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Nucleic acid fluorescence PCR detection kit for universal enterovirus, coxsackievirus A16 and enterovirus 71

A detection kit and coxsackie virus technology, which is applied in the determination/inspection of microorganisms, microorganisms, biochemical equipment and methods, etc., to achieve good adsorption effect, improve detection sensitivity, and high amplification efficiency

Inactive Publication Date: 2016-03-23
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the defects of the existing enterovirus universal type (EV), coxsackievirus A16 type (CA16), enterovirus type 71 (EV71) fluorescence quantitative PCR detection kit, and provide a kind of RNA extraction The EV / CA16 / EV71 nucleic acid fluorescent PCR detection kit with high rate, high detection sensitivity, wide detection range and accuracy can quickly and accurately detect enterovirus RNA in unknown samples such as throat swabs, and can distinguish CA16, EV71 genotype, test results can be used for auxiliary diagnosis of EV / CA16 / EV71 infection

Method used

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  • Nucleic acid fluorescence PCR detection kit for universal enterovirus, coxsackievirus A16 and enterovirus 71
  • Nucleic acid fluorescence PCR detection kit for universal enterovirus, coxsackievirus A16 and enterovirus 71
  • Nucleic acid fluorescence PCR detection kit for universal enterovirus, coxsackievirus A16 and enterovirus 71

Examples

Experimental program
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Effect test

Embodiment 1

[0064] This embodiment provides a kind of enterovirus universal type EV / Coxsackie virus A16 type / enterovirus EV71 type nucleic acid fluorescent PCR detection kit, including the following components: RNA extraction solution, RNA eluent, internal standard, PCR reaction solution, EV / CA16 / EV71 enzyme mixture, EV / CA16 / EV71 positive control substance, EV / CA16 / EV71 negative control substance, upstream and downstream primers EV-F, CA16-F for target polynucleotide amplification , EV71-F and EV-R, CA16-R, EV71-R, probes EV-P, CA16-P, EV71-P for target polynucleotide detection, upstream and downstream primers IC-F for internal standard detection With IC-R, the probe IC-P is used to detect the internal standard.

[0065] 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 sequence of 90 base pairs is as follows Show:

[0066] 5'-CAGCTTGTTGTAACAAAG...

Embodiment 2

[0109] This embodiment provides a kind of enterovirus universal type EV / Coxsackie virus CA16 type / enterovirus EV71 type nucleic acid fluorescent PCR detection kit, the composition of the kit is the same as that of Example 1, and the detection of pharyngeal swab samples EV-RNA, CA16-RNA and EV71-RNA, the detection method and steps are the same as in Example 1.

[0110] Among them, the concentration gradients of EV, CA16, and EV71 are all 4.0E+08copies / ml~4.0E+02copies / ml, and the results of EV gradient sample detection (EV channel) are as follows: image 3 As shown, the CA16 gradient sample detection (CA16 channel) results are as follows Figure 4 As shown, the EV71 gradient sample detection (EV71 channel) results are as follows Figure 5 shown. From the analysis in the figure, it can be seen 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 EV, CA16 and EV71 sampl...

Embodiment 3

[0112] This example provides the detection limit of EV-RNA, CA16-RNA and EV71-RNA in a sample of enterovirus universal EV / Coxsackie virus A16 / enterovirus 71 nucleic acid fluorescent PCR detection kit. 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.

[0113]Among them, the results of the EV reference product amplification curve (EV channel) are as follows: Figure 6 As shown, the CA16 reference product amplification curve (CA16 channel) results are as follows Figure 7 As shown, the EV71 reference product amplification curve (EV71 channel) results are as follows Figure 8 As shown, the concentrations of EV reference substance, CA16 reference substance and EV71 reference substance are all 400copies / ml. It can be seen from the analysis in the figure that the detection sensitivity of the detection kit for detecting EV, CA16 and EV71 is positive for 20 times, indicating that the detection sensitivity of the ...

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Abstract

The embodiment of the invention discloses a nucleic acid fluorescence PCR detection kit for a universal enterovirus, a coxsackievirus A16 and an enterovirus 71. The nucleic acid fluorescence PCR detection kit comprises an RNA extracting solution, an RNA eluant, internal standards, a PCR reaction solution, an EV / CA16 / EV71 enzyme mixed solution, EV / CA16 / EV71 positive reference substances and EV / CA16 / EV71 negative reference substances. According to the kit, the universal enterovirus, the coxsackievirus A16 and the enterovirus 71 can be simultaneously detected in a same sample, but other pathogen RNA cannot be detected, the detection sensitivity can reach 400 copie / ml, the detection range is 4.0 E+02-4.0E+08 copies / ml, and a reliable experimental evidence is supplied to early diagnosis on infection of the universal enterovirus, the coxsackievirus A16 and the enterovirus 71.

Description

technical field [0001] The invention relates to nucleic acid fluorescent PCR detection kits, in particular to nucleic acid fluorescent PCR detection kits for common enterovirus, coxsackievirus A16 and enterovirus 71, belonging 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 o...

Claims

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Application Information

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