Kit for detecting seasonal influenza virus H1N1 through real-time PCR

A real-time fluorescent quantitative and seasonal influenza technology, applied in the field of biomedicine, can solve the problems of seasonal influenza virus H1N1, real-time fluorescent quantitative PCR detection method kit report, etc., to achieve broad clinical application value, fast sensitivity, Simple operation effect

Inactive Publication Date: 2011-01-19
INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Kit for detecting seasonal influenza virus H1N1 through real-time PCR
  • Kit for detecting seasonal influenza virus H1N1 through real-time PCR
  • Kit for detecting seasonal influenza virus H1N1 through real-time PCR

Examples

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Embodiment 1

[0047] Example 1: Real-time fluorescent quantitative PCR detection of seasonal influenza virus H1N1 in unknown samples using the present invention

[0048] 1. Use RNeasy Mini Kit (Qiagen, Cat. No. 74104) to extract total RNA from the sample to be tested.

[0049] 2. Use the SuperScript III First-Strand Synthesis System (Invitrogen, Cat. No. 18080-051) to synthesize the first-strand cDNA.

[0050] 3. Make a 10-fold serial dilution of the standard, from 1×10 10 copies / μl diluted to 1×10 2 copies / μl.

[0051]4. Calculate the number of samples N = sample to be tested + positive standard + 2 negative controls + 2 additional sample volumes. To configure Real-time PCR Mix, add in order:

[0052]

[0053] Evenly divide the mixture into 48-well 0.2ml PCR reaction tubes Fast Optical 48-well RXN Plate (ABI Company, Cat. No. 4375816), add 2 μl of standard products of different concentrations to each tube, and attach the optical reaction cover film 48-Well Optical Adhesive Film 25P...

Embodiment 2

[0059] Example 2: Application of the present invention to real-time fluorescent quantitative PCR detection of turbinates and lung tissues of a ferret model infected with seasonal influenza virus H1N1

[0060] After 16 ferrets were infected with seasonal influenza virus H1N1, the turbinate secretions of the ferrets were scraped every day on the 1st to 4th day, and the lung tissues were collected after the ferrets were relieved on the 5th day. Quantitative PCR detection, the results are shown in the table below.

[0061]

[0062] The results showed that the detection positive rate was 100%, and the change trend of the viral load was completely consistent with the detection results of other methods, indicating that the accuracy rate was also 100%.

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Abstract

The invention discloses a kit for detecting seasonal influenza virus H1N1 through real-time polymerase chain reaction (PCR) and also discloses methods for treating samples to be detected and analyzing Real-time PCR system and conditions and results. The kit comprises forward and reverse specific primers, standard products to be detected, etc. The kit can rapidly and quantitatively detect the seasonal influenza virus H1N1, has high sensitivity, can be used as the assistant diagnosis method of seasonal influenza virus H1N1 infection and the monitoring means of the clinical effects in the basic and clinical laboratories, has relatively low requirement for the experiment operators and has practical clinical application value.

Description

technical field [0001] The invention relates to a real-time fluorescent quantitative PCR detection method and a kit for rapid quantitative detection of seasonal influenza virus H1N1 nucleic acid, belonging to the technical field of biomedicine. Background technique [0002] Influenza is an acute respiratory infectious disease caused by influenza virus, which is characterized by rapid spread and wide spread. So far, influenza viruses have caused four world influenza pandemics, causing huge losses to the economy and health of human society. [0003] Influenza virus belongs to the Orthomyxoviridae family, and its genome is segmented negative-strand RNA. According to the difference of viral nucleocapsid protein (NP) and matrix protein (M), it can be divided into three types: A, B, and C, all of which can infect people. Influenza A and B viruses are the main epidemic viruses in the population. Influenza A viruses are divided into 16 HA subtypes according to the difference of th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 许黎黎鲍琳琳秦川
Owner INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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