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Kit for detecting coxsackie virus A16-type nucleic acid and detection method thereof

A Coxsackie virus and detection kit technology, applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve problems such as high time consumption, inability to meet detection requirements during outbreaks, and cross-contamination , to achieve the effect of easy operation

Active Publication Date: 2010-05-26
IPE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional RT-PCR technology requires two-step reaction, and the whole process takes 6-8 hours, which is time-consuming and cannot meet the detection needs during the outbreak; in addition, this technology also has the risk of cross-contamination, and the testing laboratory cannot do it after a period of time. avoid false positive results

Method used

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  • Kit for detecting coxsackie virus A16-type nucleic acid and detection method thereof
  • Kit for detecting coxsackie virus A16-type nucleic acid and detection method thereof
  • Kit for detecting coxsackie virus A16-type nucleic acid and detection method thereof

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Effect test

preparation example Construction

[0057] (2) Preparation of in vitro transcription RNA quality control products

[0058] After digesting the above-mentioned extracted plasmid with SAC I enzyme from Takara Company to make it linearized, the T7 RNA polymerase from Takara Company was used to perform in vitro transcription on the above-mentioned enzyme digestion reaction solution, and the obtained in vitro transcription reaction solution was processed with DNase I from Takara Company. Digestion was carried out to remove the plasmid DNA in it, and then the RNA in the digestion solution was extracted with TransZol of Quanshijin Company, that is, the RNA was transcribed in vitro; after it was quantified with a UV spectrophotometer, it was diluted to a concentration of 10 5 copies / μL was used as a quality control for in vitro transcription RNA.

[0059] The sequence of the Coxsackie type A16-specific target fragment inserted in the recombinant plasmid is shown in SEQ ID NO.4 in the sequence listing:

[0060] TTGCAGAC...

Embodiment 1

[0066] Method for detecting Coxsackievirus A16 type nucleic acid on ABI 7300 fluorescent quantitative PCR instrument with kit of the present invention

[0067] (1) RNA extraction: using QIAamp Viral RNA Mini Kit to extract,

[0068] (2) PCR Mix preparation: according to the ratio of 22 μL: 1 μL, draw the PCR reaction liquid and PCR reaction enzyme respectively, mix thoroughly, centrifuge at 12,000 rpm for 10 seconds, and take out the PCR reaction tubes, one of which is a positive quality control product reaction tube, and the other is Negative quality control product reaction tubes, and the rest are test sample reaction tubes, add 23 μL of PCRMix prepared above to each reaction tube;

[0069] The PCR Mix preparation method is shown in the table below:

[0070]

[0071] (3) Adding samples: add 2 μL of extracted sample RNA, 2 μL of positive quality control substance and 2 μL of negative quality control substance to the three reaction tubes, mix well and centrifuge at 12,000 ...

Embodiment 2

[0091] Use the test kit of the present invention to detect unknown samples according to the method of embodiment 1, and the unknown samples are derived from samples to be determined by patients in XX hospital. The test results of embodiment 2 of the present invention are as follows: figure 2 As shown, the test result was positive, and the Ct value was 24.87.

[0092] The embodiments described above are only one of the more preferred specific implementations of the present invention, and the usual changes and replacements performed by those skilled in the art within the scope of the technical solutions of the present invention should be included in the protection scope of the present invention.

[0093] CA16-1 Sequence List_ST25.txt

[0094] SEQUENCE LISTING

[0095] Beijing Aipuyi Biotechnology Co., Ltd.

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Abstract

The invention discloses a kit for detecting coxsackie virus A16-type nucleic acid and a detection method thereof. The kit comprises PCR reaction enzyme, PCR reaction liquid, a negative quality control product and a positive quality control product, wherein the PCR reaction enzyme contains a hot start Taq enzyme, a MLV reverse transcriptase and a RNA enzyme inhibitor; the PCR reaction liquid contains DEPC treating water, dNTPs, 10*PCR Buffer, a coxsackie virus A16-type forward primer, a coxsackie virus A16-type reverse primer and a coxsackie virus A16-type probe, the sequence of the coxsackie virus A16-type forward primer is 5'-CGCTGCCGATACTGAAGCACCG-3', the sequence of the coxsackie virus A16-type reverse primer is 5'-CTGTCTCCGCGGCTTGTAG-3', the sequence of the coxsackie virus A16-type probe is 5'-ACAGATTAGGCACTGGTGTTGTACCGTA-3'; the negative quality control product is the DEPC treating water; and the positive quality control product is the prepared transcription in vitro RNA standard product. The method for fast detecting the sequence of the coxsackie virus A16-type nucleic acid by using a real-time fluorescence quantitative PCR technology has the advantages of specificity, sensitiveness, high speed as well as simple and convenient operation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and discloses a Coxsackievirus A16 nucleic acid detection kit and a detection method. Background technique [0002] Hand, foot and mouth disease (HFMD) is an infectious disease caused by enterovirus (EV). Children can cause myocarditis, pulmonary edema, aseptic meningoencephalitis and other complications. Individual critically ill children develop rapidly and 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 (Coxsackieviruses 16, CA16) and Enterovirus 71 (Enterovirus 71, EV71) are the most common. [0003] There are three specific diagnostic methods for enterovirus infection, namely virus isolation and identification, serological detection and molecular bi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 周骋姚志建刘自诚逄键涛王维
Owner IPE BIOTECHNOLOGY CO LTD
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