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Primer-pair probe combination product, kit and application of primer-pair probe combination product to detection of Enterovirus, EV

A combined product and primer pair technology, which is applied in the field of microbial detection, can solve the problems of low sensitivity, complicated operation, and difficult detection, and achieve the effect of high sensitivity, strong specificity, specificity and precise absolute quantification

Inactive Publication Date: 2020-11-20
广东省疾病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are disadvantages such as complex operation, long time-consuming, poor specificity, and low sensitivity. Moreover, due to the large number of enterovirus serotypes, the traditional virus cell culture method and serological method are difficult to detect due to their respective characteristics. clinical need
So far, there is no targeted and effective treatment for enterovirus, and its prevention and control work is mainly carried out through vaccines. Except for polio and EV71, there is no vaccine available for prevention. It is one of the important pathogens that seriously affect human health. one

Method used

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  • Primer-pair probe combination product, kit and application of primer-pair probe combination product to detection of Enterovirus, EV
  • Primer-pair probe combination product, kit and application of primer-pair probe combination product to detection of Enterovirus, EV
  • Primer-pair probe combination product, kit and application of primer-pair probe combination product to detection of Enterovirus, EV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Preparation of a Universal Enterovirus Droplet Digital PCR Detection Kit

[0069] The kit includes a one-step micro-droplet digital PCR detection reagent and several microfluidic chips; wherein, the one-step micro-droplet digital PCR detection reagent includes a general-purpose enterovirus micro-droplet digital PCR reaction solution A, a general-purpose intestinal virus Virus droplet digital PCR reaction solution B, droplet generating oil, positive control, negative control and quality control solution:

[0070] Among them, the universal enterovirus droplet digital PCR reaction solution A includes 2× nucleic acid reaction buffer, 300mMDTT, Taq enzyme, reverse transcriptase and RNase inhibitor;

[0071] Universal enterovirus droplet digital PCR reaction solution B includes primers and probes for universal enterovirus; the base sequences of the upstream primers, downstream primers and specific probes are as follows:

[0072] Upstream primer: 5'-CCCTGAATGCGGMTAA...

Embodiment 2

[0077] Example 2 A detection method for a universal enterovirus droplet digital PCR detection kit

[0078] The specific detection method includes the following steps:

[0079] 1. Extract sample RNA

[0080] 1.1 Take 200 μl of clinical samples, add 400 μl of Binding Buffer added with Poly(A), mix thoroughly, transfer to a high-purification filter tube, centrifuge at 8000 rpm for 15 s, and discard the waste liquid in the collection tube.

[0081] 1.2 Add 500 μl Inhibitor Removal Buffer to the filter tube, centrifuge at 8000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0082] 1.3 Add 450 μl Washing Buffer to the filter tube, centrifuge at 8000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0083] 1.4 Repeat step 1.3, and then centrifuge at high speed for 10s. Be sure to remove the waste liquid in this step.

[0084] 1.5 Add 50 μl Elution Buffer to the filter tube, let it stand at room temperature for 2 minutes, centrifuge at 8000...

Embodiment 3

[0096] Example 3 Universal Enterovirus Primer Probe Design and Specificity Detection

[0097] In order to utilize the microdroplet digital PCR method to detect enteroviruses in samples, the present invention designs universal primers and probes that can amplify enteroviruses according to various enterovirus gene sequences published by NCBI;

[0098] Get enterovirus-positive and negative clinical samples for experimentation, and operate according to the steps of Example 2;

[0099] Result analysis and judgment:

[0100] In this embodiment, there are 50 copies of various samples.

[0101] figure 1 It is the detection result of an EV71-positive sample in the FAM channel, and the copy number is 62.1copies / μl;

[0102] figure 2 It is the detection result of a CA16 positive sample in the FAM channel, the copy number is 496copies / μl;

[0103] image 3 It is the detection result of a CA6 positive sample in the FAM channel, and the copy number is 62.1copies / μl;

[0104] Figure ...

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Abstract

The invention relates to the technical field of microbial detection, in particular to a primer-pair probe combination product, a kit and application of primer-pair probe combination product to the detection of Enterovirus, EV. The primer-pair probe combination product can be used to detect a variety of Enterovirus, EV, has the characteristics of high sensitivity, high specificity and precise absolute quantification, and can be used for rapid quantitative and accurate detection of Enterovirus, EV.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a primer-to-probe combination product, a kit and its application in the detection of enteroviruses. Background technique [0002] Enteroviruses (Enteroviruses, EVs) belong to the small RNA virus family (Rcomoviruses), are a group of non-enveloped single-stranded RNA picornaviruses, and are one of the common pathogens of human infection. Enteroviruses include more than 70 serotypes including poliovirus, coxsackievirus, echovirus, new enterovirus, rotavirus, etc., which can cause intestinal and respiratory infections, poliomyelitis, myocarditis, Aseptic meningitis and hand-foot-mouth disease and other infections, and the onset is often a syndrome, the severity of the disease varies greatly, and infants and young children may have severe myocarditis or severe pneumonia outbreaks, especially outbreaks in the neonatal period can cause death. A syndrome can be caused by di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/6851
CPCC12Q1/6851C12Q1/701C12Q2600/166C12Q2563/159C12Q2565/629C12Q2563/107C12Q2545/113C12Q2527/127
Inventor 袁润余柯昌文曾汉日苏娟周平平武婕陆靖
Owner 广东省疾病预防控制中心
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