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Primer probe set and kit for detecting seven viruses related to porcine reproductive disorder diseases based on liquid phase chip technology and application of primer probe set and kit

Pending Publication Date: 2021-11-23
浙江省动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, the co-infection of multiple pathogens in the current cases of porcine viral reproductive disorders is becoming more and more common. At this stage, the detection methods and technologies of porcine reproductive disorders related viruses have been difficult to meet the needs of current differential diagnosis and large-scale screening and classification Therefore, it is imminent to develop a high-throughput, rapid, sensitive and economical detection method for pathogens related to porcine viral reproductive disorders

Method used

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  • Primer probe set and kit for detecting seven viruses related to porcine reproductive disorder diseases based on liquid phase chip technology and application of primer probe set and kit
  • Primer probe set and kit for detecting seven viruses related to porcine reproductive disorder diseases based on liquid phase chip technology and application of primer probe set and kit
  • Primer probe set and kit for detecting seven viruses related to porcine reproductive disorder diseases based on liquid phase chip technology and application of primer probe set and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] According to classical swine fever virus, porcine epidemic encephalitis virus, American porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine circovirus type 3, porcine parvovirus, highly pathogenic porcine reproductive and respiratory syndrome virus Synthetic primers and probes were designed and synthesized for the conserved sequences of syndrome virus and porcine pseudodog disease virus (Table 1). The 5' ends of the upstream and downstream primers were labeled with biotin, and the primers were synthesized and labeled by Sangon Biotech (Shanghai) Engineering Co., Ltd. The 5' end of the capture probe is labeled with fluorescent microspheres with different codes. The specific sequence of the above-mentioned virus capture probe was designed by our laboratory and entrusted to Shanghai Toujing Life Technology Co., Ltd. to synthesize the label and prepare the hybridization solution.

[0059] Table 1 Amplification primers, probe sequences and...

Embodiment 2

[0063] Preparation method of various porcine virus recombinant plasmid standard items

[0064] The target gene in the constructed recombinant plasmid should contain the detection gene amplification fragment, and the amplification primer of the target gene is used to construct the recombinant plasmid standard product. The specific method is as follows: according to the target gene fragment, design primers (see Table 2) to amplify the virus containing Detect the nucleic acid fragments of the gene, using various viral nucleic acids as templates, among which the classical swine fever virus, the highly pathogenic porcine reproductive and respiratory syndrome virus and the American porcine reproductive and respiratory syndrome virus were RTed using One Step PrimeScript RT-PCRKit -PCR reaction to amplify the target gene, the reaction system is as follows: 2×one step RT-PCR buffer 25μl, Takara Ex Taq HS (5U / μl) 1μl, PrimeScript RT Enzyme Mix 1μl, forward primer (20μmol / L) 1μl, reverse ...

Embodiment 3

[0068] The composition of the kit and the screening method of hybridization temperature and time in the detection process

[0069] 1. Kit composition: primer probes shown in Table 1 in Example 1, multiplex PCR reagents, and reporter solution.

[0070] 2. The screening method of hybridization temperature and time during the detection process of the kit

[0071] 1) Extraction of viral nucleic acid

[0072] Viral Nucleic Acid Extraction Kit MagMAX TM -96 Viral RNA Isolation Kit Extract viral nucleic acid according to the instructions, and store the obtained viral nucleic acid in a -80°C refrigerator for later use.

[0073] 2) Multiplex PCR amplification

[0074]Use Taqpath 1-step multiplex Master mix to carry out multiplex PCR of the above 7 viruses, the specific reaction system is as follows: 4×multiplex Master mix 5 μl, forward primer (20 μmol / L) 0.5 μl, reverse primer (20 μmol / L) 0.5 μl , 4 μl of nucleic acid template, 4 μl of RNase-free water. Total volume 20 μl. Reacti...

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Abstract

The invention provides a primer probe set and a kit for detecting seven viruses related to porcine reproductive disorder diseases based on a liquid phase chip technology and application of the primer probe group and the kit, and belongs to the technical field of virus detection. The invention provides a primer probe set. which comprises a hog cholera virus detection primer probe, an American type porcine reproductive and respiratory syndrome virus detection primer probe, a porcine circovirus type 2 detection primer probe, a porcine circovirus type 3 detection primer probe, a porcine parvovirus detection primer probe, a high-pathogenicity porcine reproductive and respiratory syndrome virus detection primer probe and a porcine pseudocanine disease virus detection primer probe. A sample is detected by adopting the primer probe set and an established method, the detection result is highly consistent with the detection result of a single virus fluorescent quantitative PCR method, and the primer probe set has the characteristics of good detection specificity, good detection result repeatability and high detection sensitivity, can be used for simultaneously detecting seven viruses related to the porcine reproductive disorder diseases, and has the characteristics of high flux and low detection cost.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and specifically relates to a primer probe group, a kit and an application thereof for simultaneously detecting seven kinds of porcine reproductive disorder-related viruses based on liquid-phase chip technology. Background technique [0002] Porcine reproductive disorder disease is mainly characterized by abortion, stillbirth, mummified fetus, inactive weak piglets, deformed babies, few piglets and infertility of male and female pigs in pregnant pigs. Virus infection is one of the main causes of this type of disease. Studies have shown that porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine pseudorabies virus, swine fever virus, porcine circovirus type 2 and type 3 can all cause the occurrence of porcine reproductive disorders. [0003] Early rapid and accurate differential diagnosis is of great significance to block the spread of porcine viral reproductive ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6837C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/6837C12Q1/686C12Q2600/16C12Q2537/143Y02A50/30
Inventor 谢荣辉赵灵燕徐辉王雅婷黄靖孙冰冰张红丽董建斐刘霞冯肖肖张璐安慧婷薛银柴娟虞一聪
Owner 浙江省动物疫病预防控制中心
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