Nucleic acid fluorescent PCR detection kit for coxsacki evirus A16 and human enter ovirus 71

A technology for coxsackie virus and detection kit, which is applied in the direction of determination/inspection of microorganisms, microorganisms, biochemical equipment and methods, etc., to achieve the effects of easy purification, high purity and yield, and improved detection sensitivity

Inactive Publication Date: 2016-03-02
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the defects of existing Coxsackievirus A16 type (CA16) and enterovirus 71 type (EV71) fluorescent quantitative PCR detection kits, and provide a kind of RNA extraction yield high, detection sensitivity high, detection range Wide and accurate CA16 / EV71 nucleic acid fluorescent PCR detection kit, which can quickly and accurately detect CA16\EV71-RNA in unknown samples such as throat swabs in one tube of reaction solution, and can distinguish CA16 and EV71 genotypes, The test results can be used for auxiliary diagnosis of CA16 / EV71 infection

Method used

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  • Nucleic acid fluorescent PCR detection kit for coxsacki evirus A16 and human enter ovirus 71
  • Nucleic acid fluorescent PCR detection kit for coxsacki evirus A16 and human enter ovirus 71
  • Nucleic acid fluorescent PCR detection kit for coxsacki evirus A16 and human enter ovirus 71

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The coxsackievirus type A16 and enterovirus type 71 nucleic acid fluorescent PCR detection kits provided in this example include the following components: RNA extraction solution, RNA eluent, internal standard, PCR reaction solution, CA16 / EV71 enzyme Mixed solution, CA16 / EV71 positive control substance, CA16\EV71 negative control substance, upstream and downstream primers CA16-F, EV71-F and CA16-R, EV71-R for target polynucleotide amplification Probes CA16-P and EV71-P for acid detection, upstream and downstream primers IC-F and IC-R for detecting internal standards, and probe IC-P for detecting internal standards.

[0058] The internal standard is a recombinant of a 90 base pair artificially synthesized DNA sequence inserted into the pUC18T vector, and its concentration is 1.00E+03copies / ml~1.00E+06copies / ml; the 90 base pair sequence for:

[0059] 5'-CAGCTTGTTGTAACAAAGCATCCGCTCCCCCATTCATGTTGCTGGGTACAGACAGTTACCTTCCACTAGGCAAATCTCAACAGGATCAG-3'.

[0060] The RNA extrac...

Embodiment 2

[0103] This embodiment provides a Coxsackievirus A16 / Enterovirus 71 nucleic acid fluorescent PCR detection kit, the composition of which is the same as in Example 1, and is used to detect CA16-RNA and EV71-RNA in throat swab samples , detection method and step are identical with embodiment 1.

[0104] Among them, the concentration gradient of CA16 and EV71 is 4.0E+08copies / ml~4.0E+02copies / ml, and the results of CA16 gradient sample detection (CA16 channel) are as follows image 3 As shown, the EV71 gradient sample detection (EV71 channel) results are as follows Figure 4 shown. It can be seen from the analysis in the figure that the target channels of the CA16 and EV71 gradient samples detected by this detection kit are all positive, indicating that the detection range of the kit for CA16 and EV71 samples is 4.0E+08copies / ml~4.0E+02copies / ml.

Embodiment 3

[0106] This example provides the detection limit of CA16-RNA and EV71-RNA in the sample of a Coxsackievirus A16 / Enterovirus 71 nucleic acid fluorescent PCR detection kit sample. The composition of the kit is the same as in Example 1, and the detection method and steps are the same as in Example 1.

[0107] Among them, the CA16 reference product amplification curve (CA16 channel) results are as follows Figure 5 As shown, the EV71 reference product amplification curve (EV71 channel) results are as follows Image 6 As shown, the concentration of CA16 reference substance and EV71 reference substance is 400copies / ml. From the analysis in the figure, it can be known that the detection kit detects CA16 and EV71 detection sensitivity reference products 20 times are all positive, indicating that the detection sensitivity of the kit to CA16 and EV71 is 400copies / ml.

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Abstract

The embodiment of the invention discloses a nucleic acid fluorescent PCR detection kit for coxsacki evirus A16 and human enter ovirus 71. The nucleic acid fluorescent PCR detection kit comprises the following components: RNA extract solution, RNA eluant, internal standard substance, PCR reaction liquid, CA16/EV 71 enzyme mixed liquid, CA16/EV 71 positive control substance and CA16/EV 71 negative control substance. According to the nucleic acid fluorescent PCR detection kit, two kinds of enteroviruses including the coxsacki evirus A16 and human enter ovirus 71 are simultaneously detected in one sample, other pathogen RNAs can not be detected, and RNAs are extracted by a paramagnetic particle method, so that the detection sensitivity, accuracy and stability are improved, the detection sensitivity achieves 400 copie/ml, the detection range is (4.0E+02)-(4.0E+08) copies/ml, and reliabile experimental evidences are provided for early diagnosis of the infection of the coxsacki evirus A16 and the human enter ovirus 71.

Description

technical field [0001] The invention relates to a nucleic acid fluorescent PCR detection kit, in particular to a coxsackievirus A16 / enterovirus 71 nucleic acid fluorescent PCR detection kit, which belongs to the field of in vitro diagnosis of hand, foot and mouth disease. Background technique [0002] Hand-foot-mouth disease (HFMD), also known as exanthematous vesicular stomatitis, is a common infectious disease caused by a group of enteroviruses. The main clinical manifestations are fever and rashes on the hands, feet, and mouth. An acute infectious disease with clinical features. Most patients with HFMD have a good prognosis, and generally recover within 5 to 7 days, which is a self-limiting disease. The virus can also invade the respiratory system and central nervous system, causing symptoms such as encephalitis, pulmonary edema, flaccid paralysis, and myocarditis. Individual critically ill children may die due to various reasons. The disease can occur throughout the ye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/6806C12Q1/701
Inventor 戴立忠黄河邓中平易晓明
Owner SANSURE BIOTECH INC
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