RPA technology-based marburg virus detection kit and application thereof

A Marburg virus and reagent technology, applied in the biological field, can solve the problems of rapid detection of sudden infectious diseases, long detection time, etc., and achieve the effects of preventing the spread of infection, rapid response, and wide temperature range

Inactive Publication Date: 2017-05-10
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current human detection of Marburg virus mainly relies on real-time fluorescent quantitative PCR technology, which requires expensive temperature control equipment, specific laboratory space and professional technicians to operate, and the detection time is long, which is not suitable for on-site rapid detection of sudden infectious diseases Therefore, the establishment of a rapid and simple nucleic acid detection technology is of great significance and value for the timely discovery and identification of Marburg virus

Method used

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  • RPA technology-based marburg virus detection kit and application thereof
  • RPA technology-based marburg virus detection kit and application thereof
  • RPA technology-based marburg virus detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the RPA detection primer and probe of Marburg virus and its detection method

[0069] 1. Design and synthesis of primers and probes for RPA detection of Marburg virus

[0070] Download the complete gene sequence of Marburg virus, use DNASTAR.Lasergene.v7.1 software to perform sequence alignment analysis, select intra-species conserved, inter-species specific nucleoprotein (NP) gene segment (length is 153bp) as detection target gene, The RPA detection primers and probes for Marburg virus are designed according to the following design requirements: (1) The length of the RPA primer is 30-35 bases, the GC content is between 40-60%, and the formation of secondary structure and hairpin structure is avoided; primers Special sequences such as long strings of polypurine or polypyrimidine should be avoided; the first 3-5 nucleotides at the 5' end should be avoided as polyguanine, preferably cytosine; (2) The length of the RPA probe is 46- 52 bases, respectively mor...

Embodiment 2

[0086] Embodiment 2, the RPA detection of Marburg virus and the sensitivity comparison of Real-time PCR detection

[0087] 1. Marburg virus RPA detection

[0088] Adopt the RPA detection method of Marburg virus in the step 2 of embodiment 1, use the Marburg pseudovirus RNA solution of 10-fold gradient dilution as template (the amount of template added is: 10 5 copies / reaction, 10 4 copies / reaction, 10 3 copies / reaction, 10 2 copies / reaction, 10 copies / reaction) for RPA amplification.

[0089]2. Marburg virus Real-time PCR detection

[0090] The RNA solution of Marburg pseudovirus diluted by 10 times was used as template (the amount of template added was: 10 5 copies / reaction, 10 4 copies / reaction, 10 3 copies / reaction, 10 2 copies / reaction, 10copies / reaction), adopt primers and probes in Table 1, utilize one-step real time RT-PCR kit (Takara, DRR064A) to carry out Real-time PCR, the reaction system in each amplification tube is as shown in the table 2 shown.

[0091]...

Embodiment 3

[0102] Example 3. Specific detection of Marburg virus RPA primers and probes

[0103] The five hemorrhagic fever-related viruses are as follows: Marburg virus (Marburg pseudovirus), Zaire Ebola virus (Zaire pseudovirus Zaire), and dengue virus type 1 (DEN-1). The viral nucleic acid RNAs of GZ01 strain, Xinjiang hemorrhagic fever virus (XHF) YL04057 strain and Hantavirus 8205 strain were used as templates, and the RPA detection method of Marburg virus in step 2 of Example 1 was used to detect the RPA primers of the present invention and specificity of the probe.

[0104] The result is as image 3 shown. The experimental results show that the RPA primers and probes designed in the present invention can specifically amplify the target gene and are compatible with other four hemorrhagic fever-related viruses (Zaire Ebola virus, dengue virus, Xinjiang hemorrhagic fever virus and Han virus). Tanzania virus) nucleic acid has no cross-reaction, indicating that the Marburg virus RPA...

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Abstract

The invention discloses an RPA technology-based marburg virus detection kit and an application thereof. An experiment proves that a target gene can be effectively amplified by an RPA primer and a probe of a marburg virus, the specificity reaches 100% and sensitivity is 1*10<2>copies/reaction; and the virus can reach the level equivalent to the sensitivity of fluorescent quantitative PCR, and has no cross reaction with a zaire ebola pseudovirus, a dengue virus, a hemorrhagic fever virus with renal syndrome and a Xinjiang hemorrhagic fever virus nucleic acid. The RPA isothermal amplification system is fast in reaction and wide in temperature range, effective amplification of a target gene can be achieved under the condition of 37-42 DEG C, the method can be used for fast field detection of an infectious nucleic acid of the marburg virus, and an available fast detection method is provided for field screening of a pathogen; and meanwhile, the marburg virus detection kit also has the important significance in prevention of infection transmission of the marburg virus in China and inspection and quarantine in affected areas and entry and exit ports.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a Marburg virus detection kit based on RPA technology and its application. Background technique [0002] Marburg virus (Marburg virus) belongs to the genus Marburg virus of the Filoviridae family, mainly prevalent in Africa, can cause severe hemorrhagic fever symptoms, is highly contagious, and has a case fatality rate of 23-90%. It is characterized by acute fever accompanied by severe bleeding. At present, there are no cases of Marburg virus infection in China. However, with the rapid development of global trade and tourism, human exchanges between different countries and regions are becoming more and more frequent. Many infectious diseases have broken the restrictions of regions and national borders, and their spread has become more and more extensive. my country has also repeatedly faced the threat of imported infectious diseases. my country has a large population and a high popu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101C12Q2563/107Y02A50/30
Inventor 杨银辉康晓平吴晓燕李裕昌姜涛曹雪峰李靖户义
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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