Application of GeXP multiple gene expression heredity analysis system in detection of nine encephalitis related viruses

A tick-borne encephalitis virus and Japanese encephalitis virus technology, applied in the field of biotechnology applications, can solve problems such as accurate diagnosis of difficult and unknown pathogenic infection cases

Inactive Publication Date: 2013-03-06
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, various molecular biology diagnostic methods based on PCR technology have been widely used and developed, including SYBR Green I real-time PCR and TaqMan PCR, etc., but t

Method used

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  • Application of GeXP multiple gene expression heredity analysis system in detection of nine encephalitis related viruses
  • Application of GeXP multiple gene expression heredity analysis system in detection of nine encephalitis related viruses
  • Application of GeXP multiple gene expression heredity analysis system in detection of nine encephalitis related viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0014] Embodiment 1: Singleplex RT-PCR Validation Primers

[0015] Single-primer PCR reactions were carried out using single-infected positive samples of known virus nucleic acids from multiple strains as templates, negative samples as negative controls, and redistilled water as blank controls. Qiagen One-step RT-PCR kit was used. 25μl PCR reaction system: 5*buffer 5ul, dNTPMix 1ul, enzyme mix 1ul, upstream and downstream chimeric primers (1 μmol / L) 1.25 μl each, upstream and downstream universal primers (10 μmol / L) each 1.25 μl, template RNA 2 μl , 0.1ul of RNase inhibitor, make up to 25μl with RNase-free water. The reaction conditions are: reverse transcription at 50°C for 30 minutes; pre-denaturation at 95°C for 15 minutes; specific primer amplification at 95°C for 30s, 55°C for 30s, and 72°C for 30s, 10 cycles; chimeric primer amplification at 95°C for 30s, 68°C for 30s, 72°C for 30s, 10 cycles; universal primer amplification at 95°C for 30s, 53°C for 30s, 72°C for 30s, ...

Embodiment approach 2

[0016] Embodiment 2: Multiple reaction system specificity verification

[0017] Prepare multiple mixed primer (Mix-Primer) working solutions, so that the final concentration of each SP-Primer in the RT-PCR system is 50nmol / L, and the rest of the components are the same as those verified by the primers. Using a single positive sample of multiple strains of known viral nucleic acids as a template, a multi-primer PCR reaction is performed.

Embodiment approach 3

[0018] Embodiment 3: Multiple detection system single template sensitivity test

[0019] Use tag-free specific primers to amplify the target nucleic acid region, connect the PCR product to the pGEM-T vector for single cloning, extract the cloned plasmid, digest it with Spe I to linearize it, and use the RiboMAX Large Scale RNA Production System-T7 kit For in vitro transcription, the RNeasy MinElute Cleanup Kit purified the in vitro transcribed RNA fragments, quantified them with a NanoDrop ND-1000 UV spectrophotometer, and calculated the RNA copy number based on molecular weight and nucleic acid concentration. In vitro transcribed RNA was serially diluted to 10 6 、10 5 、10 4 、10 2 、10 1 copies / μL, take 1 μl each as a template, and test the sensitivity of the multiplex system. Three parallel experiments on different days.

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PUM

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Abstract

The invention belongs to the biotechnological application filed, and relates to simultaneous detection and typing of the infection of nine encephalitis viruses (comprising banna viruses, GI encephalitis B viruses, GIII encephalitis B viruses, tick-borne encephalitis viruses, Tahyna viruses, Liaoning viruses, Kyasanur forest fever viruses, Sindbis viruses and Yunnan orbiviruses) of viral encephalitis patient specimens in disease prevention control mechanisms at all levels, sentinel point hospitals and the like. The nucleotide sequences of nine encephalitis related virus strains are downloaded from NCBI, the pathogen relative conservation region is determined through literature consulting and multiple sequence alignment, and multiple specific primers are designed. The single tube 13-plex multiplex PCR test is carried out to detect nine encephalitis virus conservation regions, and the consumption time of the whole reaction is less than 2h. According to the invention, the non-typing disadvantage of routine single-tube multiple fluorescent qualitative PCR detection is overcome, the disadvantages comprising complex operation, long time, high cost and the like of routine chip detection methods are overcome, a new thought is provided for an encephalitis virus typing technology, the characteristics comprising high specificity, high sensitivity and rapidness of the GeXP multiple gene expression heredity analysis system provide a strong technological support for the rapid and accurate screening and typing of the encephalitis viruses, and the GeXP multiple gene expression heredity analysis system is of important significance to the encephalitis syndrome patient infection pathogen spectrum and molecular epidemiology investigation in China.

Description

field of invention [0001] The invention belongs to the field of biotechnology application, and relates to nine kinds of encephalitis-associated viruses (including Banna virus, GI Japanese encephalitis virus, GIII B virus, etc.) Simultaneous detection and typing of encephalovirus, tick-borne encephalitis virus, Tahyna virus, Liaoning virus, Kosanur forest fever virus, Sindbis virus, and Yunnan orbivirus) infection. Specifically, the nucleotide sequences of nine representative strains of encephalitis viruses were downloaded from NCBI, and the relatively conserved regions of the pathogen were determined by consulting the literature and multiple sequence comparisons, and multiple specific primers were designed. A single-tube multiplex (13-fold) PCR was performed to detect the conserved regions of 9 encephalitis viruses, and the entire reaction took less than 2 hours. This patent overcomes the shortcomings of virus culture method, direct fluorescent antibody method and chip detect...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 马学军何玢梁国栋王环宇秦萌
Owner 中国疾病预防控制中心病毒病预防控制所
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