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A kind of encephalitis related virus detection kit and its application

A kit and virus technology, applied in the field of molecular biology, to reduce the risk of cross-contamination, high sensitivity, and save resources

Inactive Publication Date: 2019-09-20
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nucleic acid invasion test is a nucleic acid signal amplification detection method developed in recent years that does not amplify the target nucleic acid, but triggers the amplification of other signal molecules by the target nucleic acid, and indirectly detects the target nucleic acid signal, thereby avoiding the crossover of the amplified products. Contamination, and improve the sensitivity of the detection, but also solve the need for a single multiplex PCR detection of various primer concentrations and dNTP and MgCl 2 Concentrations of complex optimization problems

Method used

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  • A kind of encephalitis related virus detection kit and its application
  • A kind of encephalitis related virus detection kit and its application
  • A kind of encephalitis related virus detection kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Synthesis of primers for multiplex PCR detection of encephalitis-associated viruses and probes for nucleic acid invasion reactions

[0038]Primer design: Download the gene sequences of 5 encephalitis-associated viruses (Eastern equine encephalitis virus, Western equine encephalitis virus, Japanese encephalitis virus, West Nile virus and Nipah virus) in the NCBI Genebank database, and pass MEGA 5.1 The software performs sequence comparison, finds the conserved sequence of related pathogenic genes, eliminates homologous gene fragments through blast comparison, and finally determines the target gene fragment of the virus to be tested. Multiplex PCR amplification primers were designed by Primer 5.0. The upstream and downstream primers were designed for the genes in the conserved region for the amplification of each target gene in multiplex PCR (Table 1). Universal Invader 1.2.4 software was used to design the upstream and downstream probes required for the nuclei...

Embodiment 2

[0044] Example 2 Encephalitis-associated virus detection kit detection method

[0045] S1 sample selection: EEEV virus E2 gene, WEEV virus E1 gene, WNV virus non-structural protein 5 gene and NiPA virus nucleoprotein gene were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. and cloned into pUC57 vector. JEV virus strain was used for this experiment Room separation, identification, preservation.

[0046] S2 Nucleic Acid Extraction: Nucleic acid extraction was performed according to the requirements of Roche’s MagNA Pure LC Automatic Nucleic Acid Extractor and MagNAPure LC Total Nucleic Acid Isolation Kit. Nucleic acid was extracted and stored at -80°C.

[0047] S3 Reverse transcription: According to the operation method of Invitrogen’s SuperScript III First-Strand Synthesis SuperMix manual, use the random primers provided by the kit to reverse-transcribe the JEV viral nucleic acid into cDNA and store it at -20°C.

[0048] S4 Using the multiplex PCR primers in Tabl...

Embodiment 3

[0050] Embodiment 3 utilizes the kit of the present invention to detect the specificity of 5 kinds of encephalitis-associated viruses

[0051] A multiplex PCR system was used to amplify the nucleic acid and plasmid samples of five encephalitis viruses, and the reaction products were detected using the nucleic acid invasion reaction and nano-gold chromogenic method described in Example 2. The results are shown in Table 3. When using multiplex PCR reaction technology combined with nucleic acid invasion reaction and nano-gold color development for detection, only when the corresponding primer probes are used, the nano-gold will be positive after color development. When detecting other pathogens, the results All were negative, indicating that this method has high specificity for detecting respiratory pathogens.

[0052] Table 3 Detection specificity results of multiplex PCR combined with nucleic acid invasion reaction and nano-gold chromogenic technology

[0053]

[0054]

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Abstract

The invention discloses an encephalitis-related virus detection kit and application thereof. The kit is used on the basis of the principles of multiplex PCR (polymerase chain reaction) combined nucleic acid intrusive reaction and nano gold color development. The kit comprises a combination of five types of primers and probes, and can be used for simultaneously detecting eastern equine encephalitis virus, western equine encephalitis virus, epidemic encephalitis b virus, West Nile virus and Nipah virus. When being used for detecting the five viruses, the kit has the advantages of high specificity and high sensitivity, and does not need any expensive apparatus; the detection result can be observed by naked eyes; and thus, the kit can be applied to the basic level.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV), Japanese Encephalitis Virus (JEV), West Nile Virus (WNV) and Nipah (NiPA) Multiplex PCR primers for simultaneous detection of virus infection, nucleic acid invasion reaction probe, kit and application. Background technique [0002] Viral encephalitis is a different degree of central nervous system infection caused by viral infection. Its clinical manifestations are: fever, headache, vomiting, confusion, stiff neck, and respiratory failure. Common viruses that may induce encephalitis and meningitis include arboviruses such as Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, Japanese Encephalitis Virus and West Nile Virus, Paramyxovirus Konipahendra The Nipah virus of the virus genus, etc., these five viruses are all RNA viruses, and they are all pathogens of severe ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
Inventor 戚宇华樊欢朱政葛以跃崔仑标周明浩
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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