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Degenerate primers and rt-pcr detection method for detection of arenaviridae viruses

A degenerate primer, arena virus technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. The effect of annealing temperature, reducing primer degeneracy, and improving specificity

Inactive Publication Date: 2015-11-18
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 (synthesized by Handsome Biotechnology Co., Ltd.) shown in SEQ ID NO: 5 was artificially synthesized, and cloned separately with pGEM-TEasy vector. Amplify the T7 promoter and insert sequence by PCR, and use MEGAscriptT7Kit as a template for in vitro transcription to generate and purify the target RNA, which is the positive control RNA template; use the RNA reverse transcription kit (purchased from Promega) to carry out the RNA template RNA reverse transcription to obtain PCR template, the specific reverse transcription method is carried out according to the instructions; carry out PCR reaction according to 25μL PCR reaction system: 10×PCR buffer 2.5μL, concentration of 25mM MgCl 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 primer solution with a concentration of 25 μM...

Embodiment 2

[0017] It is basically the same as in Example 1, except that the Lassa fever virus nucleotide fragment is replaced with the Flack virus nucleotide fragment as shown in SEQIDNO:6, and the results show that the Flack virus positive plasmid amplification can obtain a 408bp size fragment bands, blank and negative controls without bands.

Embodiment 3

[0019] It is basically the same as in Example 1, except that the Lassa fever virus nucleotide fragment is replaced by the Lahuning virus nucleotide fragment as shown in SEQIDNO:7, and the results show that the positive plasmid amplification of the Lahuning virus can obtain 411bp Bands of large and small fragments, no bands appeared in the blank and negative control.

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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, Arenavirusgenus, 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. and New World Arenaviruses (New World Arenaviruses), Old World Arenaviruses are distributed all over the world, including Lassa Virus (LASV), Mopeia Virus (Mopeia Virus, MOPV), Ippy Virus (IppyVirus) , IPPYV), Mobala virus (MobalaVirus, MOBV), lymphocytic choriomeningitis virus (Lymphocyticchoriomeningitisvirus, LCMV); New World arenaviruses are divided into A, B, and C strains according to their gene sequences. A strains include viruses: Piritalvirus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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