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Real-time fluorescent nucleic acid thermostatic amplification detection kit of influenza virus A (H1N1) (2009)

A technology of influenza virus and H1N1, applied in the direction of fluorescence/phosphorescence, measurement/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as complicated operation, hindering large-scale promotion and use, and contamination of amplicons

Active Publication Date: 2014-01-22
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are reverse transcriptase-polymerase chain reaction (Reverse Transcriptase-PCR, RT-PCR for short) and real-time fluorescent quantitative polymerase chain reaction (Real Time Fluorescent Quantified PCR, FQ PCR for short), RT-PCR adopts "gene amplification The operation mode of "amplification + amplicon detection" is easy to cause contamination of the amplicon, which often results in false positive or false negative of the experimental results.
Although real-time fluorescence quantitative PCR solves the above problems technically, its sensitivity and accuracy are high, but the operation is complicated and the detection cost is relatively expensive, which hinders its large-scale promotion and use in developing countries.

Method used

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  • Real-time fluorescent nucleic acid thermostatic amplification detection kit of influenza virus A (H1N1) (2009)
  • Real-time fluorescent nucleic acid thermostatic amplification detection kit of influenza virus A (H1N1) (2009)
  • Real-time fluorescent nucleic acid thermostatic amplification detection kit of influenza virus A (H1N1) (2009)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Embodiment 1, be used for the design of the special primer and probe of real-time fluorescent nucleic acid constant temperature amplification detection type A H1N1 influenza virus (2009)

[0101] The present invention selects the H1N1 (2009) virus HA gene with no secondary structure and a highly conserved segment as the amplified target sequence region (its nucleotide sequence is shown in sequence 1 in the sequence table), and according to the principle of primer probe design, use DNAATAR, DNAman software and artificially designed special primers and probe sequences for real-time fluorescent nucleic acid constant temperature amplification to detect influenza A (H1N1) virus (2009) obtained the following specific sequences:

[0102] (1) a capture probe (TCO, Target Capture Oligo) that can specifically combine with the target nucleic acid (H1N1 (2009) RNA) sequence of the influenza A H1N1 influenza virus (2009) shown in sequence 1 in the sequence listing, said The nucleoti...

Embodiment 2

[0106] Example 2, preparation of influenza A H1N1 virus (2009) real-time fluorescent nucleic acid constant temperature amplification detection kit

[0107] Using the special primers and probes provided in Example 1, a real-time fluorescent nucleic acid constant temperature amplification detection kit for influenza A (H1N1) virus (2009) of the present invention was obtained. The kit contains capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, H1N1 (2009) detection probe, M-MLV reverse transcriptase and T7 RNA polymerase; when there is an internal standard in the kit, Internal standard detection probes are also included.

[0108] The capture probe exists in the nucleic acid extraction solution, the T7 primer, nT7 primer, H1N1 (2009) detection probe, and internal standard detection probe exist in the H1N1 (2009) detection solution, and the M-MLV reverse Recording enzyme and T7 RNA polymerase exist in the SAT enzyme solution. Specifically, the kit is divided into A ...

Embodiment 3

[0141] Embodiment 3, the detection sensitivity of real-time fluorescent nucleic acid constant temperature amplification of influenza A (H1N1) virus (2009)

[0142] With the kit of the present invention (see Example 2 for the composition, there is no H1N1 (2009) internal standard in the kit, and there is no internal standard detection probe in the detection solution) the measured concentration is 1 × 10 4 copies / reaction, 1×10 3 copies / reaction, 1×10 2 Copies / reaction, 10copies / reaction of Influenza A H1N1 virus (2009) positive control RNA to determine the lower limit of sensitivity. Specific steps are as follows:

[0143] (1) dilution

[0144] Will 1×10 4 Influenza A H1N1 virus (2009) positive reference RNA of copies / reaction, 10-fold serial dilution to 1×10 3 copies / reaction, 1×10 2 copies / reaction, 10copies / reaction.

[0145] (2) Nucleic acid extraction

[0146] 2.1 Add 200 μl lysate (containing HEPES 35mM, (NH 4 ) 2 SO 4 20mM), 200 μl influenza virus positive re...

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Abstract

The invention discloses a real-time fluorescent nucleic acid thermostatic amplification detection kit of influenza virus A (H1N1) (2009). The detection kit comprises a capture probe, H1N1 (2009) detection primers of a T7 primer and an nT7 primer, an H1N1 (2009) detection probe, an M-MLV (moloney murine leukemia virus) reverse transcriptase, T7 RNA (ribonucleic acid) polymerase and the like. By adopting the kit disclosed by the invention, the H1N1 (2009) RNA in a swab can be detected; the detection kit has the characteristics of high specificity, high sensitivity (the sensitivity can achieve 100 copies / reaction), low pollution (an amplification product RNA is easily degraded in a natural environment) and fast detection, and plays an important role in clinical diagnosis of early injection of influenza A (H1N1).

Description

technical field [0001] The invention relates to the biological detection technology of viruses, in particular to primers and probes used in the real-time fluorescent nucleic acid constant temperature amplification detection of Influenza A H1N1 influenza virus (2009) which combines specific target capture technology and real-time fluorescent nucleic acid constant temperature amplification detection technology. Needles and related kits. Background technique [0002] Influenza viruses are divided into three types: A, B, and C according to nucleoprotein (NP) and matrix protein (M). The influenza A virus genome consists of 8 negative-strand RNA segments, which are divided into 16 HA subtypes and 9 NA subtypes according to the surface hemagglutinin (HA) and neuraminidase (NA). [0003] Influenza A virus is a common influenza virus, easy to mutate, and the most aggressive. It can cause human influenza and sometimes pneumonia and other complications, mainly necrosis and shedding o...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
CPCC12Q1/6844C12Q1/70C12Q2561/113C12Q2563/107
Inventor 方亮冯金梦于明辉居金良
Owner SHANGHAI RENDU BIOTECH
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