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Nucleic acid composition for detecting four enteroviruses simultaneously and kit and detection method

A nucleic acid composition, enterovirus technology, applied in the field of diagnosis, can solve problems such as unfavorable viruses, false positives, and mutual interference of primers

Inactive Publication Date: 2019-03-22
深圳市艾伟迪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional detection methods or detection products can only detect one virus type at a time. When multiple virus types are detected at one time, there will be mutual interference between multiple primers, resulting in problems such as primer dimers and false positives. Conducive to the study of the above four viruses

Method used

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  • Nucleic acid composition for detecting four enteroviruses simultaneously and kit and detection method
  • Nucleic acid composition for detecting four enteroviruses simultaneously and kit and detection method
  • Nucleic acid composition for detecting four enteroviruses simultaneously and kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] A kit for the simultaneous detection of four enteroviruses is provided.

[0098] The kit includes RT-PCR reaction solution, nucleic acid composition for simultaneous detection of four enteroviruses, enzyme mixed solution, positive control and negative control.

[0099] Wherein, the RT-PCR reaction solution includes 250mM Tris-base, 0.25% TritonX-100, 25mmol / L MgCl 2 .

[0100] Nucleic acid compositions include:

[0101] The A6-type Coxsackievirus amplification primer pair whose sequences are shown in SEQ ID No.1 and SEQ ID No.2;

[0102] The A10-type Coxsackievirus amplification primer pair whose sequences are shown in SEQ ID No.3 and SEQ ID No.4;

[0103] The A16-type Coxsackievirus amplification primer pair whose sequences are shown in SEQ ID No.5 and SEQ ID No.6;

[0104] The EV71 enterovirus amplification primer pair whose sequences are shown in SEQ ID No.7 and SEQ ID No.8;

[0105] The A6-type Coxsackievirus detection probe whose sequence is shown in SEQ ID No...

Embodiment 2

[0114] Measure the sensitivity of the test kit of four kinds of enteroviruses of simultaneous detection of embodiment 1

[0115] (1) A6 type Coxsackie virus positive standard, A10 type Coxsackie virus positive standard, A16 type Coxsackie virus positive standard, EV71 type enterovirus positive standard in the kit of embodiment 1 are mixed And dubbed a mixed solution to obtain a positive test article.

[0116] (2) Perform a 10-fold serial dilution of the positive sample to be tested (i.e. 10copies / mL~10 5 copies / mL), using the kit of Example 1 to perform multiple fluorescent quantitative PCR detection on the positive test items under each gradient, the system of multiple fluorescent quantitative PCR reactions is shown in Table 1. The reaction conditions of the multiplex fluorescent quantitative PCR reaction were: reverse transcription at 50°C for 15min; pre-denaturation at 95°C for 3min; denaturation at 95°C for 15s, annealing, extension, and signal acquisition at 55°C for 40s...

Embodiment 3

[0126] (1) A6 type Coxsackie virus positive standard, A10 type Coxsackie virus positive standard, A16 type Coxsackie virus positive standard, EV71 type enterovirus positive standard in the kit of embodiment 1 are mixed And make a mixed solution to obtain a positive test article; the normal saline not containing the above four enterovirus positive control substances is a negative test article.

[0127](2) The experiment is divided into an experimental group, a control group 1 and a control group 2, and the kit of the experimental group is the kit of Example 1. The kit of control group 1 is roughly the same as the kit of Example 1, except that the sequence of the EV71 enterovirus amplification primer pair is as shown in SEQ ID No.21 and SEQ ID No.22, and is compatible with EV71 The sequence of the EV71 type enterovirus detection probe corresponding to the type enterovirus amplification primer pair is shown in SEQ ID No.23. Specifically, the sequence shown in SEQ ID No.21 is: 5'...

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Abstract

The invention relates to a nucleic acid composition for detecting four enteroviruses simultaneously and a kit and a detection method. The nucleic acid composition comprises type A6 coxsackievirus amplification primer pairs as shown in SEQ ID No.1 and SEQ ID No.2, type A10 coxsackievirus amplification primer pairs as shown in SEQ ID No.3 and SEQ ID No.4, type A16 coxsackievirus amplification primerpairs as shown in SEQ ID No.5 and SEQ ID No.6 and type EV71 enterovirus amplification primer pairs as shown in SEQ ID No.7 and SEQ ID No.8. The nucleic acid composition can detect type A6, type A10 and type A16 coxsackieviruses and type EV71 enterovirus simultaneously.

Description

technical field [0001] The invention relates to the technical field of diagnosis, in particular to a nucleic acid composition, a kit and a detection method for simultaneously detecting four enteroviruses. Background technique [0002] Enterovirus (human enterovirus, HEV) belongs to Picornavirus (Picornavirus) enterovirus genus (Enterovi ridae), is a class of single-stranded positive-sense RNA virus, divided into four groups (A ~ D). Enteroviruses currently mainly studied include coxsackievirus A16 (CVA16) and enterovirus 71 (enterovirus71, EV71), while coxsackievirus A6 (coxsackievirus A6, CVA6) and Coxsackievirus A10 (CVA10) has also received more and more attention. [0003] In order to better guide the research on the above four viruses, it is necessary to develop a product capable of detecting A6-type Coxsackieviruses, A10-type Coxsackieviruses, A16-type Coxsackieviruses and EV71-type enteroviruses. However, traditional detection methods or detection products can only ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 邓春兴李泓彦姚飞平杨彪张敏詹馨惠
Owner 深圳市艾伟迪生物科技有限公司
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