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Primer group, kit with primer and application

A technology of primer set and set of primers, which is applied in the field of primer sets and can solve the problems of low sensitivity

Inactive Publication Date: 2018-02-13
HANGZHOU HUAJIN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, domestic routine etiological diagnosis methods mainly include single-plex, multiplex RT-PCR, enzyme-linked immunosorbent assay, virus isolation and identification, virus antigen detection, agar diffusion test, complement fixation test, immunodiffusion precipitation test, virus in And test, indirect hemagglutination test, latex agglutination test, etc. These methods may take a long time to obtain test results, and the sensitivity is low, etc.

Method used

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  • Primer group, kit with primer and application
  • Primer group, kit with primer and application
  • Primer group, kit with primer and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Embodiment 1 RT-nPCR experiment

[0110] Primer design:

[0111] Coat primers:

[0112] M1 (sequence shown in SEQ ID No.1): 5'-GTCTCGCCCAGTACTACACACAGTAC-3'(26bp)

[0113] M2 (sequence shown in SEQ ID No.2): 5'-GCTGAAATTGGCCAACCACAGAC-3' (23bp)

[0114] Inner set of primers:

[0115] N1 (sequence shown in SEQ ID No.3): 5'-TTCATGCGGAGTGGGACACAG-3'(21bp)

[0116] N2 (sequence shown in SEQ ID No.4): 5'-TGGTCTACGTCCTCCTGTACAG-3'(22bp)

[0117] Extraction of total RNA:

[0118] Total RNA was extracted from the inactivated foot-and-mouth disease virus cell strains (type O, type A and AsiaI), normal cells (negative control) and samples to be tested by the Trizol method. The specific operation is as follows:

[0119] For about 100mg of cells or tissues, add 1.5mL of Trizol to homogenate. (Soak the homogenizer with 75% ethanol for 5 minutes, and wash the homogenizer with 75% ethanol and DEPC water for each homogenized tube).

[0120] Centrifuge at 10,000 rpm at 4°C for ...

Embodiment 2

[0141] Embodiment 2 specificity and universality test

[0142] Swine vesicular disease virus (SVDV), swine vesicular stomatitis virus (VSV), attenuated classical swine fever virus (CSFV), attenuated highly pathogenic porcine blue ear disease attenuated virus (HPRRSV), attenuated porcine blue ear disease classic attenuated virus (PRRSV) by Trizol method ), porcine pseudorabies virus (PRV), negative control, O type, A type, Asia I type FMDV carry out total RNA extraction according to the method for embodiment 1, carry out RT-nPCR amplification, and product carries out agarose gel electrophoresis identification, Validate the specificity and generality of the method.

[0143] Experimental result analysis: O-type, A-type, Asia I-type FMDV total RNA is amplified with the established RT-nPCR method, and there is only one target band of about 1000bp, while swine vesicular disease virus (SVDV), porcine vesicular Stomatitis virus (VSV), attenuated swine fever virus (CSFV), attenuated h...

Embodiment 3

[0144] Embodiment 3 Sensitivity comparison test

[0145] The multiplex RT-PCR kit contains 3 pairs of primers, the amplification lengths are 630bp, 485bp and 282bp respectively. As long as more than one target band is amplified, it can be judged as positive. O-type, A-type, Asia I-type FMDV samples were mixed at a ratio of 1:1:1, and after the RNA was extracted according to the method in Example 1, 10× serial dilution was used as a template, and the dilution gradient was 10 0 ~10 -9 °. The RT-nPCR method established according to the method of Example 1 and the FMDV multiplex RT-PCR kit were respectively used for detection, and the products were identified by agarose gel electrophoresis, and the sensitivity of the two methods was compared.

[0146] Analysis of experimental results: use the established RT-nPCR method to perform 10× serial dilution amplification of type O, type A, and Asia I type FMDV RNA, and identify the products by agarose gel electrophoresis, and the seven...

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Abstract

The invention relates to the technical field of biology, in particular to a primer group, a kit with the primer and application. The primer group comprises an outer nested primer and an inner nested primer; a sequence of a forward primer of the outer nested primer is shown as SEQ ID NO.1; a sequence of a reverse primer of the outer nested primer is shown as SEQ ID NO.2; a sequence of a forward primer of the inner nested primer is shown as SEQ ID NO.3; a sequence of a reverse primer of the inner nested primer is shown as SEQ ID NO.4. The invention further provides a foot and mouth disease virusamplification method. A nested RT-PCR (RT-nPCR) method is quick and high in sensitivity, stability and specificity, and simultaneous amplification of O-type, A-type and AsiaI-type foot and mouth disease viruses can be realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer set, a kit containing the primer set and applications thereof. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious infectious disease common to cloven-hoofed animals, and its pathogen is foot-and-mouth disease virus (FMDV). Foot-and-mouth disease virus belongs to the genus Foot-and-Mouth Disease Virus of the Picornaviridae family. The genome is a single-stranded positive-strand RNA with a total length of about 8500 nucleotides. The disease has many transmission routes, strong infectivity, and a variety of animals, which seriously endangers the productivity of livestock and the quality of livestock products, and causes large economic losses to animal husbandry and import and export trade. [0003] There are seven serotypes of foot-and-mouth disease virus: A, O, C, Asia I, SAT1, SAT2 and SAT3, more than 80 subtypes and many disti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
CPCC12Q1/701C12Q1/6848C12Q2549/119
Inventor 唐自军俞保彬姚小飞周天琼张治国
Owner HANGZHOU HUAJIN PHARMA
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