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Hand-foot-mouth disease virus EV71 nucleic acid detection kit

A detection kit and hand-foot-mouth technology, applied in the field of hand-foot-mouth virus EV71 nucleic acid detection kit, can solve the problem of low sensitivity

Inactive Publication Date: 2012-09-19
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no amplification process in this signal amplification process, so there will be no contamination of amplification products, and the specificity is high, but its sensitivity is low, and the detection limit is only 1012 copies / ul

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] The design of embodiment 1 hand-foot-mouth virus EV71 nucleic acid detection kit

[0015] A hand-foot-mouth virus EV71 nucleic acid detection kit is designed by adopting the following experiments:

[0016] (1) RNA extraction: get influenza A virus HINI nutrient solution (negative control) 1ml, breast cancer cell MCF-7 nutrient solution (negative control) 1ml, hand, foot and mouth disease virus EV71 nutrient solution (positive control) 1ml, DEPC water (blank) Control) 1ml; RNA was extracted using TAKARA virus nucleic acid extraction special kit, and the extraction method was carried out according to the instructions. The concentration of the extracted nucleic acid samples is measured with an ultraviolet spectrophotometer, and the number of copies per microliter is obtained after conversion.

[0017] (2) Primer design: Download the nucleic acid sequence from Genebank, select the 5′ non-coding region of enterovirus EV71, and design and synthesize NASBA primers. The Tm val...

Embodiment 2

[0024] The composition of embodiment 2 hand-foot-mouth virus EV71 nucleic acid detection kits

[0025] A hand, foot and mouth virus EV71 nucleic acid detection kit includes: N.BstNBI enzyme 500U, T7 RNA polymerase 3200U, AMV reverse transcriptase 400U, RNase H 80U, dNTPs 1mol, NTP 1mol, ITP 0.5mol, RNasin 600U, AMV reverse transcriptase Transcriptase Buffer 500μL, T7 RNA polymerase Buffer 500μL, RNase H Buffer 500μL, DMSO 500μL, sorbitol 1mol, NEB buffer 3500μL, P1 and P2 primers 0.4mmol each, RIDA probe 5000pmol, DEPC water 1ml, influenza A virus HINI 1 mg of RNA, 1 mg of total RNA of breast cancer cell MCF-7, and 1 mg of EV71 RNA; the 5' end of the RIDA probe is labeled with a fluorescent group of FAM, and the 3' end of which is labeled with a fluorescent quencher group of TAMARA.

Embodiment 3

[0026] The detection effect of embodiment 3 hand-foot-mouth virus EV71 nucleic acid detection kits

[0027] Get 8 routine hand, foot and mouth disease patient serum samples provided by Hebei Provincial Center for Disease Control and Prevention and 18 routine healthy volunteer serum samples according to the detection method of embodiment 1 step (3) and the judging method of step (4), the sample is detected, Under the condition that the negative and blank control test tubes were light red, the positive control test tubes and 8 cases of hand, foot and mouth disease patient sample test tubes all emitted green fluorescence under ultraviolet light, and the serum sample test tubes of 18 healthy volunteers all emitted green fluorescence under ultraviolet light. It is light red. This experiment proves that the detection accuracy of the kit can reach 100%.

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Abstract

The invention discloses a hand-foot-mouth disease virus EV71 nucleic acid detection kit, and belongs to the technical field of virus detection. The kit consists of N.BstNBI enzyme, T7 RNA (Ribonucleic Acid) polymerase, AMV (Avian Myeloblastosis Virus) reverse transcriptase, RNase H, NASBA (Nucleic Acid Sequence Based Amplification) reaction solution, a NASBA reaction primer, an RIDA (Radioisotope Dilution Assay) probe, DEPC (Diethypyrocarbonate) water, influenza virus a HINI RNA, breast cancer cell MCF-7 total RNA and EV71 RNA. According to the kit, a hand-foot-mouth disease virus EV71 nucleic acid sequence is detected by combining NASBA and RIDA technologies; and the kit has the advantages of sensitivity, specificity, easiness, convenience, high speed, high efficiency, no dependency on special instruments and the like.

Description

technical field [0001] The invention belongs to the technical field of virus detection, in particular to a nucleic acid detection kit for hand, foot and mouth virus EV71. Background technique [0002] Hand, foot and mouth disease is an infectious disease caused by enterovirus. It mostly occurs in children under 5 years old. It can cause herpes on the hands, feet, mouth and other parts. A small number of children can cause myocarditis, pulmonary edema, aseptic meningoencephalitis, etc. complication. In some severely ill children, if the disease develops rapidly, it will lead to death. There are more than 20 kinds of enteroviruses that cause hand, foot and mouth disease. Coxsackie virus types 16, 4, 5, 9, and 10 in group A, types 2 and 5 in group B, and enterovirus type 71 are all hand, foot and mouth disease. Among the more common pathogens, Coxsackievirus A16 (Cox A16) and Enterovirus 71 (EV 71) are the most common. [0003] There are three specific diagnostic methods for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 徐东刚洪明王圆圆邢微微
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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