeat step (3); (5) discard the supernatant, add 1ml of 75% absolute ethanol, mix upside down, repeat step (3), discard the liquid, blow dry, add 20 μl DEPC water, - Store at 80°C for later use.

[0071] Prepare cDNA by reverse transcription: 10ulRNA, 2ulprimemix, 0.5ulM-MLVRT, 0.5ulRRI, 5xReverseTranscriptaseM-MLVBuffer4ul, 2.5mMdNTP2ul, ddH2O1ul. 1h at 42°C, 15min at 75°C, 4, ...

Embodiment 3

[0081] Example 3 Microsphere coupled probe

[0082]The probe BN-Probe of 44# microspheres coupled with BVDV; the probe PM1-Probe of 25# microspheres coupled with PEDV; the probe PVP6-Probe of 20# microspheres coupled with PRoV; the probe of 15# microspheres coupled with TGEV Probe TGEV-Probe, the coupling steps are as follows:

[0083] (1) Take 44# microspheres, 25# microspheres, 20# microspheres, and 15# microspheres (Luminex company) out of the 4-degree refrigerator and return to room temperature, vortex for 20 s, and sonicate for 20 s; take out 100 μl of microspheres Into a 1.5ml imported centrifuge tube;

[0084] (2) Centrifuge at 12000xg for 2 minutes, take out the centrifuge tube and put it on a magnetic stand, carefully remove the supernatant, resuspend the microspheres with 50 μl MES, vortex and shake to mix the microspheres completely;

[0085] (3) Add 10 μm probe BN-Probe 2 μl to 44# microspheres;

[0086] Add 10 μm probe PM1-Probe 2 μl to 25# microsphere;

[008...

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Abstract

Disclosed is a liquid chip detection method for four types of pathogens of porcine virus diarrhea. The method includes firstly, designing and synthesizing corresponding upstream primers, downstream primers and probes to gene N<pro> of virus BVDV, gene N of virus TGEV, gene M of virus PEDV and gene VP6 of virus PRoV respectively, subjecting 5'-ends of all the downstream primers to biotin labeling, and modifying 5'-ends of all the probes with amino group (NH2(CH2)12); choosing microspheres with different color coding and coupling the same with the probes of BVDV, TGEV, PEDV and PRoV respectively, mixing the four types of probes which are coupled with the microspheres to obtain a probe mixture; hybridizing amplified products obtained by biotinylation with the probe mixture coupled with the microspheres to obtain a hybridized product, subjecting the hybridized product to liquid chip detection, and determining detection results according to MFI values. The method can be applied to detection of four types of pathogens of porcine virus diarrhea simultaneously and has the advantages of good specificity, good repeatability and stability and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of virology, and specifically relates to a liquid-phase chip detection method for four pathogens of porcine viral diarrhea, and more particularly relates to a liquid-phase chip detection method capable of simultaneously detecting four pathogens of porcine viral diarrhea. Background technique [0002] Porcine viral diarrheal disease is a highly contagious diarrheal disease caused by a variety of viruses with diarrhea, vomiting and dehydration as the main clinical symptoms. Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (PRoV) and porcine bovine viral diarrhea virus (BVDV) are the main pathogens causing the disease. Can be infected, the case fatality rate is as high as 100%. [0003] In recent years, the incidence and mortality of porcine viral diarrhea diseases have gradually increased, and the infection of a single pathogen has developed into a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6837C12Q1/701C12Q2563/149C12Q2531/113C12Q2563/107Y02A50/30
Inventor 刘惠莉熊年年陶洁张春玲李本强
Owner SHANGHAI ACAD OF AGRI SCI
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