LAMP primer composition for detecting hand-foot-and-mouth disease related pathogens and related application thereof

A primer composition and hand, foot and mouth disease technology, applied in the field of infectious disease diagnosis, can solve the problems of expensive detection equipment, few detection targets, cumbersome operation, etc., and achieve high accuracy, good specificity, and improved detection speed Effect

Active Publication Date: 2017-08-18
SHANGHAI IGENETEC DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the fluorescent PCR method is mostly used, but this detection method has the disadvantages of few detection targets, low sensitivity, cumbersome operation, time-consuming detection, and expensive detection equipment.

Method used

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  • LAMP primer composition for detecting hand-foot-and-mouth disease related pathogens and related application thereof
  • LAMP primer composition for detecting hand-foot-and-mouth disease related pathogens and related application thereof
  • LAMP primer composition for detecting hand-foot-and-mouth disease related pathogens and related application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Design and preparation of the LAMP primer composition for detecting hand-foot-mouth disease-related pathogens

[0029] 1.1 Sequence acquisition:

[0030] (1) Obtaining the sequence of the enterovirus 5'UTR region: download the nucleic acid sequence of the enterovirus 5'UTR region (as shown in SEQ ID NO.26) from the GeneBank public database.

[0031] (2) Acquisition of the VP1 gene sequence: download the enterovirus EV71 type gene sequence (as shown in SEQ ID NO.27), the CA16 type gene sequence (as shown in SEQ ID NO.28), and the CA10 type gene sequence from the GeneBank public database. The gene sequence (as shown in SEQ ID NO.29) and the VP1 gene sequence of CA6 type (as shown in SEQ ID NO.30).

[0032] (3) Acquisition of the internal reference HBB gene sequence: download the HBB gene nucleic acid sequence (as shown in SEQ ID NO.25) from the GeneBank public database.

[0033] 1.2 Primer design

[0034] (1) Specific gene primers: compare the above-mentioned...

Embodiment 2

[0048] Example 2 Preparation and use of a kit for detecting enterovirus-associated pathogens

[0049] The clinical samples of enterovirus EV, enterovirus EV71, enterovirus CA16, enterovirus CA10, enterovirus CA6, influenza A virus and hepatitis B virus in the following examples are all from Fudan University Affiliated Children's Hospital.

[0050] 2.1 Preparation of kits for detection of enterovirus-associated pathogens

[0051] The kit for detecting enterovirus-associated pathogens provided by the present invention consists of the following:

[0052] 2.1.1 Isothermal amplification buffer

[0053] The solvent of the constant temperature amplification buffer is water, and the solute and concentration are as follows: 12mM dNTPs, 10×Isothermal Amplification reaction buffer, 150mM MgSO4 aqueous solution, and 150μM HNB.

[0054] 2.1.2 Constant temperature amplification enzyme solution

[0055] The solvent of the constant temperature amplification enzyme solution is water, and t...

Embodiment 3

[0095] Example 3: Detecting the Sensitivity of the Thermostatic Amplification Microfluidic Chip

[0096] Using the isothermal amplification microfluidic chip prepared in 2.1 of Example 2 to conduct a sensitivity experiment

[0097] Enterovirus universal type, enterovirus EV71 type, enterovirus CA16 type, enterovirus CA10 type, and enterovirus CA6 type nucleic acids prepared in 2.2.2 of Example 2 were diluted respectively and mixed to obtain a new mixed system. In the new mixed system, the nucleic acid concentrations of enterovirus universal type, enterovirus EV71 type, enterovirus CA16 type, enterovirus CA10 type, and enterovirus CA6 type are all 102 TU / μl, and the resulting mixed system is named For mixed nucleic acid LP.

[0098] The mixed nucleic acid LP was mixed with the constant temperature amplification system, and then injected into the constant temperature amplification microfluidic chip, and the constant temperature amplification reaction was carried out according t...

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Abstract

The invention provides a LAMP primer composition for detecting hand-foot-and-mouth disease related pathogens and related application thereof. The LAMP primer composition is high in sensitivity and accuracy. Microfluidic chip technology based on a LAMP method is applied in detection of the hand-foot-and-mouth disease related pathogens, and the advantages of high flux, quickness, sensitivity and high specificity of a microfluidic chip are utilized, so that detection speed, flux and sensitivity of the hand-foot-and-mouth disease related pathogens are increased, cost is lowered greatly, and detection time is shortened.

Description

technical field [0001] The invention relates to the technical field of diagnosis of infectious diseases, in particular to a LAMP primer composition for detecting pathogens related to hand, foot and mouth disease and related applications thereof. Background technique [0002] Hand-foot-mouth disease (HFMD) is a common infectious disease in children caused by a variety of human enteroviruses. Most patients have mild symptoms, with fever and rash or herpes on the hands, feet, mouth and other parts as the main symptoms. A small number of patients may develop aseptic meningitis, encephalitis, acute flaccid paralysis, neurogenic pulmonary edema, and myocarditis, and individual severe cases progress rapidly and may lead to death. [0003] The viruses that cause HFMD belong to the Enterovirus genus of the family Picornaviridae, including types 2, 4, 5, 7, 9, 10, and 16 of Coxsackievirus A (CVA), and types B (Coxasckievirus B , CVB) types 1, 2, 3, 4, 5, etc.; Enterovirus 71 (Human ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/159C12Q2545/101
Inventor 方雪恩李杨严俊闵建峰孔继烈
Owner SHANGHAI IGENETEC DIAGNOSTICS CO LTD
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