Swine flu virus real-time fluorescent RT-PCR typing kit

A technology of RT-PCR and swine influenza virus, applied in the direction of fluorescence/phosphorescence, material excitation analysis, biochemical equipment and methods, etc., to achieve good sensitivity, improve reliability and accuracy

Active Publication Date: 2010-11-17
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, no real-time fluorescent RT-PCR typing kit for swine influenza virus detection has been developed at home and abroad. Therefore, the de

Method used

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  • Swine flu virus real-time fluorescent RT-PCR typing kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: the development of swine influenza virus typing detection reagent

[0051] 1. Design of primers and probes: by comparing and analyzing the nucleic acid sequences of the reported swine influenza viruses, the H1 and H3 HA genes and the N1 and N2 NA genes of swine influenza viruses were respectively used as amplification targets According to the basic principles of primer and probe design, multiple pairs of primers and probes are designed by software and manually.

[0052] 2. Selection of clinical samples: According to relevant domestic and foreign literature reports, respiratory samples, serum, lung tissue and other samples can be used.

[0053] 3. Establishment and optimization of the reaction system

[0054] Sample preparation: The constructed plasmids containing the amplified fragments of various purposes were used as positive quality control products, and the subtypes of avian influenza virus H5 / H7 / H9, porcine reproductive and respiratory syndrome virus...

Embodiment 2

[0063] Embodiment 2: Swine influenza virus typing detection kit and its use

[0064] 1. Prepare a kit comprising the following components: 1 tube of RNA extraction solution (50ml / tube), 1 tube of H1 subtype primer-probe mixture (50 μl / tube), 1 tube of H3 subtype primer-probe mixture (50 μl / tube) ) 1 tube, 1 tube of N1 subtype primer-probe mixture (50 μl / tube), 1 tube of N2 subtype primer-probe mixture (50 μl / tube), 1 tube of RT-PCR reaction solution (1440 μl / tube), RT-PCR enzyme system (300μl / tube) 1 tube, negative quality control (200μl / tube) 1 tube, H1 subtype positive quality control (100μl / tube) 1 tube, H3 subtype positive quality control (100μl / tube) tube) 1 tube, N1 subtype positive quality control (100 μl / tube) 1 tube, N2 subtype positive quality control (100 μl / tube) 1 tube.

[0065] 2. Specimen collection, transportation and storage

[0066] Specimen collection is carried out according to the actual situation. Specimens that can be tested include: respiratory tract ...

Embodiment 3

[0085] Example 3: Application of swine influenza virus typing detection kit to detect samples

[0086] With positive quality control substance, negative quality control substance, specific reference substance in embodiment 1 as samples to be tested, detect these samples respectively according to the method of embodiment 2, positive quality control substance detection result is positive, negative quality control substance and The test results of specific reference products were all negative (see attached figure 1 ,2).

[0087] Take H1N1 type swine influenza virus, H3N2 type swine influenza virus, and H1N2 type swine influenza virus samples as samples to be tested respectively, and detect these samples respectively according to the method in Example 2, H1N1 type swine influenza virus, H3N2 type swine influenza virus, H1N2 type All swine influenza viruses were accurately typed (see attached image 3 , 4, 5).

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Abstract

The invention relates to a swine flu virus real-time fluorescent RT-PCR isotyping kit, in particular to a kit for performing isotyping of swine flu viruses by using a one-step method real-time reverse transcription-polymerase chain reaction (RT-PCR) technique. The kit has high sensitivity and specificity. Through the isotyping of the swine flu viruses in respiratory tract specimens and serum and lung tissue samples and other samples by using the kit of the invention, the early-period quick diagnosis of the swine flu viruses is realized, and the trend of the swine flu viruses can be monitored.

Description

technical field [0001] The invention relates to a swine influenza virus typing kit, in particular to a kit for swine influenza virus typing by one-step real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) technology. The kit has high sensitivity and specificity. The swine influenza virus in respiratory samples, serum, lung tissue and other samples can be typed by the kit of the present invention, so as to realize early and rapid diagnosis of swine influenza virus and monitor its prevalence. trend. Background technique [0002] Swine influenza (SI) is a porcine respiratory disease caused by swine influenza virus. This disease occurs all over the world, causing serious harm, huge economic losses, and a threat to human health. Swine influenza virus (SIV) belongs to the family Orthomyxoviridae and the genus Influenza virus A. Typical virus particles are spherical, with a diameter of 80nm to 120nm, and are enveloped. There are many radially arranged p...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 李明高秀杰陈华云何蕴韶
Owner DAAN GENE CO LTD
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