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Real-time fluorescence nucleic acid constant temperature amplification detection kit of general influenza a virus (IAV)

A technology of influenza A virus and kits, applied in the field of probes and related kits, and primers, which can solve the problems of amplification contamination, long detection cycle, and false positive test results, etc.

Active Publication Date: 2014-01-08
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems that the existing general-purpose influenza A virus (IAV) detection method has low sensitivity, long detection cycle, easy to cause contamination of amplified products, false positive or false negative of experimental results and high detection cost, the present invention Provides a real-time fluorescent nucleic acid constant temperature amplification detection technology for general-purpose influenza A virus (IAV) with short detection cycle, high sensitivity, high specificity, low pollution, stable reaction, low detection cost, and easy popularization and application. Primers, probes, kits and their use

Method used

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  • Real-time fluorescence nucleic acid constant temperature amplification detection kit of general influenza a virus (IAV)
  • Real-time fluorescence nucleic acid constant temperature amplification detection kit of general influenza a virus (IAV)
  • Real-time fluorescence nucleic acid constant temperature amplification detection kit of general influenza a virus (IAV)

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Effect test

Embodiment 1

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

[0101] The present invention selects no secondary structure and a highly conserved segment in the IAV virus M gene 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, DNA ATAR, DNAman software and artificial design are used for real-time fluorescent nucleic acid constant temperature amplification to detect the special-purpose primer and probe sequence of universal influenza A virus (IAV), obtain following specific sequence:

[0102] (1) a capture probe (TCO, Target Capture Oligo) that can be specifically combined with the target nucleic acid (IAV RNA) sequence of the general type influenza A virus (IAV) shown in sequence 1 in the sequence listing (TCO, Target Capture Oligo...

Embodiment 2

[0106] Embodiment 2, prepare the real-time fluorescent nucleic acid constant temperature amplification detection kit of universal influenza A virus (IAV)

[0107]Using the special primers and probes provided in Example 1, a real-time fluorescent nucleic acid constant temperature amplification detection kit for general-purpose influenza A virus (IAV) of the present invention was obtained. The kit includes capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, IAV detection probe, internal standard detection probe, M-MLV reverse transcriptase and T7 RNA polymerase.

[0108] The capture probe exists in the nucleic acid extraction solution, the T7 primer, nT7 primer, IAV detection probe, and internal standard detection probe exist in the IAV detection solution, and the M-MLV reverse transcriptase and T7 RNA polymerase The enzyme exists in the SAT enzyme solution. Specifically, the kit is divided into A box (specimen processing unit) stored at 2-30°C and B box (nucleic a...

Embodiment 3

[0141] Example 3. Real-time fluorescent nucleic acid constant temperature amplification detection of clinical sample throat swab

[0142] Use the kit of the present invention (see embodiment 2 for composition) to detect the general type influenza A virus (IAV) in the throat swab of clinical sample, and specific method comprises the following steps:

[0143] (1) Sample collection, transportation and storage

[0144] The clinician will collect the specimens according to the actual situation. The test specimens are throat swabs. The collection method is: wipe the posterior pharyngeal wall and the pharyngeal tonsils on both sides with a special sampling cotton swab, invade the swab into 3-5mL normal saline, and send it sealed for inspection. Within 48 hours after the sample is collected, store it at 4°C and send it to the Influenza Surveillance Network Laboratory (or store it at -70°C for testing, and deliver it within one week).

[0145] (2) Nucleic acid extraction

[0146] 2.1...

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Abstract

The invention discloses a real-time fluorescence nucleic acid constant temperature amplification detection kit of general influenza a virus (IAV). The kit comprises a capture probe, an IAV amplifier primer T7 primer, an nT7 primer, an IAV detection probe, M-MLV reverse transcriptase, T7 RNA polymerase and other reagents. The kit can detect the IAV RNA in a swab, has the advantages of being high in specificity and sensitivity (100 copies / reaction), low in pollution (due to the fact that amplification product RNA is easy to degrade in a natural environment) and quick in detection (due to the fact that detection can be finished in 50 minutes conventionally), plays an important role in clinical diagnosis of early infection of general influenza a and is wide in application prospect.

Description

technical field [0001] The present invention relates to the biological detection technology of virus, in particular to the primers used in the real-time fluorescent nucleic acid constant temperature amplification detection of general-purpose influenza A virus (IAV) which combines specific target capture technology and real-time fluorescent nucleic acid constant temperature amplification detection technology, Probes 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, the most aggressive, can cause human influenza and sometimes pneumonia and other complications, mainly the necrosis and shedding of cili...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 方亮于明辉冯金梦居金良
Owner SHANGHAI RENDU BIOTECH
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