NASBA kit for detecting monkey pox virus and application thereof

A technology of monkeypox virus and kit, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., to achieve the effect of simple operation, guaranteed specificity and high purity

Inactive Publication Date: 2015-02-11
浙江国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Judging from the prevalence of monkeypox in humans, monkeypox is weaker in pathogenicity and infectivity than smallpox, but monkeypox is susceptible to many different animals and has a wide distribution range, which is easy to cause epidemics, and once the virus is in the human body Recombination mutation occurs, and the virulence of the virus is similar to smallpox or exceeds that of smallpox, the consequences will be disastrous

Method used

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  • NASBA kit for detecting monkey pox virus and application thereof
  • NASBA kit for detecting monkey pox virus and application thereof
  • NASBA kit for detecting monkey pox virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the preparation of the NASBA kit that detects monkeypox virus and the establishment of reaction system

[0047] One, the preparation of the NASBA kit that detects monkeypox virus

[0048] 1. Design of primers and probes

[0049] According to the conserved sequence (48332-48780 of GenBank: AF380138, sequence 4) in the whole monkeypox virus genome (GenBank: AF380138), a set of primers and probes were designed, which were forward primer P1, reverse primer P2 and probe The gene sequences of needles, primers and probes are as follows:

[0050] Primer P1 (sequence 1):

[0051] 5'-AATTCTAATACGACTCACTATAGGGAGAAGGCAGCATCACCTATAGATGAC-3'

[0052] Primer P2 (SEQ ID NO: 2): 5'-TACAGAGCTTTATTAACTTCTCGCTT-3'

[0053] Probe (Sequence 3): 5'- CATCG GAACGACGAACCACCAGAGGATGA CGATG -3'

[0054] Among them, the 32nd-51st position of primer P1 (sequence 1) is a specific sequence complementary to the 3' end of monkeypox virus conservative sequence (396-415th position o...

Embodiment 2

[0078] Embodiment 2, the specific detection of the NASBA kit that detects monkeypox virus

[0079] Samples to be tested: positive control plasmid (pGSI-MPV plasmid), sheeppox vaccine, chickenpox vaccine, vaccinia vaccine, chickenpox vaccine. Wherein, the above-mentioned poxvirus vaccines use DNA extraction kits to extract genomic DNA according to the instructions for future use.

[0080] Using the reaction solution I and the enzyme mixture II in step one and two of embodiment 1, measure according to the operation method described in step two of embodiment 1, and determine the result according to the detection result determination method in step two of embodiment 1 ( I) and (II) judge the results respectively, so as to test the specificity of the NASBA kit. At the same time, the experiment was set up with sterilized double distilled water as the negative control of the sample to be tested.

[0081] The results of agarose gel electrophoresis of RNA amplification products were ...

Embodiment 3

[0082] Embodiment 3, detect the sensitivity detection of the NASBA kit of monkeypox virus

[0083] The positive control plasmid (pGSI-MPV plasmid) was used as the sample to be tested, and the sensitivity of the NASBA kit prepared in Example 1 was tested. At the same time, the experiment was set up with sterilized double distilled water as the negative control of the sample to be tested. The specific operation is as follows:

[0084] Take the positive control plasmid (pGSI-MPV plasmid plasmid) of known concentration, and dilute it by 10 times to the following serial concentrations: 2.76, 2.76×10 1 , 2.76×10 2 , 2.76×10 3 , 2.76×10 4 , 2.76×10 5 , 2.76×10 6 , 2.76×10 7 Copy number / μL. Using the positive control plasmid (pGSI-MPV plasmid) of the obtained serial concentrations as a template, use the reaction solution I and the enzyme mixture II in Step 1 and 2 of Example 1, according to the steps described in Step 2 of Example 1 The operation method is to measure, and the...

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Abstract

The invention discloses an NASBA kit for detecting a monkey pox virus and the application thereof. The NASBA kit contains a composition used for detecting the monkey pox virus, the composition is composed of a primer pair and a probe, the primer pair is composed of two single-stranded DNA molecules shown in the first sequence and the second sequence in a sequence list, and the sequence of the probe is the third sequence in the sequence list. The NASBA kit is used for detecting the monkey pox virus, and the advantages of being simple, convenient, efficient, quick and peculiar are achieved. The NASBA kit can quickly screen the monkey pox virus and has great significance to screening port input diseases, technical support and strategic reserve are well prepared for preventing introduction of the diseases, and the demand of current health quarantine is met.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a NASBA kit for detecting monkeypox virus and an application thereof. Background technique [0002] Monkeypox virus is a double-stranded DNA virus. It belongs to the poxviridae family like smallpox virus. The symptoms of infection are similar to those of smallpox, such as fever, headache, swollen lymph nodes, cough and extreme pain all over the body. It is commonly known as "monkey smallpox". One of the dangerous viruses in our country, which can be transmitted through direct contact with patients or infected animals, or through the bodily fluids of patients. [0003] Monkeypox virus was first discovered in African monkeys in a laboratory in Copenhagen, Denmark in 1958. The world's first human case of monkeypox was reported in the Congo in 1970. Since then, such cases have been reported in Nigeria, Cameroon and other African countries. After the eradication of smallpox in 1978, the WH...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 吕沁风罗鹏何蕾杨永耀
Owner 浙江国际旅行卫生保健中心
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