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Zika virus detection kit based on high resolution melting analysis and detection method thereof

A high-resolution melting and Zika virus technology, applied in the field of Zika virus detection kits, can solve the problem that serological analysis cannot provide specificity, and achieve the effect of reducing the risk of contamination, simple operation, and fast speed

Inactive Publication Date: 2019-09-13
山东国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serological analysis does not provide suitable specificity due to extensive antibody cross-reactivity with other flaviviruses

Method used

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  • Zika virus detection kit based on high resolution melting analysis and detection method thereof
  • Zika virus detection kit based on high resolution melting analysis and detection method thereof
  • Zika virus detection kit based on high resolution melting analysis and detection method thereof

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Embodiment 1

[0039] In this embodiment, a set of Zika virus-specific detection primers based on high-resolution melting curves is designed, and the sequence is as follows:

[0040] NS3-F: 5'-GCTATGGGATGTGCCTGCTC-3'

[0041] NS3-R: 5'-TCCCTTCACCGCTTCTCA-3'.

[0042] Utilize above-mentioned detection primer, provide a kind of Zika virus detection kit based on high-resolution melting curve, comprise following composition:

[0043] 1) Specific primers for Zika virus NS3 gene

[0044] NS3-F: 5'-GCTATGGGATGTGCCTGCTC-3'

[0045] NS3-R: 5'-TCCCTTCACCGCTTCTCA-3'.

[0046] 2) PCR mixture: FastStart Taq DNA polymerase, reaction buffer, dNTP mixture, ResoLight saturated fluorescent

[0047] light dyes;

[0048] 3) The reverse-transcribed cDNA of the Zika virus genome;

[0049] 4) ddH2O;

[0050] 5) 25 mM MgCl2.

Embodiment 2

[0052] The present embodiment provides a Zika virus detection method based on a high-resolution melting curve, which includes the following steps:

[0053] 1) Preparation of high-resolution melting curve template Zika virus genomic RNA

[0054] Zika virus genomic RNA was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 kit (TaKaRa Company), referring to the kit instructions.

[0055] 2) Reverse transcription of Zika virus genomic RNA into cDNA

[0056] Zika virus genomic RNA was reverse-transcribed into cDNA using PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa Company), referring to the kit instructions.

[0057] 3) Verify the designed specific primers by PCR

[0058] The 30 μL reaction system includes: FastStart Taq DNA polymerase 0.5 μL, Zika virus genome reverse transcription cDNA template 2 μL, dNTP mixture 2 μL, ResoLight saturated fluorescent dye 1 μL, NS3-F primer 1 μL, NS3-R primer 1 μL, reaction buffer 3 μL, 25 mM MgCl 2 3.6 μL, ddH...

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Abstract

The invention relates to the field of nucleic acid detection, and discloses a Zika virus detection kit based on high resolution melting analysis and a detection method thereof. The invention is characterized in that specific primers are designed according to Zika virus NS3 gene sequences, Zika virus nucleic acid is detected by using an optimized high resolution melting reaction system, so that theZika virus detection kit is simple in operations, fast in speed and free of the need of post-processing on PCR products and capable of detecting the difference of single base and truly realizing theclosed-tube operation.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a Zika virus detection kit and a detection method based on a high-resolution melting curve. Background technique [0002] Zika virus belongs to the Flaviviridae family and is closely related to other mosquito-borne flaviviruses such as dengue, West Nile and Japanese encephalitis viruses. Zika virus has a positive single-stranded RNA genome of approximately 11 kb in length, with a single open reading frame at the 5′ and 3′ ends flanked by two untranslated regions. A single ORF encodes a polyprotein that, upon cleavage, produces 3 structural types (capsid, membrane precursor, and envelope protein) and 7 nonstructural proteins. Zika virus strains can be divided into two distinct phylogenetic lineages (African and Asian lineages). Similar to other flaviviruses, Zika virus is primarily transmitted by Aedes mosquitoes, including the urban and suburban mosquitoes Aedes...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2527/107C12Q2563/107C12Q2521/107
Inventor 滕新栋陈晓光郑健董梅程晓兰梁洁张瑾张涛姜华刘海江朱可张娟郭宁顾长美
Owner 山东国际旅行卫生保健中心
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