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Reagent kit for quickly detecting Zika viruses

A Zika virus and kit technology, applied in the field of molecular biology, can solve the problems of low sensitivity and specificity, difficult to popularize and apply, etc., and achieve the effect of reducing the difficulty of experimental operation and low cost

Active Publication Date: 2019-12-31
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Colloidal gold immunochromatography is often used in the on-site rapid detection of Zika virus. The sensitivity and specificity of this method are low, and it is difficult to popularize and apply in practical work.

Method used

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  • Reagent kit for quickly detecting Zika viruses
  • Reagent kit for quickly detecting Zika viruses
  • Reagent kit for quickly detecting Zika viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] In this embodiment, Chelex-100, betaine, Sigma company; Manganese chloride, magnesium sulfate, potassium chloride, sodium hydroxide, EDTA, ammonium sulfate, Sinopharm Chemical Reagent Co., Ltd.; Tris-HCl, Shanghai Shengke Biotechnology Co., Ltd.; TritonX-100, Beijing Milebo Medical Technology Co., Ltd.; dNTP, Pharmacia Corporation.

[0063] According to the NS1 gene published by GeneBank No.: KU321639.1, a specific primer pair for rapid detection of Zika virus was designed. The nucleotide sequence of the primer pair is as follows:

[0064] FP: 5'-GCCCATGTCCTGTATGGGTGGGGCGCATTGAGAGAATCCG-3'; SEQ ID NO.1;

[0065] BP: 5'-GCCCATGTCCTGTATGGGTTCCACGTCCCAAGGCTTTGAC-3'; ID NO.2;

[0066] Ft: 5'-CTCGTGAACGGGGTTGTTAGA-3'; SEQ ID NO.3;

[0067] Bt: 5'-CAAGAAACCATGTTTCTGCATGTTC-3'; SEQ ID NO.4.

[0068] Dilute the Zika virus solution by 10 times in a centrifuge tube to obtain a titer of 10 -2 TCID 500 / ml, 10 -1 TCID 500 / ml, 10 0 TCID 500 / ml, 10 1 TCID 500 / ml, 10 2 ...

Embodiment 2

[0083] Validation of Specificity of Zika Virus Rapid Screening

[0084]In this embodiment, the kit and detection method in Example 1 are used to detect dengue virus type Ⅰ, dengue virus type Ⅱ, dengue virus type Ⅲ, dengue virus type Ⅳ, yellow fever virus, and Japanese encephalitis virus respectively. , Chikungunya virus, SFTS virus, Hantaan virus, measles virus, rubella virus, parainfluenza virus, West Nile virus, Echo virus, yellow fever virus vaccine strain, norovirus and parvovirus (kept in our laboratory ) for detection. Among them, 200 μL of each virus solution was taken, and 2 mL of Chelex lysate was added, placed in a nucleic acid amplification instrument at 95°C and heated for 10 minutes. The detection method is carried out, and the detection results are shown in Table 2. From Table 2, it can be seen that the kit of this embodiment has better specificity, and the detection time is short, convenient and quick.

[0085] Table 2 The specificity verification results of Z...

Embodiment 3

[0088] Mock sample detection: Zika virus RNA and a plasmid cloned with the Zika virus NS5 protein gene are mixed into blood, feces, sputum and other samples as mock samples for detection. Using turbidity method and chromogenic method to detect respectively, the detection results are as follows: Figure 4-7 .

[0089] The results show that Zika virus-positive samples such as simulated blood, feces, and sputum can all be detected, indicating that the kit has good applicability and the detection is not interfered by other components in environmental samples.

[0090] The present invention provides a kit for rapid detection of Zika virus. The sample can be subjected to nucleic acid amplification detection after simple on-site processing. The result is intuitive and can be interpreted by naked eyes, reducing the traditional virus PCR detection from 4 hours to less than 1 hour. , which not only reduces the difficulty of experimental operation, but also requires professional quality...

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Abstract

The invention discloses a reagent kit for quickly detecting Zika viruses. The reagent kit comprises reaction fluid of which the pH is 8.8, Bst DNA polymerase, AMV reverse transcriptase, FP and BP primers, Ft and Bt primers, an indicator and a sealant. The reagent kit provided by the invention adopts a unique buffer system, and has the advantage of being high in amplifying compatibility; high-efficient sensitive specific amplifying can be performed without thoroughly removing impurities including proteins and the like; the operation is easy: a reaction can be completed only through placing a reaction system into a 64-67 DEG C thermostatic water-bath pot; the result determination is simple and convenient: the color development result can be observed by naked eyes, and the result can also bejudged through a fluorescence amplifying detection instrument or through a turbidity instrument; and the reagent kit is not only suitable for in-situ monitoring, suitable for detection requirements ofin-site quarantine checking under the background of large people stream and quick clearance, and is also suitable for quick detection in a laboratory.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for rapid detection of Zika virus. Background technique [0002] Zika virus disease is a self-limiting acute disease caused by Zika virus (ZIKV) and transmitted by mosquitoes. Zika virus is mainly transmitted through the bite of the Aedes aegypti mosquito. The clinical features are mainly fever, rash, conjunctivitis or joint pain, and rarely cause death. The World Health Organization believes that neonatal microcephaly and Guillain-Barré syndrome may be related to Zika virus infection. [0003] Zika virus disease is mainly prevalent in tropical and subtropical regions of the world. In 1952, the virus was isolated from humans in Uganda and Tanzania. Since then, sporadic cases have been reported in many countries. In 2007, the Zika virus outbreak first occurred on Yap Island in Micronesia, a country in the Western Pacific. As of November 2016, evidence of Zika virus tran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2563/107C12Q2561/113Y02A50/30
Inventor 王静杨宇刘威林楠张乔张晓龙施琦孙筱霞
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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