Method for diagnosing avian influenza virus by means of RT-PCR (reverse transcription-polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) and kit using method

A bird flu virus and diagnostic method technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve problems such as blanks, achieve the effects of eliminating pollution, facilitating automation, and strong specificity

Inactive Publication Date: 2012-06-13
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above method is still a blank in the detection of AIV.

Method used

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  • Method for diagnosing avian influenza virus by means of RT-PCR (reverse transcription-polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) and kit using method
  • Method for diagnosing avian influenza virus by means of RT-PCR (reverse transcription-polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) and kit using method
  • Method for diagnosing avian influenza virus by means of RT-PCR (reverse transcription-polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) and kit using method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Extraction of genomic RNA in the sample Take ground clinical pathological tissue or throat swab, anal swab sample 300 μL, add 800 μL TRIzol, and extract RNA according to the operation steps of the TRIzol kit.

[0029] Design specific upstream primer M1 and downstream primer M2 according to the conserved M gene of influenza virus, wherein the sequence of the upstream primer M1 used is shown in Seq ID No: 1, wherein a biotin label is used: 5'BIOTIN-TTCTAACCGAGGTCGAAAC-3' , the sequence of the downstream primer M2 is shown in Seq ID No: 2, wherein the digoxigenin label is used: 5'DIG-AAGCGTCTACGCTGCAGTCC-3', all of which are directly synthesized using the existing technology.

[0030] The RT-PCR reaction was carried out with the PrimerScript One Step RT-PCR kit. The reaction system was buffer 25 μL, Enzyme Mix 2 μL, upstream primer M1 (20 μM) 1 μL, downstream primer M2 (20 μM) 1 μL, template 6 μL, plus DEPC water to 50 μL. The RT-PCR reaction program was: 50°C for 30 min,...

Embodiment 2

[0043] Sensitivity of RT-PCR ELISA to Determination of EID 50 RT-PCR detection was carried out after the RNA template was extracted from the virus and serially diluted. A part of the RT-PCR product was detected by 1.5% agarose gel electrophoresis, and a part was used for 1:100, 1:200, 1:400, 1:800, 1 : 1600, 1: 3200, 1: 6400 dilution, and further use the ELISA method established in the present invention for detection. The result is as figure 2 As shown, it was found that RT-PCR ELISA method can detect 0.001EID 50 RT-PCR ELISA detection found that the positive product was diluted to 1:6400 and could still be detected, which was more than 100 times more sensitive than agarose gel electrophoresis (detected by agarose gel electrophoresis, AIV positive The product was undetectable when diluted to 1:40).

Embodiment 3

[0045] For the specificity of RT-PCR ELISA, use the TRIzol kit to extract the RNA of AIV H9N2, H5N1, H4N6, NDV, and IBV according to the instructions, and use them as templates for specificity detection using the established RT-PCR ELISA method. The test results are shown in the table below. The OD450 values ​​of the first three serotypes of AIV virus are all greater than 0.65, and the OD450 values ​​of NDV and IBV viruses are all lower than the negative control value, indicating that this method has strong specificity and no cross-reaction with some viruses, and can be used for AIV differential diagnosis.

[0046] Table. Specificity test of RT-PCR ELISA method

[0047]

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Abstract

The invention provides a special prime designed according to conservative M genes of influenza virus. By the aid of the principle of specific binding of biotin and streptavidin, biotin-labeled PCR (polymerase chain reaction) products are bound on a microtiter plate, anti-digoxin peroxidase is added, and finally TMB (tetramethylbenzidine) color development solution is added, so that positive products can develop colors. On the basis, a special kit is successfully prepared. The method combines PCR with a high-sensitivity digoxin detection system, the sensitivity of the method is 100 times higher than that of a conventional agarose gel electrophoresis detection method, and the method is high in specificity and overcomes difficulty in detecting AIV (avian influenza virus) of a low content in a tissue sample. Compared with a virus isolation method, the method has the advantages that coincidence rate can reach more than 95%, pollution of chemical agents (such as ethidium bromide) is eliminated, automation is benefited, the method is suitable for detecting a large number of samples, and a new approach is provided for early diagnosis of avian influenza and molecular epidemiological investigation.

Description

technical field [0001] The invention relates to the field of animal virology, in particular to diagnostic technology in the field of biotechnology, and more specifically to a high-throughput rapid diagnosis of avian influenza virus using RT-PCR ELISA technology and a kit prepared by applying the diagnostic method. Background technique [0002] Avian influenza is an acute contact infectious disease caused by type A influenza virus, which can infect many animals including humans, poultry, pigs, and horses. Avian influenza not only brings catastrophic damage to the livestock industry, but also poses a serious threat to public health. Avian influenza virus (AIV) mutates rapidly. Although it has not yet acquired the ability to effectively spread among humans, its increasingly frequent human infection events indicate that AIV virus is undergoing a complex process of mutation to adapt to the human body. Once again, the human A flu outbreak is already brewing. Long-term epidemiolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N21/33
Inventor 杨少华张秀美黄艳艳胡北侠许传田颜世敢张琳
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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