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Multiple-fluorescence reverse transcription-polymerase chain reaction (RT-PCR) kit for rapid detection of novel H7N9 subtype avian influenza virus and detection method thereof

A RT-PCR and avian influenza virus technology, applied in the field of molecular detection, can solve problems such as virus mutation, fragment recombination, and immune preparations cannot play a protective role

Active Publication Date: 2013-09-04
珠海出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, it has been proved by experiments that when different subtypes of avian influenza viruses infect a cell at the same time, the viral genomes of different subtypes will undergo fragment recombination, resulting in virus mutation.
According to reports by Chen et al., the new H7N9 subtype avian influenza virus is a new type of reassortant virus, and its internal gene comes from the H9N2 subtype avian influenza virus that exists in China. reasons for protection

Method used

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  • Multiple-fluorescence reverse transcription-polymerase chain reaction (RT-PCR) kit for rapid detection of novel H7N9 subtype avian influenza virus and detection method thereof
  • Multiple-fluorescence reverse transcription-polymerase chain reaction (RT-PCR) kit for rapid detection of novel H7N9 subtype avian influenza virus and detection method thereof
  • Multiple-fluorescence reverse transcription-polymerase chain reaction (RT-PCR) kit for rapid detection of novel H7N9 subtype avian influenza virus and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Establishment of multiple fluorescent RT-PCR detection system for novel H7N9 subtype avian influenza virus

[0032] 1. Materials

[0033] Antigens: H1N2, H3N2, H5N1, H5N9, H6N2, H7N3 and H9N2 avian influenza antigens were purchased from Harbin Veterinary Research Institute. The novel H7N9AIV nucleic acid is cDNA after reverse transcription of RNA, from the Chinese Center for Disease Control and Prevention.

[0034] Reagents and instruments: DNA / RNA magnetic bead extraction kit, purchased from Shenzhen Yirui Biotechnology Co., Ltd.; One Step RT-PCR kit, DNA gel recovery kit, plasmid extraction kit, DL2000DNA Mark, pMD18-T Purchased from Bao Bioengineering (Dalian) Co., Ltd.; Ultraviolet Spectrophotometer NanoDrop ND-1000, Thermo Fisher Scientific, USA; ABI7500Fast Real-Time PCR System, Applied Biosystems, USA.

[0035] 2. Experimental methods and results

[0036] 2.1 Primers and probes

[0037] Download the hemagglutinin antigen (HA) gene sequence (GenBank:...

Embodiment 2

[0049] Example 2 Specificity Experiment

[0050] Take H1N2, H3N2, H5N1, H5N9, H6N2, H7N3, H9N2 avian influenza virus RNA and the cDNA of the new H7N9 as templates respectively, identify with the multiple fluorescent RT-PCR system established in Example 1, and verify the specificity of the established method , see the test results image 3 . Depend on image 3 It can be seen that only the H7 and N9 of the H7N9 sample amplified two standard S-shaped curves. In the other samples, only the H7 detection of the H7N3 sample showed specific amplification, and other reference viruses had no fluorescence growth, indicating that the established method can detect All new and old H7 subtype viruses and new N9 subtype viruses have the ability to distinguish the original N9 subtype viruses.

Embodiment 3

[0051] Example 3 Sensitivity experiment

[0052] 1×10 respectively 0 ~1×10 7 copies / μL of eight concentration gradients of positive standard plasmids were used as templates for multiplex fluorescence RT-PCR. Add 5 μL of reaction template to 25 μL of RT-PCR system. Depend on Figure 4 It can be seen that the lowest concentration at which both H7 and N9 have obvious S-shaped amplification curves is 10 copies, that is, the minimum detection limit of this method for both H7 and N9 is in the order of 10 copies.

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Abstract

The invention discloses a multiple-fluorescence reverse transcription-polymerase chain reaction (RT-PCR) kit for rapid detection of a novel H7N9 subtype avian influenza virus and a detection method thereof. The kit comprises a group of specific primers and probes for detecting all H7 subtype avian influenza virus and a group of specific primers and probes for detecting the novel N9 subtype avian influenza virus. The two groups of primers are designed for gene sequences of hemagglutinin antigen (HA) and neuraminidase antigen (NA) of the H7N9 subtype avian influenza virus. Thus, in the actual detection and application process, leak detection on the H7 subtype avian influenza virus is avoided, and the novel N9 subtype avian influenza virus can be detected and judged. A test proves that the kit has favorable specificity and sensitivity and plays an important role in early detection of the H7N9 subtype virus.

Description

technical field [0001] The invention belongs to the field of molecular detection, and in particular relates to a multiplex fluorescence RT-PCR kit and method for rapidly detecting novel H7N9 subtype avian influenza virus. Background technique [0002] At the end of March 2013, outbreaks of avian influenza virus infecting people occurred in Shanghai and Anhui successively. It was confirmed by the National Avian Influenza Reference Laboratory that the avian influenza was caused by the H7N9 new influenza virus discovered for the first time in the world. As of 16:00 on May 1, 2013, a total of 127 confirmed cases have been reported in the mainland of the country, of which 26 people died, and the mortality rate reached 20.47%. Among the subtypes of avian influenza virus, its mortality rate is second only to H5N1 (2003.1 ~ 2013.5). , 30 / 45, 66.67%). Due to the sudden nature and high mortality of this subtype virus and the lack of effective preventive and treatment measures, it has...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 罗宝正莫秋华薄清如徐海聂杨泽李儒曙廖秀云沙才华
Owner 珠海出入境检验检疫局检验检疫技术中心
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