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Method for detecting Zika virus by using RPA technology and special complete set of reagent for method

A Zika virus and a complete set of technology, applied in the biological field, can solve the problems of inapplicable rapid detection of sudden infectious diseases, large instrument size, long detection time, etc., and achieve great practical value, high sensitivity, and good specificity

Pending Publication Date: 2019-10-29
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescent quantitative PCR technology has high sensitivity, but it requires expensive temperature control equipment, specific laboratory space and professional technicians to operate, and the detection time is long, and the instrument is large in size, so it is not suitable for on-site rapid detection of sudden infectious diseases

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, the preparation of the kit reagent that detects Zika virus

[0064] 1. Synthesis of primers and probes

[0065] The whole genome sequence of Zika virus was downloaded from NCBI, and the MEGA5.0 software was used for comparative analysis. After analysis, the NS1 gene (Genebank number: KU509998.1) was selected as the target gene. According to the nucleotide sequence of the NS1 gene, primers and probes based on RPA technology are designed. The design principles are as follows: (1) The length of the primer is 30-35bp; (2) The length of the probe is 46-52bp, from 5' to 3' Include DNA fragment A, tetrahydrofuran (THF) (tetrahydrofuran is the cleavage site of exo) and DNA fragment B in turn; DNA fragment A is a single-stranded DNA molecule composed of 30-35 nucleotides; DNA fragment A and DNA fragment B are partially identical to the two segments on the NS1 gene, and the two segments have no overlap on the NS1 gene; the deoxyribonucleotide immediately upstream ...

Embodiment 2

[0077] Embodiment 2, sensitivity

[0078] The PBS buffers in the following examples are all pH 7.2, 0.01 mM PBS buffers.

[0079] 1. Detection of virus titer

[0080] 1. Inoculate the Zika virus into a culture flask lined with C6 / 36 cells and place it at 33°C, 5% CO 2 cultured in an incubator. When the proportion of cytopathic cells in the culture flask reached 75%, the culture flask was frozen and thawed once at -70° C., and then the cell culture supernatant was collected.

[0081] 2. Take the cell culture supernatant collected in step 1, and perform a 10-fold serial dilution with PBS buffer to obtain a supernatant dilution.

[0082] 3. Take a 96-well plate containing C6 / 36 cells growing to the logarithmic phase (each well contains 10 4 C6 / 36 cells), add 100 μL supernatant dilution obtained in step 2 to each well (repeat 4 wells for each concentration), place at 33°C, 5% CO 2 Cultured in the incubator for 5 days.

[0083] Take a 96-well plate containing C6 / 36 cells grow...

Embodiment 3

[0098] Embodiment 3, specificity

[0099] Japanese encephalitis virus SC04-17 strain was recorded in the following literature: Zheng Yang, Kang Xiaoping, Li Yuchang, Wu Xiaoyan, Zhang Xiaosong, Yang Yinhui, Expression and purification of Japanese encephalitis virus EDⅢ protein and its application in Array-ELISA method, Zhonghua Journal of Microbiology and Immunology, 2013, 33, 954-959. Japanese encephalitis virus SC04-17 strain is hereinafter referred to as Japanese encephalitis virus.

[0100] Dengue virus is described in Microarray hybridization for assessment of the genetic stability of chimeric West Nile / dengue 4virus, Laassri M, Bidzhieva B, Speicher J, Pletnev AG, Chumakov L., J.Med.Vorol., 2011 May; 83(5 ); 910-920.

[0101] Tick-borne encephalitis virus MDJ01 strain was recorded in the following literatures: Huo Naifan, Kang Xiaoping, Hu Yi, Li Yuchang, Li Jing, Zhang Yu, Ran Xin, Jia Jia, Cao Xuefeng, Yang Yinhui, Tick-borne encephalitis virus envelope glycoprotein c...

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PUM

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Abstract

The invention discloses a method for detecting Zika virus by using an RPA technology and a special complete set of reagent for the method. The complete set of reagent consists of a primer pair X and aprobe X. The primer pair X consists of a primer F as shown in a sequence 3 in a sequence table and a primer R as shown in a sequence 2 in the sequence table. The probe X comprises a DNA fragment A, tetrahydrofuran and a DNA fragment B in sequence from 5' to 3'. The DNA fragment A and the DNA fragment B are respectively single-stranded DNA molecules as shown in a sequence 4 and a sequence 5 in thesequence table. Experiments prove that the complete set of reagent provided by the invention can detect Zika virus by performing RPA, and has good specificity and high sensitivity. The reagent has great practical value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting Zika virus by using RPA technology and a set of special reagents thereof. Background technique [0002] Zika virus (ZIKV) belongs to the Flaviviridae family and is transmitted by mosquito bites. Zika virus was first discovered in the blood of rhesus monkeys in Uganda's Zika forest in 1947. Symptoms of Zika virus infection are usually mild, and Zika virus infection has been ignored for a long time. Since 2015, Zika virus has caused a worldwide epidemic, with more than 1 million cases of Zika virus infection in Brazil. In China, several cases of Zika virus infection have also been found. Research has shown that Zika virus infection can cause microcephaly in newborns and has also been linked to other birth defects. The development of rapid detection reagents for Zika virus is crucial to controlling the spread of Zika virus and adopting effective pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12R1/93
CPCC12Q1/701C12Q1/6844
Inventor 康晓平秦成峰姜涛李裕昌邓永强吴晓燕户义李靖
Owner ACADEMY OF MILITARY MEDICAL SCI
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