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Nucleic acids of liquid-phase gene chip for synchronously detecting five porcine viruses and detection method thereof

A liquid-phase gene chip and simultaneous detection technology, applied in the biological field, can solve problems such as difficult and false positive results

Active Publication Date: 2015-02-04
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] In summary, the research on high-throughput liquid-phase chip detection technology in animal disease virus detection is still in its infancy, and there are few reports on liquid-phase chip high-throughput detection of porcine disease virus, mainly due to the need to realize multiple To detect viruses at the same time, it must be ensured that the primers, probes and amplification products used for the detection of these pathogens cannot cross-react with each other, otherwise, false positive results are prone to occur, especially the more virus factors are detected, the primers should be designed , the more difficult the probe is

Method used

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  • Nucleic acids of liquid-phase gene chip for synchronously detecting five porcine viruses and detection method thereof
  • Nucleic acids of liquid-phase gene chip for synchronously detecting five porcine viruses and detection method thereof
  • Nucleic acids of liquid-phase gene chip for synchronously detecting five porcine viruses and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 Design and preparation of amplification primers and hybridization probes in the five-fold liquid-phase gene chip reaction system

[0101] Firstly, the specific target genes of the five viruses were screened separately. According to the purpose of detection, multiple viral gene sequences were downloaded from GenBank for each virus, compared and analyzed, and conserved regions were selected, and Array Designer 4.0 software was used to assist in the design of amplification primers and suitable A hybridization probe for a liquid-phase gene chip reaction system.

[0102] Liquid-phase gene chip detection is based on ordinary PCR detection, and further through the design of hybridization probes, PCR amplification products are melted and then hybridized with the hybridization probes, and the fluorescence value after the hybridization reaction is detected to determine the detection results. The premise of chip detection is to carry out PCR amplification reaction, the p...

Embodiment 2

[0111] Example 2 Establishment and optimization of five-fold liquid-phase gene chip detection reaction method

[0112] 1. Optimization of primer concentration in the five-fold one-step asymmetric RT-PCR amplification reaction system

[0113] In the amplification reaction stage, the key lies in the ratio of labeled primers to unlabeled primers, which is a key factor in the liquid-phase chip reaction system and has a great impact on detection sensitivity. In this step, the inventor has done a lot of optimization experiments. First, the five viruses were screened for the dosage ratio of labeled primers and unlabeled primers one by one. Different ratios were added to the reaction system, and the rest of the reaction conditions remained the same. The final detection MFI values ​​corresponding to different primer concentration ratios were compared, and the primer ratio that could achieve the highest MFI value and lower background value and negative sample value was preferred. Add f...

Embodiment 3

[0139] Example 3 Five-fold liquid-phase gene chip detection method specificity, sensitivity, repeatability detection test and evaluation

[0140] 1 specific detection test

[0141] In order to verify whether the detection nucleic acid and detection method of the present invention are specific to the five kinds of viruses detected, the inventors of the present invention can only detect and identify these five kinds of porcine viruses, and will not cross-react with other viruses, resulting in false positives or For non-specific amplification, a specificity test was carried out, as follows:

[0142] Adopt the method of the present invention (specifically can adopt the preferred reaction condition of embodiment 2) to the various strains of above-mentioned five kinds of target viruses and vaccine strain sample and other various common swine pathogen samples, whole genome nucleic acid of porcine tissue, commonly used passaging cell The whole genome nucleic acid samples were tested,...

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Abstract

The invention provides a set of nucleic acids of a liquid-phase gene chip for synchronously detecting five porcine viruses, which comprise forward and reverse primers and hybrid probes for porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV) and porcine parvovirus (PPV). The invention also provides a multiplex liquid-phase chip high-flux molecular biology detection method of the five porcine viruses. According to the method, porcine virus nucleic acids in the sample to be detected are extracted to perform multiplex unsymmetric nucleic acid amplification / multiplex liquid-phase gene chip (suspension chip) combined detection, thereby synchronously and accurately detecting and identifying the five porcine viruses in the sample to be detected. The method has the advantages of high specificity, high sensitivity, high stability, high flux and high detection speed, and is simple to operate.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, it provides a group for simultaneous rapid detection and identification of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV-2), porcine pseudorabies virus (PRV), and classical swine fever virus (CSFV). ) and porcine parvovirus (PPV) a total of five kinds of porcine virus five-fold liquid gene chip high-throughput detection of nucleic acid and its detection method. Background technique [0002] (1) Several important porcine viruses that cause diseases such as pig reproductive disorders [0003] Porcine reproductive and respiratory syndrome virus (PRRSV). Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infects pigs and causes Porcine Reproductive and Respiratory Syndrome (PRRS), which is characterized by reproductive disorders in sows and respiratory symptoms in piglets and fattening pigs. Highly immunosuppressive disease and highl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6816C12Q1/701C12Q1/705C12Q2600/16C12Q2531/107C12Q2537/143C12Q2563/107C12Q2563/149
Inventor 陈茹曹永长高小博宋长绪于晓璐邱杨田纯见朱道中罗长保薛春宜
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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