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Degenerate primer for detecting virus belonging to virus family Arenaviruses and RT-PCR detection method

A technology for arenaviruses and degenerate primers, applied in biochemical equipment and methods, microbe measurement/testing, DNA/RNA fragments, etc., can solve the problems of no public reports on virus general-purpose detection primers, and improve the quality of primers. Effects of annealing temperature, reduction of primer degeneracy, and improvement of specificity

Inactive Publication Date: 2013-07-31
中华人民共和国大榭出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the current gene detection method or using specific primers only for a certain virus in the arenaviridae, such as the invention patent application CN102719557, which discloses multiple PCR reagents including detection of Lassa fever virus, but for There are no public reports on universal detection primers for all arenaviridae viruses

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The nucleotide fragment of Lassa fever virus shown in SEQ ID NO:5 (synthesized by Handsome Biotech Co., Ltd.) was artificially synthesized, and cloned separately with pGEM-T Easy vector. The T7 promoter and insert sequence were amplified by PCR, and the MEGAscript T7 Kit was used as a template for in vitro transcription to generate and purify the target RNA, which was the positive control RNA template; the RNA reverse transcription kit (purchased from Promega) was used for The template is subjected to RNA reverse transcription to obtain a PCR template. The specific reverse transcription method is carried out according to the instruction manual; PCR reaction is carried out according to the 25 μL PCR reaction system: 10×PCR buffer 2.5 μL, MgCl concentration of 25 mM 21.5 μL solution, 0.5 μL dNTP solution with a concentration of 10 mM, 0.5 μL Taq enzyme solution with a concentration of 5 U / μL, 1 μL upstream primer solution with a concentration of 25 μM, 1 μL downstream prim...

Embodiment 2

[0017] It is basically the same as Example 1, except that the Lassa fever virus nucleotide fragment is replaced with the Flack virus nucleotide fragment shown in SEQ ID NO: 6, and the results show that the Flack virus positive plasmid amplification can obtain 408bp Bands of large and small fragments, no bands appeared in the blank and negative control.

Embodiment 3

[0019] It is basically the same as in Example 1, except that the Lassa fever virus nucleotide fragment is replaced with the Lahuning virus nucleotide fragment as shown in SEQ ID NO:7, and the results show that the positive plasmid amplification of the Lassa fever virus can The band of the 411bp fragment was obtained, and no band appeared in the blank and negative controls.

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PUM

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Abstract

The invention discloses a degenerate primer for detecting virus belonging to the virus family Arenaviruses and a RT-PCR detection method. The degenerate primer can sensitively detect the virus belonging to the virus family Arenaviridae, and is a universal primer which is specific for the virus belonging to the virus family Arenaviridae, and can be used for detecting lymphocytic choriomeningitis virus which is widely distributed, and also can be used for detecting epidemic strains of other Arenaviruses, thereby preventing foreign epidemic strains from affecting our country, and the primer also can be used for detecting unknown viruses which is homologous to the expansion gene, thereby discovering new virus. Because a codehop primer design method is used, the degeneracy of the primer is reduced and the primer annealing temperature is increased simultaneously, thereby improving the singularity of PCR reaction of the designed degenerate primer.

Description

technical field [0001] The invention relates to the detection of arenaviruses, in particular to a degenerate primer and RT-PCR detection method for detecting arenaviridae viruses. Background technique [0002] Arenaviridae belong to single-stranded negative-sense RNA. There is only one genus in the Arenavirus family, namely the Arenavirus genus, and more than 20 species of viruses are known. According to the different antigenic substances and geographical distribution of arenaviruses, arenaviruses are divided into Old World arenaviruses (Old World World Arenaviruses) and New World Arenaviruses (New World Arenaviruses), Old World Arenaviruses are distributed all over the world, including Lassa Virus (LASV), Mopeia Virus (MOPV) ), Ippy Virus (IPPYV), Mobala Virus (Mobala Virus, MOBV), Lymphocytic choriomeningitis virus (LCMV); New World arenaviruses are divided into A, B, C three species. A strains include viruses: Pirital virus (PIRV), Fleck virus (Flexal virus, FLEV), Pa...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 胡群马思杰邹春颖童淑梅
Owner 中华人民共和国大榭出入境检验检疫局
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