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Nucleic acid for detecting Zika virus, real-time fluorescence RPA kit and method

A Zika virus and real-time fluorescence technology, applied in the field of biotechnology detection, can solve the problems of difficult to achieve detection results, difficult design of primers and probes, etc., to avoid non-specific amplification problems, overcome long detection time, and simplify operations The effect of the process

Active Publication Date: 2017-02-01
淮安市疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulty in designing primers and probes, it is generally difficult to achieve the desired detection effect.

Method used

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  • Nucleic acid for detecting Zika virus, real-time fluorescence RPA kit and method
  • Nucleic acid for detecting Zika virus, real-time fluorescence RPA kit and method
  • Nucleic acid for detecting Zika virus, real-time fluorescence RPA kit and method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The design of embodiment 1 primer, probe

[0036] In the present invention, adopting recombinase polymerase amplification technology (RPA) to detect Zika virus, the design of primers and RPA probes is particularly critical, and the primers and probes of this technology are designed without special software. It can only rely on artificial design, and mark a fluorescent group and a fluorescent quenching group on the two thymine nucleotides in the middle of the RPA probe, and design an abasic site between the two groups ( dSpacer), this site can be recognized and cut by exonuclease III with 3'-5' exonuclease activity, freeing the fluorescent group, thereby emitting a fluorescent signal and then captured by a fluorescent detection instrument. The design of RPA probes is particularly important, which is related to the success of the entire experiment. The success of its design not only needs to be designed based on experience, but also requires a large number of experimental...

Embodiment 2

[0043] The construction of embodiment 2 plasmid

[0044] Compare the whole genome sequence of Zika virus in Genebank, select a relatively conservative sequence segment (membrane protein gene, position 1584-2107), and insert the SP6 promoter sequence at the 5' end of position 1584: ATTTAGGTGACACTATAG, in A 6×His tag sequence was introduced at the 3' end of the 2107 site. The reason for the primer SP6 promoter is that after the successful construction of the plasmid, the above-mentioned recombinant sequence was amplified by PCR method, and the artificially synthesized sequence was transcribed into RNA in vitro to simulate the nucleic acid of Zika virus by using the transcription kit. The purpose of using the 6×His tag sequence is to distinguish the positive quality control from the real Zika virus nucleic acid in the sample.

[0045] details as follows:

[0046] (1) The artificially synthesized sequence (SEQ ID No.4) is as follows:

[0047] 5′- ATTTAGGTGACACTATAG CATTGGTTGG...

Embodiment 3

[0057] Embodiment 3 Zika virus isothermal amplification detection method

[0058] In the method established by the present invention, in order to avoid the failure or contamination of the reagents used, a positive control and a negative control reagent are provided, and the negative control uses nuclease-free water;

[0059] The positive control adopts the plasmid DNA carrying the amplified product, and the nucleotide sequence of the amplified product contains the sequence shown in SEQID No.4.

[0060] The design of the negative control can effectively verify whether the reagents used are contaminated and avoid the occurrence of false positives. The design of the positive control can effectively verify the effectiveness of the reagents used and avoid the occurrence of false negatives.

[0061] After multiple optimization experiments, the following method was determined to be used for detection.

[0062] The method for detecting Zika virus by isothermal amplification comprises...

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Abstract

The invention discloses nucleic acid for detecting Zika virus, a real-time fluorescence RPA kit and a detection method thereof. The real-time fluorescence RPA kit is convenient to use, the quantity of reagents adopted is small, the cost is low, the compatibility of required instruments is high, reaction can be performed on a real-time fluorescence PCR instrument and instruments with a fluorescent trapping function, and the greatest advantage of the kit lines in performing isothermal amplication and being capable of detecting fluorescence signals in real time within 10 to 30 min. The detection method greatly simplifies the operation process, reduces steps of repetitive operation, saves time, reduces labor force consumed by repetitive operation, and effectively lowers the cost, and a detection result shows that the method is high in specificity and sensitivity, and rapid and accurate screening of Zika virus is realized.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, in particular to a real-time fluorescent RPA kit and a detection method for Zika virus detection. Background technique [0002] Zika virus disease, also known as Zika fever, is a viral disease caused by Zika virus (ZIKV) and transmitted by mosquitoes. Zika virus was first discovered in Uganda in 1947. Scientists isolated a virus from rhesus monkeys used for yellow fever surveillance and named it "Zika virus". In 1952, human infection with Zika virus was found in Uganda and Tanzania. Only sporadic reports or small epidemics of the disease have subsequently been reported. Until 2007, an outbreak of Zika virus occurred on the island of Yap, Federated States of Micronesia in the South Pacific region. The virus was imported from Southeast Asia, and it was the first time Zika virus was found to spread outside the Asian and African continent. In 2013, a Zika virus outbreak occurred in French Po...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2561/113C12Q2521/507C12Q2563/107
Inventor 杨鹏飞燕清丽李兵兵邢亚东刘纯成李双姝胡锦流姚海波何南江
Owner 淮安市疾病预防控制中心
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