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Double fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting dengue virus and zika virus

A dual-fluorescence, Zika virus-based technology for cost-saving applications in microbial-based methods, microbial assays/tests, resistance to vector-borne diseases, etc.

Active Publication Date: 2017-09-12
HUZHOU NO 1 PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently, there are no safe and effective vaccines approved for use

Method used

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  • Double fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting dengue virus and zika virus
  • Double fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting dengue virus and zika virus
  • Double fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting dengue virus and zika virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A dual real-time fluorescent quantitative RT-PCR detection kit for detecting DENV and ZIKV, comprising: quantitative RT-PCR reaction solution, enzyme mixture, primer-probe mixture, standard (DENV, ZIKV), strong positive control ( DENV, ZIKV), weak positive control (DENV, ZIKV), negative control. The box body of the kit is provided with container holes corresponding to each component of the kit. The box body is provided with container holes, which are respectively used to place the quantitative RT-PCR reaction solution tube (label 1), the enzyme mixed solution tube (label 2), the primer-probe mixed solution tube (label 3), and the negative control tube (label 4). ), standard quality control (including DENV, ZIKV standard substance, a total of 2 tubes, label 5), strong positive control quality control (including DENV, ZIKV positive control substance, a total of 2 tubes, label 6), strong positive control quality control (including DENV, ZIKV positive control substances, a...

Embodiment 2

[0055] 1 Materials and methods

[0056] 1.1 Clinical specimens and viral nucleic acids:

[0057] The positive clinical samples of DENV and ZIKV were provided by Zhejiang Huzhou Center for Disease Control and Prevention and Zhejiang Provincial Center for Disease Control and Prevention. Plasma, serum and / or urine samples from suspected patients in a hospital, and the samples are collected and shipped to the laboratory. In addition, the positive nucleic acids of other viruses related to fever and rash, such as measles virus, rubella virus, Dow virus 71, Coxsackie virus A16 and Coxsackie virus A6, as well as influenza A and B viruses, were obtained from Provided by the Center for Disease Control and Prevention of Huzhou City, Zhejiang Province and the First People's Hospital of Huzhou City, Zhejiang Province.

[0058] 1.2 Primers and probes

[0059] Multiple gene sequences covering DENV subtypes and ZIKV genotypes at home and abroad were downloaded from the NCBI gene bank. Usi...

Embodiment 3

[0086] Detection of clinical samples using this kit: positive clinical samples of DENV (4 copies) and ZIKV (2 copies) were provided by the Huzhou Center for Disease Control and Prevention of Zhejiang Province and the Zhejiang Provincial Center for Disease Control and Prevention, and the suspected samples were from Huzhou, Zhejiang Province A total of 325 plasma, serum and / or urine samples were collected from the First People's Hospital of the city, Huzhou Central Hospital of Zhejiang Province, and several other hospitals in Zhejiang Province. The collected samples were verified by double real-time fluorescent quantitative RT-PCR in this method, and the detection results were as follows: 4 samples were positive for DENV, with a positive rate of 1.2%; 2 samples were positive for ZIKV, with a positive rate of 0.6%. The positive test results were 100% consistent with the reported results.

[0087] Huzhou First People's Hospital

[0088] A Dual Fluorescent Quantitative RT-PCR Ki...

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Abstract

The invention provides a double fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting dengue virus and zika virus. The kit comprises a quantitative RT-PCR reaction liquid, an enzyme mixed liquid, a primer probe mixed liquid, standard products (DENV and ZIKV), strong-positive reference products (DENV and ZIKV), weak-positive reference products (DENV and ZIKV), and negative reference products. The kit has the advantages that primer probes with high specificity for DENV and ZIKV are designed according to high conservancy of DENV and ZIKV and the polyprotein protein area with smaller difference between types; the dengue virus and the zika virus can be simultaneously detected through one-time reaction in a single tube by a single tube one-step type double real-time fluorescent quantitative RT-PCR; the specificity and sensitivity are high, and the kit can be used for the laboratory emergency diagnosis of epidemic outbreak caused by dengue virus and zika virus, quick screening, clinical diagnosis, and study of epidemiology of fever eruption virus.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a fluorescent quantitative RT-PCR detection kit, in particular to a one-step dual real-time fluorescent quantitative RT-PCR for detecting dengue virus and Zika virus in human or mosquito specimens in the same reaction tube The nucleic acid detection method can be applied to laboratory emergency diagnosis, rapid screening, clinical diagnosis and epidemiological research of dengue virus and Zika virus outbreaks caused by dengue virus and Zika virus. Background technique [0002] Both dengue virus (DENV) and Zika virus (Zika virus, ZIKV) belong to the family Flaviviridae, the genus Flavivirus, and their genomes are single-stranded positive-sense RNA. DENV is divided into four serotypes according to their antigenicity: DENV-1, DENV-2, DENV-3, and DENV-4. Different strains of the same type may have antigenic differences, and there are certain differences in antigenicity between each...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2545/113C12Q2561/101Y02A50/30
Inventor 王翔温晓红崔大伟郭慧慧
Owner HUZHOU NO 1 PEOPLES HOSPITAL
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