Fluorescent quantitative kit capable of detecting coxsackievirus type A2, A5

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, delayed treatment of patients, unclear detection of pathogens of hand, foot and mouth disease, etc. cost effect

Active Publication Date: 2014-05-14
ZHEJIANG UNIV
View PDF5 Cites 5 Cited by
  • Summary
  • 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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fluorescent quantitative kit capable of detecting coxsackievirus type A2, A5
  • Fluorescent quantitative kit capable of detecting coxsackievirus type A2, A5
  • Fluorescent quantitative kit capable of detecting coxsackievirus type A2, A5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] A dual real-time fluorescence quantitative RT-PCR detection kit for detecting Coxsackievirus A2 and A5 types, comprising: quantitative RT-PCR reaction solution, enzyme mixture, primer-probe mixture, standard products (CVA2, CVA5), Strong positive control substances (CVA2, CVA5), weak positive control substances (CVA2, CVA5), negative control substances. The quantitative RT-PCR reaction solution contains PCR reaction buffer (containing magnesium chloride and deoxyribonucleotide triphosphate mixture, etc.), the enzyme mixture contains heat-resistant Taq DNA polymerase, RNase inhibitor and MMLV reverse transcriptase, primers The probe mixture contains two sets of CVA2 and CVA5 primers and corresponding CVA2 and CVA5 two different fluorescently labeled probes. The negative control is high temperature and high pressure sterilized DEPC (diethyl pyrocarbonate) treated water, and the positive control is CVA2, Positive plasmid samples for CVA5. The strong positive control plasm...

Embodiment 2

[0054] 1 Materials and methods

[0055] 1.1 Clinical specimens and viral nucleic acids:

[0056] The clinical samples of Coxsackievirus A2 and A5 came from the stool samples of confirmed and suspected patients with HFMD 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 A5, Coxsackievirus A10, 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 A2 and A5 types at home and abroad were downloaded from the NCBI gene bank. Homology comparison was carried out by using DNAman softw...

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 collected clinical samples were mainly from the First Affiliated Hospital of Zhejiang University School of Medicine and several other hospitals in Zhejiang Province between May 2013 and July 2013. A total of 367 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: 23 samples were positive for Coxsackievirus A2, with a positive rate of 6.3%; 11 samples were positive for Coxsackievirus A5, positive The rate is 3.0%. The positive test results are in full agreement with their reported results.

[0089] Zhejiang University

[0090] A Fluorescent Quantitative Kit for Detecting Coxsackieviruses A2 and A5 ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a fluorescent quantitative kit capable of detecting coxsackievirus type A2, A5. The fluorescent quantitative kit comprises a quantitative RT-PCR reactant liquor, an enzyme mixture solution, a primer probe mixture solution, CVA2 and CVA5 standard substances, CVA2 and CVA5 strong positive reference substances, CVA2 and CVA5 weak positive reference substances, and CVA2 and CVA5 negative reference substances. The one-step method real-time fluorescent quantitative RT-PCR technology is adopted, and the highly specific CVA2 and CVA5 primers and fluorescence labeling probes are used, so that the presence of CVA2 and CVA5 can be simultaneously detected from dejecta samples. Compared with the single fluorescent quantitative RT-PCR method, the fluorescent quantitative kit is quicker and more convenient, and the cost is saved. The fluorescent quantitative kit can detect the CVA2 and CVA5 in real time and accurately quantify the CVA2 and CVA5, so as to provide the early diagnosis for clinical, provide reference for the formulation of clinical treatment scheme. The fluorescent quantitative kit can be applied to experiment emergency diagnosis, quick screen and clinical diagnosis of outbreak caused by coxsackievirus type A2, A5, and can also be applied to epidemiologic research on hand-foot-mouth diseases.

Description

Technical field [0001] The present invention is the biotechnology field, involving fluorescent quantitative RT-PCR detection kits, which involves a one-step method of dual practical time fluorescence quantitative RT-PCR detecting patients in patients in patients in the same reaction tube.The detection method can be applied to the study of the laboratory emergency diagnosis, rapid screening, clinical diagnosis, and epidemiological research on the laboratory of the Kocci virus A2 and A5. Background technique [0002] Hand, Foot and Mouth Disease (HFMD) is a human intestinal virus (Human Enterovirus, [0003] HEV) is common infectious diseases that are caused by rash symptoms in the hands, feet, mouth, and hips. They are mainly milder, but there are also reports of severe illnesses and death cases.Human intestinal virus is divided into 4 groups of A, B, C, and D. There are currently more than 90 genotypes. The most important pathogen is the intestinal virus type 71 (EV71) and the Co...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap