Fluorescent quantitative kit for detecting coxsackie viruses A6 and A10

A coxsackie virus, fluorescence quantitative technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of waste of diagnostic reagents, unclear detection of hand, foot and mouth disease pathogens, missed detection, etc., and achieve cost savings Effect

Active Publication Date: 2014-05-14
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the pathogen diagnosis market of hand, foot and mouth disease in my country is still mainly based on the detection of common enterovirus or enterovirus 71 and coxsackie virus A16, which leads to unclear or missed detection of the pathogen of hand, foot and mouth disease, etc. delays and waste of diagnostic reagents, etc.

Method used

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  • Fluorescent quantitative kit for detecting coxsackie viruses A6 and A10
  • Fluorescent quantitative kit for detecting coxsackie viruses A6 and A10
  • Fluorescent quantitative kit for detecting coxsackie viruses A6 and A10

Examples

Experimental program
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Effect test

Embodiment 1

[0037] A dual real-time fluorescent quantitative RT-PCR detection kit for detecting Coxsackievirus A6 and A10 types, including: quantitative RT-PCR reaction solution, enzyme mixture, primer-probe mixture, standard products (CVA6, CVA10), Strong positive control substances (CVA6, CVA10), weak positive control substances (CVA6, CVA10), negative control substances.

[0038] The quantitative RT-PCR reaction solution contains PCR reaction buffer, magnesium chloride and deoxyribonucleotide triphosphate mixture, the enzyme mixture contains heat-resistant Taq DNA polymerase, RNase inhibitor and MMLV reverse transcriptase, primer probe mixture The solution contains two groups of specific primers CVA6 and CVA10 and corresponding CVA6 and CVA10 two different fluorescently labeled specific probes. The negative control is high temperature and high pressure sterilized DEPC (diethyl pyrocarbonate) treated water. Positive control substances and CVA6, CVA10 weak positive control substances are...

Embodiment 2

[0054] 1 Materials and methods

[0055] 1.1 Clinical specimens and viral nucleic acids:

[0056] The clinical samples of Coxsackievirus A6 and A10 were obtained from the stool samples of confirmed and suspected patients with hand, foot and mouth disease in the First Affiliated Hospital of Zhejiang University School of Medicine and several other hospitals in Zhejiang Province. The samples were collected and transported to the laboratory. In addition, other enteroviruses such as Enterovirus 71, Coxsackievirus A16, Coxsackievirus A2, Coxsackievirus A5, Coxsackievirus B1, Coxsackievirus B3, Eck The positive nucleic acids of virus type 30, influenza A virus, respiratory syncytial virus, and Boca virus were provided by the State Key Laboratory of Diagnosis and Treatment of Infectious Diseases.

[0057] 1.2 Primers and probes

[0058] Multiple gene sequences covering Coxsackievirus A6 and A10 types at home and abroad were downloaded from the NCBI gene bank. Homology comparison was...

Embodiment 3

[0088] The detection of clinical samples using this kit is mainly based on the "Twelfth Five-Year Plan" major project - infectious disease pathogen detection technology platform project (2012ZX10004-210). The clinical samples collected were mainly from the First Affiliated Hospital of Zhejiang University School of Medicine and several other hospitals in Zhejiang Province between March 2013 and June 2013. A total of 419 stool samples were collected from patients with HFMD and suspected patients. The collected specimens were verified by double real-time fluorescent quantitative RT-PCR in this method, and the detection results were as follows: 171 samples of Coxsackievirus A6 were positive, with a positive rate of 40.8%; 27 samples of Coxsackievirus A10 were positive, positive rate of 6.4%. The positive test results were 100% consistent with the reported results.

[0089] Zhejiang University

[0090] A Fluorescent Quantitative Kit for Detecting Coxsackieviruses A6 and A10

...

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Abstract

The invention provides a fluorescent quantitative kit for detecting coxsackie viruses A6 and A10. The fluorescent quantitative kit is composed of a quantitative reverse transcription-polymerase chain reaction (RT-PCR) reaction liquid, enzyme mixed liquor, primer probe mixed liquor, CVA6 and CVA10 standard substances, CVA6 and CVA10 strong positive reference substances, CVA6 and CVA10 weak positive reference substances, and a negative reference substance. Existence of CVA6 and CVA10 is simultaneously detected from a stool specimen by adopting a one-step real-time fluorescent quantitative RT-PCR technology, and CVA6 and CVA10 primers and fluorescently-labeled probes with high specificity, compared with a single fluorescent quantitative PCR method, the fluorescent quantitative kit is more convenient and faster, real-time in detection, and accurate to quantitate; the cost is saved; early diagnosis is provided clinically; a reference frame is provided for formulation of a clinical treatment scheme; the fluorescent quantitative kit can be applied to laboratory emergency diagnosis, rapid screening and clinical diagnosis of epidemic outbreak caused by coxsackie viruses A6 and A10, and research of epidemiology of a hand-foot-and-mouth disease.

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 nucleic acids of Coxsackievirus A6 and A10 types in patient stool specimens in the same reaction tube The detection method can be applied to laboratory emergency diagnosis, rapid screening, clinical diagnosis and epidemiological research of hand, foot and mouth disease caused by Coxsackievirus A6 and A10 outbreaks. Background technique [0002] Hand, foot and mouth disease (HFMD) is caused by human enterovirus (human enterovirus, [0003] HEV) is a common infectious disease in children. There are currently more than 90 genotypes of human enteroviruses, which are divided into four groups: A, B, C, and D. The most important pathogens are enterovirus 71 (EV71) and Coxsackie in the HEV-A group. Virus A16 type (Coxsackie virus A16, CVA16). How...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 陈瑜李兰娟谢国良崔大伟
Owner ZHEJIANG UNIV
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