Degenerate reverse transcription-polymerase chain reaction (RT-PCR) detection reagent and kit for hantavirus group

An RT-PCR and hantavirus technology, applied in the field of hantavirus detection, can solve the problems of inability to detect other hantavirus viruses, public reports, and unavoidable introduction, and achieves high sensitivity and improved specificity. , good specificity

Inactive Publication Date: 2012-03-21
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

At present, in the RT-PCR detection method of Hantaan virus, only the Hantaan virus type and / or Seoul virus type in Hantaan virus are used for RT-PCR detection with specific primers, such as the invention patent with the authorized announcement number CN101294226, A kit with two pairs of primers designed for the genotypes of Hantaan virus and Seoul-type virus is disclosed, which is used to detect Hantaan virus and / or Seoul-type virus; the kit cannot detect Hantaan virus Other viruses within the genus Virus
Although most of the domestic epidemic areas are currently infected by Hantaan virus and Seoul type virus, with the increase of import and export trade volume, foreign epidemic strains (Pumala type virus, Dobraval type virus, Tula type virus , Andean virus, and Sotopalaya virus, etc.) are inevitable, so it is particularly important to prevent the introduction of foreign epidemic strains and the detection of unknown types of viruses. Published report on a general-purpose PCR detection reagent for viruses

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1, RT-PCR detection reagent and kit detect Hantaan virus

[0014] 1. Total RNA extraction: Vero cells were cultured with 5% inactivated calf serum DMEM at 37°C, routinely digested and subcultured into a monolayer, and then inoculated with Hantavirus 76-118 strains (Hantan type, courtesy of Zhejiang Center for Disease Control and Prevention) ), after 2 hours of adsorption, use 0.5% calf serum DMEM (purchased from Hyclone) to maintain the solution and place it at 37°C, CO 2 Cultured in an incubator. After 7-10 days of positive IFA detection, the cells were harvested and the total RNA of the cells was extracted with an RNA extraction kit (purchased from Qiagen) as positive RNA (the specific extraction method was in accordance with the instructions). Use a micro-spectrophotometer to measure the RNA concentration and purity, OD260 / 280≥1.9, indicating that the RNA is of high purity without DNA and protein contamination, take 50ng for RT-PCR reaction;

[0015] 2. ...

Embodiment 2

[0020] The actual detection of embodiment 2, RT-PCR detection reagent and kit to mouse Hantavirus

[0021] 253 mice collected in Daxie Port Area from 2008 to 2009 were killed by cervical dislocation after ether anesthesia, and 10-25 mg of lung tissue was collected, homogenized and extracted with RNA extraction kit for total RNA from lung tissue (specific extraction method According to the instructions), measure the RNA concentration and purity with a micro-spectrophotometer, OD260 / 280≥1.9, indicating that the RNA purity is high, without DNA and protein contamination, take 50ng for RT-PCR reaction; then proceed according to step 2 of Example 1 RNA reverse transcription, step 3 for PCR reaction, and step 4 for agarose gel electrophoresis, the results showed that 253 mouse lung samples were detected, and 2 of them were amplified to obtain bands of 478bp fragments. The test results were consistent with the results of the "National Detection Program for Hemorrhagic Fever with Renal...

Embodiment 3

[0022] Embodiment 3, RT-PCR detection reagent and kit detection detect Seoul type virus

[0023] It is basically the same as steps 1-4 of Example 1, except that the Hantavirus 80-39 strain virus (Seoul type, gifted by Zhejiang Center for Disease Control and Prevention) was inoculated, and the Hantavirus 80-39 strain Vero cells were detected A band with a size of 478 bp appeared in the culture, and the sequence of the sequencing result is shown in SEQ ID NO:5.

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PUM

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Abstract

The invention discloses a degenerate reverse transcription-polymerase chain reaction (RT-PCR) detection reagent and a kit for hantavirus group. The detection reagent comprises a pair of degenerate heterozygous oligonucleotide primers, and the sequences of the degenerate heterozygous oligonucleotide primers are respectively G1: GCAACAGCAACATGGTTTcartaytayac and G2: CTTCTTCATTCATATTTCCATGCarnccyttytc; the non-merger consensus sequence of the 5' ends in the primers plays a role in stabilizing the combination of a 3' merger core area and a template under the condition that the degeneracy of the primers is not increased, so that the specificity of the degenerate PCR reaction is improved, various viruses in hantavirus can be amplified and detected, the homologous unknown viruses of the extendedgenes can also be detected and the amplified target fragments can be sequenced by the detection reagent; and the kit are relatively high in sensitivity and good in hantavirus group specificity, can be used for detecting domestic popular Hantaan and Seoul viruses, and can also be used for detecting other oversea epidemic strains. The degenerate RT-PCR detection reagent and the kit for the hantavirus group are high in sensitivity, good in hantavirus specificity and universal.

Description

technical field [0001] The invention relates to a detection technology for hantaviruses, in particular to a degenerate RT-PCR detection reagent and kit for hantaviruses. Background technique [0002] The genus Hantavirus has only one virus group, the Hantavirus group (referred to as Hantavirus), which belongs to the Bunyaviridae family. According to the molecular structure and antigenicity of Hantaan virus gene, Hantaan virus can be divided into at least 34 serotypes / genotypes, including Hantaan virus (Hantaan virus), Seoul virus (Seoul virus), Puumala type Puumala Virus, Dobrava Virus, Tula virus, Thottapalayam virus and Andes virus etc. Hantavirus can infect wild rodents and humans, causing human hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS); these diseases spread to more than 70 countries in Asia, Europe, Africa, America, Oceania, etc The country, with its wide prevalence and serious harm, has become a global public health problem....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 胡群郭利平马思杰谢东华韩辉
Owner 中华人民共和国大榭出入境检验检疫局
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