Nucleic acid dual fluorescence PCR (Polymerase Chain Reaction) detection kit for influenza A/B virus

A type of influenza B virus and detection kit technology, applied in the biological field, can solve the problems of time-consuming, complicated and expensive gene sequence determination methods, reduce the chance of pollution, and achieve fast, objective and repeatable detection results Effect

Inactive Publication Date: 2011-11-23
JIANGSU BIOPERFECTUS TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005]1) Virus isolation and culture have high technical requirements, are expensive, and time-consuming, and the positive rate of isolation varies from laboratory to laboratory, which cannot meet the requirements of virus epidemic at the same time. The need to handle a large number of samples, currently only used for experimental research
[0006]2) Serological methods cannot process samples with high throughput, and cannot accurately reflect whether they are currently infected or carry the virus
[0008] At present, most of the products adopt the detection method for a single virus. For the clinical detection of multiple viruses, it is necessary to perform multi-tube detection on one sample, which increases the detection Cost and complexity of operation
[0009] From the above, it can be seen that the commonly used virus isolation method is time-consuming, the serological diagnosis method has hysteresis, and the gene sequence determination method is also relatively complicated and cumbersome.

Method used

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  • Nucleic acid dual fluorescence PCR (Polymerase Chain Reaction) detection kit for influenza A/B virus
  • Nucleic acid dual fluorescence PCR (Polymerase Chain Reaction) detection kit for influenza A/B virus
  • Nucleic acid dual fluorescence PCR (Polymerase Chain Reaction) detection kit for influenza A/B virus

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Influenza A / B nucleic acid detection kit

[0040] The double fluorescent quantitative PCR detection kit of influenza A / B nucleic acid of the present embodiment comprises RNase inhibitor, RT-PCR reaction solution, enzyme mixture, double reaction solution of influenza A / B virus, positive control and negative control,

[0041] Wherein, the RNase inhibitor is DEPC water; the RT-PCR reaction solution includes 10× buffer, MgCl2 and dNTPs;

[0042] The Influenza A / B Duplex Reaction Solution includes the following components:

[0043]Component (1): composed of a pair of primers for detecting influenza A virus and a probe for detecting influenza A virus; wherein, the base sequences of the two primers are SEQ ID No.1 and SEQ ID No.2 respectively shown; the base sequence of the probe is shown in SEQ ID No.3, the 5' end of the probe is marked with a fluorescent reporter group, and the 3' end is marked with a fluorescent quencher group; the primer for detecting influenza...

Embodiment 2

[0063] Embodiment 2 Sensitivity test

[0064] The positive reference product is the inactivated virus culture solution, which comes from the Jiangsu Provincial Center for Disease Control and Prevention.

[0065] The negative reference product is RNase-free water. Weigh 1g of DEPC with an electronic balance, add purified water to 1000ml and mix well, then sterilize at 121°C / 20 minutes in a sterilizing pot, mark it, and store it at room temperature.

[0066] The kit of the present invention is used for detection.

[0067] The test results show that the kit of the present invention has good sensitivity, and the CT value changes in a gradient as the concentration decreases ( Figure 4 , Figure 5 ). The test result shows that the kit of the present invention has high sensitivity for the diagnosis of type A / type B influenza virus.

[0068]

Embodiment 3

[0069] Embodiment 3 specificity test

[0070] In order to detect the specificity of the type A / B influenza virus detection kit of the present invention, the type A / B influenza virus detection kit of the present invention is used to detect respiratory syncytial virus, human adenovirus, and human parainfluenza virus.

[0071] The test results show that: the FAM channel only amplifies influenza A virus ( Figure 6 ), the HEX channel only amplifies influenza B virus ( Figure 7 ). It shows that the detection kit of the present invention can specifically amplify influenza virus without cross-reaction with other viral nucleic acids.

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Abstract

The invention provides a nucleic acid dual fluorescence PCR (Polymerase Chain Reaction) detection kit for an influenza A/B virus. The detection kit comprises an RNA (Ribonucleic Acid) enzyme inhibitor, an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) liquid, an enzyme mixed liquor, an influenza A/B virus dual reaction liquid, a positive control and a negative control, wherein the influenza A/B virus dual reaction liquid comprises a component (1) which consists of a pair of primers for detecting the influenza A virus and a probe for detecting the influenza A virus and a component (2) which consists of a pair of primers for detecting the influenza B virus and a probe for detecting the influenza B virus; and the enzyme mixed liquor comprises a Taq enzyme and a reverse transcription enzyme. By adopting the detection kit, the influenza A and B viruses can be detected at the same time, and the problem that only one influenza virus can be detected in one reaction in the conventional product is solved. The detection kit has the advantages of easiness and convenience for operating, high repeatability, quick and objective detection result and the like, and has a great application prospect in the field of in-vitro diagnosis of influenza viruses.

Description

technical field [0001] The invention relates to a dual fluorescent PCR detection kit for simultaneous detection of type A / type B influenza virus nucleic acid, which belongs to the field of biotechnology. Background technique [0002] Influenza is a seasonal disease. In temperate regions, influenza prevails throughout the winter. In tropical regions, influenza viruses exist throughout the year, and are more common in rainy seasons. Common influenza A, commonly known as cold, is an acute respiratory infectious disease caused by influenza A virus as the pathogen, and the main mode of transmission is air droplet transmission. The severity of influenza is related to the individual immune status. Generally, only about 50% of infected patients will develop typical clinical symptoms of influenza. Typical symptoms of influenza include sudden fever, dizziness, headache, myalgia, mild systemic symptoms, and may be accompanied by sore throat and cough, nasal congestion, runny nose...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 张旭王国强刘中华魏赵延严浩荣
Owner JIANGSU BIOPERFECTUS TECH CO LTD
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