RT-PCR detection method, primer and probe as well as kit of Zika virus, dengue virus and chikungunya virus

A technology of dengue virus and chikungunya fever, which is applied in the field of molecular biology detection of insect-borne infectious diseases, can solve the problems of detection products that have not been reported, and achieve the goals of shortening detection time, improving detection efficiency and saving costs Effect

Inactive Publication Date: 2017-05-31
深圳澳东检验检测科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, single nucleic acid detection kits related to Zika virus, dengue virus, and chikungunya virus have been developed on the ma...

Method used

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  • RT-PCR detection method, primer and probe as well as kit of Zika virus, dengue virus and chikungunya virus
  • RT-PCR detection method, primer and probe as well as kit of Zika virus, dengue virus and chikungunya virus
  • RT-PCR detection method, primer and probe as well as kit of Zika virus, dengue virus and chikungunya virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Primer and probe design

[0060] The design of primers and probes mainly refers to the gene sequences of all Zika virus, dengue virus and chikungunya virus in the GenBank database, using the BLAST tool on the NCBI website, DNAStar, ABI’s PrimerExpress and other software and self-designed analysis The software analyzes the conserved sequences of Zika virus, dengue virus and chikungunya virus, and designs specific primers and probes based on the obtained conserved sequences, analyzes the interaction between primers, optimizes and screens primer-probe combinations, Minimize the cross-reaction between flaviviruses to ensure the high specificity, high sensitivity and high genotype coverage of the reagent. The resulting primers and probes are shown in Table 1:

[0061] surface Zika virus, dengue virus and chikungunya virus and internal standard detection primer probe sequences

[0062]

[0063] The 5' end of SEQ ID No.3 is marked with a fluorescent reporter group, and...

Embodiment 2

[0065] Detection of Zika virus, dengue virus, chikungunya virus triple real-time fluorescent RT-PCR in clinical samples

[0066] 1. Sample processing: Take a certain amount of serum sample and add 3 times the volume of methanol, mix well and let it stand for five minutes, centrifuge at 3000r / min for 15min, and select the supernatant for nucleic acid extraction of viral RNA. Viral RNA was extracted using the ReansyMini Kit from QIAGEN, Germany, and extracted according to the kit instructions to obtain viral RNA. 10-fold serial dilutions were performed with LB buffer and labeled.

[0067] 2. Reagent preparation:

[0068]

[0069] RT-PCR reaction solution consists of: RT-PCR buffer, magnesium chloride, deoxyribonucleotide triphosphate mixture and PCR enhancer; PCR buffer includes Tris-HCl (pH8.0), ammonium sulfate, potassium chloride and Triton X-100; the PCR enhancer is selected from one or more of tetramethylammonium chloride, carnitine, trehalose and non-ionic detergent N...

Embodiment 3

[0089] Comparison of Analytical Sensitivity of Zika Virus, Dengue Virus, Chikungunya Virus Triple Real-Time Fluorescent RT-PCR and Single-plex Conventional Reagents

[0090] The standard strain nucleic acid sample of Zika virus is diluted to the nucleic acid concentration of 0.001 LD50; Carry out Zika virus, dengue fever virus, chikungunya fever virus triple real-time fluorescent RT-PCR and single multiplex according to the method and steps in Example 2 For conventional reagent amplification detection comparison, each concentration was repeated 50 times. The results are shown in Table 3 and figure 1 shown.

[0091] Table 3 Analysis sensitivity results

[0092] Detection method Total number of samples (sets) Positive samples (parts) The detection rate(%) Triple Real-Time Fluorescent RT-PCR Kit 50 48 96 Conventional Singleplex RT-PCR Kit 50 44 88

[0093] from Table 3 and figure 1 From the results shown, it can be seen that the analytical ...

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Abstract

The invention relates to the technical field of molecular biological detection of an insect-borne infectious disease, and discloses a triple real-time fluorescence RT-PCR detection method, primer and probe as well as kit of Zika virus, dengue virus and chikungunya virus. The primer comprises the sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.7 and SEQ ID No.8; the probe comprises the sequences of SEQ ID No.3, SEQ ID No.6, and SEQ ID No.9; the kit comprises a primer probe mixed solution, an RT-PCR reaction solution, an enzyme mixed solution, and a positive standard substance. The Zika virus, the dengue virus and the chikungunya virus can be fast detected in the reaction solution of a same tube by use of a triple real-time fluorescence RT-PCR amplification technology by optimizing the reaction solution formula and the primer probe sequences, the single detection is unnecessary; compared with the traditional PCR detection method, the operation step is reduced, whether the reaction solution contains the three virus can be fast and accurately detected, the detection time is shortened, the detection efficiency is improved, and the cost is saved.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection of insect-borne infectious diseases, in particular to a triple real-time fluorescent RT-PCR detection method, primers, probes and kits for Zika virus, dengue virus and chikungunya virus. Background technique [0002] Zika virus disease, also known as Zika fever (Zika fever), is a viral disease caused by Zika virus (Zika Virus, ZIKV) and transmitted by mosquitoes. Symptoms of Zika virus disease: mild fever, rash, conjunctivitis, muscle and joint pain, malaise, and headache, etc. These symptoms usually last for 2 to 7 days. Studies have shown that Zika virus disease may cause neurological and autoimmune complications, such as Guillain-Barr syndrome (Guillian-Barr syndrome), and Zika virus infection in pregnant women may cause microcephaly in babies. Since May 2015, after the first confirmed case of Zika was found in Brazil, more than 1.5 million people in more than 40 countries ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2521/107C12Q2537/143C12Q2563/107Y02A50/30
Inventor 翟建新吴德曾华书张利吴晓卫钟文杨潘艳萍
Owner 深圳澳东检验检测科技有限公司
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