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Method for detecting coxsackie group A 16-type virus in mixed infection of hand-foot-and-mouth disease

A technology of mixed infection and hand, foot and mouth disease, applied in the biological field, can solve the problems of high cost, increased cost, long time, etc., and achieve the effect of reducing cost

Inactive Publication Date: 2017-07-21
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This requires two RT-PCRs, which not only increases the cost, but also takes a long time. For large-scale epidemic investigations, the cost is huge, so it is necessary to improve the detection method of CV-A16 in enterovirus mixed infection

Method used

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  • Method for detecting coxsackie group A 16-type virus in mixed infection of hand-foot-and-mouth disease
  • Method for detecting coxsackie group A 16-type virus in mixed infection of hand-foot-and-mouth disease
  • Method for detecting coxsackie group A 16-type virus in mixed infection of hand-foot-and-mouth disease

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Embodiment

[0020] 1. Sample processing

[0021] Take 1 gram of stool sample, add 5mL of PBS to shake and mix well, centrifuge at 3000r / min for 30min, take the supernatant, and store it at -80°C;

[0022] 2. Viral RNA extraction

[0023] The extraction of viral RNA was performed according to the instructions of the kit (Qiamp viral RNA mini Kit), as follows: Take 560 AVL and add it to a 1.5 mL centrifuge tube, then add 140 μL of supernatant, mix by inverting for 15 sec, and place at room temperature for 10 min; add 560 μL of absolute ethanol, invert Mix for 15 sec; add 630 μL of the above solution to the Qiamp adsorption column, centrifuge at 8000 rpm for 1 min, pour off the waste liquid in the collection tube; add 500 μL AW1, centrifuge at 8000 rpm for 1 min, pour off the waste liquid in the collection tube; add 500 μL AW2, and centrifuge at 14000 rpm for 3 min , pour off the waste liquid in the collection tube; put the adsorption column into an empty collection tube, centrifuge at 1400...

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Abstract

The invention discloses a method for detecting coxsackie group A 16-type virus in mixed infection of hand-foot-and-mouth disease (HFMD). The method comprises the following steps: extracting a viral RNA; performing RT-PCR amplification with universal primers EntAF and EntARo of enterovirus A group; then using primers EntAF and EntARi for semi-nested PCR amplification; designing an upstream primer CA16AFO and a downstream primer CA16ARi of specific CV-A16 according to the universal primers of the enterovirus A group and a CV-A16 sequence; using a RT-PCR product as a template for nested PCR amplification; and taking an amplification product for 1% agarose gel electrophoresis to realize identification. The method provided by the invention has advantages of simple operation, high detection speed, high accuracy, low cost and the like, and is particularly suitable for large-scale epidemic disease survey.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection method for coxsackie group A type 16 enterovirus in clinical samples of hand, foot and mouth disease. Background technique [0002] Coxsackievirus A16 (CV-A16) is one of the main pathogens causing hand-foot-mouth disease (HFMD). HFMD is a common infectious disease that seriously endangers children. Most patients are characterized by fever and rashes or herpes on the hands, feet, and mouth. Meningitis, etc., even death. The prevalence of HFMD will become one of the major public health problems. [0003] At present, there are more than 20 kinds of pathogens that can cause hand, foot and mouth disease, mainly Coxsackievirus A group 16, 4, 5, 7, 9, 10, B group 2, 5 , 13, ECHO viruses and human enterovirus 71 (enterovirus 71, EV-A71), among which EV-A71 and Coxsackievirus A16 (Coxsackievirus A16, CV-A16) are the most common . The study found that during the ep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2549/119
Inventor 马绍辉杨昭庆赵义林黄小琴孙浩刘洪波张海浩
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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