Method for preparing RNase-resistant dengue virus nucleic acid detection quality control product

A technology for dengue virus and quality control material, applied in the field of infectious disease detection, can solve the problems of cumbersome purification method and general purification effect.

Inactive Publication Date: 2015-06-17
张瑾
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, armored RNA quality control products are mostly purified by polyethylene glycol precipitation or cesium chloride ultracentrifugation. The purification method is cumbersome and the p

Method used

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  • Method for preparing RNase-resistant dengue virus nucleic acid detection quality control product
  • Method for preparing RNase-resistant dengue virus nucleic acid detection quality control product
  • Method for preparing RNase-resistant dengue virus nucleic acid detection quality control product

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0022] Example 1: Obtainment and identification of armored RNA containing histidine tag and 3'URT gene of dengue virus

[0023] 1. Material

[0024] Type 1 dengue virus RNA, D-pET32a plasmid with protein tag removed, and MS2 phage RNA are all kept in this room.

[0025] 2. Method

[0026] 1) Primer design

[0027] According to the analysis of the entire genome sequence of dengue virus types I to IV published by GenBank, the sequence of the highly conserved 3'non-coding region of the virus was selected as the target fragment for amplification. Primer 5.0 software designed primers DFVF / DFVR with a fragment size of 320 bp. HindIII / NotI restriction sites were introduced into the amplification primers. In order to amplify the coat protein gene and the mature enzyme protein gene of MS2 phage, the primer pair MSF and MSR were designed, and BamHI and Hind III restriction sites were added at the 5'end, respectively. The primer pair MS-HISF / MS-HISR is used to insert the sequence encoding 6 his...

Example Embodiment

[0050] Example 2: Verification of the stability of armored RNA quality control products

[0051] 1. Stability test under different storage conditions

[0052] The virus-like particle solution purified in Example 1 was diluted with a buffer containing 15% glycerol to 105 copies / mL, and each tube was divided into 50uL and placed at room temperature, 4°C, -20°C for 5d, 30d, 60d, 90d. , 120d, 150d, 180d, take samples for testing and observe its stability. Another tube of 600 uL virus particles was placed at -70°C and 37°C repeatedly freeze-thaw 10 times to test its stability. The data of the test group and the control group (-70℃) were analyzed by t test, which showed that the difference between the test group and the control group was not statistically significant (P>0.5). The standard substance can be stored at -20℃ and 4℃ for at least 6 months Above, the room temperature storage period is at least 14 days, and repeated freezing and thawing has little effect on the stability of the...

Example Embodiment

[0055] Example 3: Practical application of quality control products in dengue fever nucleic acid detection

[0056] 1. Reagent

[0057] Nucleic acid extraction reagents: Viral RNA Mini RNA Extraction Kit

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Abstract

The invention discloses a method for preparing a RNase-resistant dengue virus nucleic acid detection quality control product. An armored RNA quality control product carrying a histidine tag and a dengue virus 3'URT gene is prepared by utilizing an MS2 phage armor technology and a site-directed mutagenesis technology; a dengue virus armored RNA quality control product is obtained through nickel column affinity chromatography purification, the quality control product is easy to purify, stable, high in concentration and free of biological infectivity, resists RNase, and can be used for carrying out quality control on the whole processes of virus detection, reverse transcription, PCR and the like in dengue virus nucleic acid detection.

Description

technical field [0001] The invention relates to the technical field of infectious disease detection, and relates to a preparation method of an RNase-resistant dengue virus nucleic acid detection quality control product. Background technique [0002] Dengue virus belongs to the Flaviviridae family (Flaviviridae). It is a single-stranded positive-sense RNA virus and is the most important arbovirus for humans. It can cause dengue fever and dengue hemorrhagic fever, and is mainly transmitted by Aedes aegypti and Aedes albopictus. The disease is widely prevalent in tropical and subtropical regions, and is a human infectious disease with wide distribution, frequent incidence and great harm. In recent years, global warming, population flow and other factors have led to a significant increase in the incidence of dengue virus infection in tropical and subtropical regions of Asia, Africa and South America, and there are often small-scale outbreaks in southern my country . Therefore, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/70C12N15/40C12N7/04C12R1/93
CPCC12Q1/701C12Q2600/166Y02A50/30
Inventor 张瑾薛晓宁徐翮飞陈晓光张娟张齐林元朱可
Owner 张瑾
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