Application of GeXP multiplex gene expression genetic analysis system in genotyping of 16 common respiratory viruses

A respiratory and viral technology, applied in the field of biotechnology applications, can solve problems such as limited applications

Inactive Publication Date: 2012-12-26
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

Due to the obvious shortcomings of traditional viral diagnostic techniques, its clinical application is limited

Method used

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  • Application of GeXP multiplex gene expression genetic analysis system in genotyping of 16 common respiratory viruses
  • Application of GeXP multiplex gene expression genetic analysis system in genotyping of 16 common respiratory viruses
  • Application of GeXP multiplex gene expression genetic analysis system in genotyping of 16 common respiratory viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0015] Embodiment 1: Singleplex RT-PCR Validation Primers

[0016] Single-primer PCR reactions were performed using single-infected positive specimens of multiple strains of known viral nucleic acids as templates, negative clinical 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, dNTP Mix 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, 72°C for 30s, 10 cycles; chimeric primer amplification at 95°C for 30s, 65°C for 30s, 72°C for 30s, 10 cycles; universal primer amplification at 95°C for 30s, 48°C for 30s, 72°C for 3...

Embodiment approach 2

[0017] Embodiment 2: Multiple reaction system specificity verification

[0018] 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

[0019] Embodiment 3: Multiple detection system single template sensitivity test

[0020] 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 RiboMAX TM Large Scale RNA Production System-T7 reagent The RNA fragments transcribed in vitro were purified by the RNeasy MinElute Cleanup Kit, quantified by a NanoDrop ND-1000 UV spectrophotometer, and the copy number of the RNA was calculated according to the 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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Abstract

The invention belongs to the biotechnology application field, and relates to simultaneous detection and genotyping of infections of 16 respiratory viruses (including FluA, FluB, sH1N1, PIV1, PIV2, PIV3, RSVA, RSVB, HRV, HMPV, HBoV, CoV NL63, CoV OC43, CoV 229E, CoV HKU1 and Adv) of nasopharyngeal extract specimens of patients of respiratory-related diseases of all levels of disease prevention and control institutions and sentinel hospitals. Specifically, nucleotide sequences of representative strains of the 16 respiratory viruses are downloaded from NCBI; through literature review and multiple sequence alignment, pathogen relatively-conservative regions are determined, and multiplex specific primers are designed. Single-tube multiplex (18 stages) PCR detection is carried out for detecting the 16 respiratory virus conservative regions, and an entire reaction takes less than 2 hours. According to the invention, a defect that genotyping cannot be carried out with conventional single-tube multiplex fluorescent qualitative PCR detections can be overcome, and defects of complicated operations, long time, and high cost of conventional chip detection methods can be overcome. With the application provided by the invention, a novel idea is provided for respiratory virus genotyping technologies. With characteristics of high specificity, high sensitivity, and high speed, powerful technological support is provided for rapid and accurate screening and genotyping of the respiratory-disease-related viruses. The invention has important significance upon the researches of respiratory-tract patient infection pathogen spectrum of out nation, and upon molecular epidemiological investigations.

Description

field of invention [0001] The invention belongs to the field of biotechnology application, and relates to 16 kinds of respiratory viruses (including FluA, FluB, sH1N1, PIV1, PIV2, PIV3) used in nasopharyngeal extract samples of patients with respiratory related diseases in disease prevention and control institutions at all levels, sentinel hospitals, etc. Simultaneous detection and typing of , RSVA, RSVB, HRV, HMPV, HBoV, CoV NL63, CoV OC43, CoV 229E, CoV HKU1 and Adv) infections. Specifically, the nucleotide sequences of 16 representative strains of respiratory viruses were downloaded from NCBI, and the relatively conserved regions of the pathogen were determined through literature review and multiple sequence comparisons, and multiple specific primers were designed. Perform single-tube multiplex (18-fold) PCR to detect the conserved regions of 16 respiratory viruses, and the entire reaction takes less than 2 hours. This patent overcomes the shortcomings of virus culture met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 马学军李瑾毛乃颖许文波谭文杰
Owner 中国疾病预防控制中心病毒病预防控制所
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