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Test kit for coxsackie virus A6 nucleic acid and test method

A technology for coxsackie virus and detection kits, which is applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of waste, high cost, reduce the probability of pollution and low requirements , highly specific and sensitive effects

Inactive Publication Date: 2015-09-02
安阳市疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a detection method is provided, because it is a dual-channel product, the cost is high, and there is serious waste when it is applied to the special detection of Coxsackievirus A6

Method used

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  • Test kit for coxsackie virus A6 nucleic acid and test method
  • Test kit for coxsackie virus A6 nucleic acid and test method
  • Test kit for coxsackie virus A6 nucleic acid and test method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The present invention searches for the VP1 gene sequence of the Coxsackievirus A6 strain in the Genebank database for the Coxsackievirus A6 type, uses DNAMAN 7.0 software to compare multiple sequences, and finds out the conserved segment. Use GenScript and Oligo 7.0 software to design specific primers and Taqman probes in this segment, and perform BLAST comparison on the NCBI database, and design five sets of primers and probes respectively. The sequences of primers and probes are shown in Table 1 Show. Two CVA6 clinical positive samples were respectively amplified by PCR, and the amplification curves were as follows: figure 1 and figure 2 .

[0057] Table 1 Primer and Probe Sequences

[0058]

[0059] Depend on figure 1 and figure 2 The results show that the amplification curves of the fifth group of primers and probes are the most typical, with obvious exponential phase and plateau phase, higher fluorescence intensity (ordinate value), and smaller CT value (...

Embodiment 2

[0061] For the fifth set of primers and probes, the molar ratio of "primer:probe" is 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1 and 2:1 , to test the CVA6 clinically positive samples to investigate the effect of different molar ratios of "primer:probe" on the amplification curve. The results are shown in Table 2 and image 3 .

[0062] Table 2 The effect of the molar ratio of "primer:probe" on the amplification curve

[0063] Primer probe ratio

[0064] CT value

[0065] From Table 2 and image 3 It can be seen from the results that the results of the above-mentioned ratio experiments are all good, and can be interpreted normally. However, when the molar ratio of primer to probe is 3.5:1, the curve is the best, and the curve rises higher (ordinate) than the other six ratios, and the CT is relatively smaller (abscissa), indicating that the fluorescence intensity is large The exponential phase of the response comes earlier and the curve rises earlier.

Embodiment 3

[0066] Embodiment 3: the nucleic acid detection kit of Coxsackievirus A6 type of the present invention

[0067] The kit of the invention comprises a real-time fluorescent PCR reaction solution, a reverse transcription system, a primer mixture, a specific fluorescent probe, a Coxsackie virus A6 standard product and DEPC treated water.

[0068] The real-time fluorescence PCR reaction liquid of the present invention comprises 2×PCR buffer, heat-resistant Taq DNA polymerase, magnesium sulfate, and deoxyribonucleotide triphosphate mixture.

[0069] The reverse transcriptase in the reverse transcription system of the present invention is MMLV reverse transcriptase.

[0070]In the primer mixture of the present invention, the base sequence of the forward primer is shown in SEQ ID No.1, the base sequence of the reverse primer is shown in SEQ ID No.2, the forward primer and reverse primer The molar ratio of the primers is 1:1 for SEQ ID No.1:SEQ ID No.2.

[0071] The base sequence of ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a real-time fluorescence quantification reverse transcription PRC test kit for a rapid coxsackie virus A6 and a test method. The test kit for coxsackie virus A6 nucleic acid comprises a coxsackie virus A6 forward primer shown by a nucleotide sequence such as SEQ ID NO.1, a reverse primer shown by a nucleotide sequence such as SEQ ID NO.2 and a special fluorescence probe shown by a nucleotide sequence such as SEQ ID No.3. The test kit for the coxsackie virus A6 nucleic acid is high in sensitivity, strong in specificity, rapid, easy and convenient to operate, precise and stable in result and wide in application range, and suitable for screening of a coxsackie virus A6 crowd.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a nucleic acid detection kit, in particular to a rapid Coxsackievirus A6 type real-time fluorescent quantitative reverse transcription PCR detection kit, which can be applied to public health units and clinical medical units to detect Rapid, qualitative or quantitative detection of hand, foot and mouth disease outbreaks, clustered cases and clinical cases caused by Coxsackievirus A6. Background technique [0002] Hand, foot and mouth disease is an acute infectious disease with rash and fever caused by a variety of human enteroviruses (HEV), mainly infecting infants and young children. The disease was first reported in the late 1950s, and two pathogens, Coxsackievirus A16 and Enterovirus 71, were successfully isolated from clinical samples in 1958 and 1969, respectively. In the following decades, Coxsackievirus A16 and Enterovirus 71 circulated alternately in different countrie...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/70C12Q1/6851C12Q1/701C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 李洋张相萍徐耀宇翟明强包小兵胡秀杰宋凤燕宋艳文侯晓惠林媛媛张栓虎梁玉清于杰
Owner 安阳市疾病预防控制中心
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