Nested RT-PCR method, primers and reagent kit for detecting Zika virus

A RT-PCR, Zika virus technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve high sensitivity, stable results, and easy operation

Inactive Publication Date: 2016-08-17
THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no nested RT-PCR assay for Zika virus

Method used

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  • Nested RT-PCR method, primers and reagent kit for detecting Zika virus
  • Nested RT-PCR method, primers and reagent kit for detecting Zika virus
  • Nested RT-PCR method, primers and reagent kit for detecting Zika virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] This embodiment provides two sets of primer pairs for detecting Zika virus and its application.

[0056] Design of two sets of primer pairs: use the existing 71 ZIKV genome sequences in GenBank for multiple sequence alignment, and select the conserved region (536bp, SEQ.ID.No.5) of the ZIKV envelope protein (envelope protein) as the target Two sets of primer pairs were designed for the sequence.

[0057] The specific sequences of the two sets of primer pairs are shown in Table 1:

[0058] Table 1

[0059]

[0060] In Table 1, ZIKV / 1st / F represents the upstream primer of the first round of nested RT-PCR, ZIKV / 1st / R represents the downstream primer of the first round of nested RT-PCR, and ZIKV / 2nd / F represents the nested The upstream primer for the second round of RT-PCR, ZIKV / 2nd / R represents the downstream primer for the second round of nested RT-PCR. M in the primer sequence represents A or C, R represents A or G, Y represents C or T, and S represents C or G.

...

Embodiment 2

[0066] This embodiment provides a kit and application thereof, the kit comprising:

[0067] (1) two groups of primer pairs of embodiment 1;

[0068] (2) PCR amplification reagents.

[0069] The PCR amplification reagents include 5×PCR Buffer and Ex Taq HS DNA polymerase included in the TaKaRa RNA PCRKit Ver.3.0 kit with the article number RR019A from TaKaRa Company.

[0070] Experiments have proved that the combined use of the above-mentioned PCR amplification reagents and two sets of primer pairs has a more superior effect, which is specifically manifested in the characteristics of good repeatability, strong specificity, and high sensitivity.

[0071] The test kit can further include:

[0072] (3) positive control and negative control;

[0073] (4) RNA extraction reagents;

[0074] (5) Reverse transcription reagents.

[0075] The positive control is a plasmid inserted with artificially synthesized ZIKV E protein gene cDNA (synthesized by Shanghai Sangon Bioengineering Co...

Embodiment 3

[0079] This embodiment provides a method for detecting Zika virus, such as detecting the presence of Zika virus in a biological sample. The method uses two sets of primer pairs in Example 1 and the kit in Example 2.

[0080] The above-mentioned biological sample is blood, urine, cells or tissues containing DNA and / or RNA, or is a plasmid, or is DNA, cDNA, mRNA or cDNA fragments, or is a reagent containing DNA, cDNA, mRNA or cDNA fragments.

[0081] When the biological sample is DNA, cDNA or cDNA fragments, or a reagent containing DNA, cDNA or cDNA fragments, or a plasmid, it is directly used as a template for nested RT-PCR reaction with the two sets of primer pairs.

[0082] When the biological sample is blood, cells or tissues containing DNA and / or RNA, or mRNA, or a reagent containing mRNA, it is converted into DNA, cDNA or cDNA fragments and then nested with the two sets of primer pairs RT-PCR reaction.

[0083] The method for detecting Zika virus comprises the following s...

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Abstract

The invention provides two primer pairs. One primer pair includes nucleotide sequences shown as SEQ ID NO:1 and SEQ ID NO:2, and the other primer pair includes nucleotide sequences shown as SEQ ID NO:3 and SEQ ID NO:4. The invention further provides application of the two primer pairs in preparing products for detecting the Zika virus. The invention further provides a reagent kit comprising the two primer pairs and application. As proved by experiments, the two primer pairs are good in specificity and repeatability and high in sensitivity, and a Zika virus detecting method established based on the two primer pairs can be used for sensitively, accurately, stably and rapidly detecting whether the Zika virus exists or not and has great significance on clinical rapid diagnosis and public prevention and control over the Zika virus in China.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a nested RT-PCR method, primers and kit for detecting Zika virus. Background technique [0002] Zika virus (Zika virus, ZIKV) was first discovered in Uganda from rhesus monkeys in 1947, and the virus was isolated from humans in Uganda and Tanzania in 1952. ZIKV is transmitted to humans through the bite of infected mosquitoes of the genus Aedes, mainly involving Aedes aegypti in tropical regions. It is the same mosquito that transmits dengue, chikungunya and yellow fever. However, there have been two cases of sexual transmission of Zika virus and the presence of Zika virus in semen in another case. [0003] In recent years, the number of ZIKV infection cases and outbreak countries and regions has increased significantly. In 2007, Zika virus outbreaks occurred in the Pacific island country of Micronesia, Yap Island for the first time. In 2013 and 2015, they occurred in French ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6848C12Q2549/119C12Q2531/113
Inventor 高福刘映霞王强毕玉海杨扬郑海霞
Owner THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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