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A kind of multiplex fluorescent PCR detection kit of hand, foot and mouth disease virus, and application

A hand-foot-and-mouth disease virus and detection kit technology, applied in the field of kits, can solve the problems of time-consuming and energy-consuming, inability to effectively distinguish infections, and high detection costs, to prevent false negatives, avoid PCR reaction inhibition, and achieve good specificity. Effect

Active Publication Date: 2021-02-19
NANJING MOKOBIO BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional fluorescent real-time PCR can only amplify the nucleic acid of one pathogen at a time. It takes multiple attempts to accurately identify the causative pathogen, which consumes a lot of time and energy, and it cannot effectively distinguish infections caused by multiple pathogens. In addition, it can only detect EV71, CA16 and other enteroviruses with a single index in different reaction tubes, and cannot detect all viruses at one time. Not only the detection efficiency is low, but also the detection cost is high

Method used

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  • A kind of multiplex fluorescent PCR detection kit of hand, foot and mouth disease virus, and application
  • A kind of multiplex fluorescent PCR detection kit of hand, foot and mouth disease virus, and application
  • A kind of multiplex fluorescent PCR detection kit of hand, foot and mouth disease virus, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1 PCR detection kit

[0063] Described Enterovirus 71 type (EV71), Coxsackie virus A16 type (Cox A16), Dow virus universal type (EVU), internal standard gene primers and probes are as shown in Table 1, wherein in Table 1 All primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;

[0064] Table 1 Enterovirus 71 (EV71), Coxsackievirus A16 (CoxA16), Dowvirus Universal (EVU), internal standard gene primers, and probes

[0065]

[0066] The kit is composed of PCR reaction buffer, enzyme system, positive control, negative control, and all primer pairs and probes in Table 1;

[0067] Table 2 is the EV71 / CA16 / EVU primer-probe mixture components (batch: 1000 parts)

[0068] serial number raw material name Specification Dosage 1 EV71F 100μmol / L 50μl 2 EV71R 100μmol / L 50μl 3 EV71P 100mmol / L 25μl 4 CA16F 100μmol / L 80μl 5 CA16R 100μmol / L 80μl 6 CA16P 100μmol / L 40μl 7 EV...

Embodiment 2

[0075] Example 2 The method for detecting Enterovirus 71 (EV71), Coxsackievirus A16 (CoxA16), and Enterovirus Universal (EVU)

[0076] (1), extraction of viral nucleic acid

[0077] Use the QIAamp MinElute Virus Spin Kit (Cat. No.: 57704) from QIAGEN or the Viral Genomic DNA / RNA Extraction Kit from Tiangen Biochemical Technology Co., Ltd. (Cat. No.: DP315) to extract nucleic acid from the sample;

[0078] (2), the configuration of the reaction system

[0079] According to the number of reaction tubes n, take PCR reaction buffer 16 μL×n, enzyme system 2 μL×n, EV71 / CA16 / EVU primer and probe mixture 2 μL×n, ensure PCR reaction buffer, EV71 / CA16 before use The mixture of / EVU primers and probes is fully dissolved and mixed, and the enzyme system is centrifuged before use to ensure that all enzymes are concentrated at the bottom.

[0080] Dispense the PCR reaction solution into PCR reaction tubes according to 20 μL / tube, and move the reaction tube containing the PCR reaction solu...

Embodiment 3

[0103] Embodiment 3 kit performance analysis

[0104] 1. Positive coincidence rate: Take the positive reference product P1-P10 as the sample to be tested, and pass the positive coincidence rate of the positive reference sample of the ABI7500 real-time fluorescent quantitative PCR instrument detection kit. The samples P1-P10 to be tested are shown in Table 8, and the test results Show, P1-P10 are all positive, coincidence rate is 100% (10 / 10), wherein, P1, P2, P7, P8, P9, P10 are EV71 positive, such as Figure 1a ; P3, P4, P7, P8, P9, P10 are negative for CA16, such as Figure 1b Shown; P1-P9 are all EVU positive, such as Figure 1c shown;

[0105] Table 8 Samples of positive reference products to be tested

[0106]

[0107]

[0108] 2. Negative coincidence rate: Take the negative reference products N1~N10 as the samples to be tested, as shown in Table 9, test on the ABI7500 real-time fluorescent quantitative PCR instrument, analyze the coincidence rate of the negative ...

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Abstract

The present invention relates to a multiplex fluorescent PCR detection kit for hand, foot and mouth disease virus and its application, including the following primer pairs for amplifying common hand, foot and mouth disease pathogens, and primers and probes for internal standard genes for quality control, The primer and probe sequences are shown in SEQ ID NO.1-SEQ ID NO.12. The present invention can detect three common pathogens that cause intestinal system infection simultaneously in one reaction, shorten the detection time, and have high accuracy. and detection laboratories provide powerful detection tools; the present invention has good specificity to the primers and probes used for enterovirus type 71, coxsackievirus A16 and enterovirus general type, and avoids different primers / There is a problem of cross-linking between probes; in addition, the kit of the present invention is designed with an internal standard, and the internal standard template is a housekeeping gene that participates in sample extraction and PCR amplification, which can effectively monitor the entire reaction process and prevent false negatives; And it can effectively avoid the inhibition of PCR reaction by some inhibitors.

Description

technical field [0001] The invention relates to a kit, in particular to a multiple fluorescent PCR detection kit for hand, foot and mouth disease virus, and its application. Background technique [0002] Hand, foot and mouth disease (HFMD) is one of the common infectious diseases caused by a variety of enteroviruses. The disease has the characteristics of rapid spread and high prevalence, and can cause large-scale epidemics in a short period of time. It is more common in children under 5 years old, and the incidence rate is highest in the age group younger than 3 years old. From April to September every year, infants and young children are more likely to be infected with hand, foot and mouth disease, and the total number of patients generally reaches its peak in May to July. The main modes of transmission are: close contact with people, through contact with virus-contaminated toys, towels, milk utensils, underwear, bedding, etc.; the virus in the patient's throat secretions...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686
Inventor 范建徐璐程成
Owner NANJING MOKOBIO BIOTECH
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