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
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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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