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H7N9 subtype avian influenza virus detection kit, and application method thereof

A technology for detecting kits and influenza viruses, applied in the field of RT-PCR-ELISA kits, can solve the problems of time-consuming and laborious, expensive test instruments and reagents, and high technical requirements, and achieve easy operation, low cost, and high throughput. Effect

Inactive Publication Date: 2016-05-25
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gene sequencing is the gold standard for molecular diagnosis, but it is time-consuming and labor-intensive. It usually takes 24-48 hours. It requires high technical requirements for experimenters. At the same time, the test equipment and reagents are expensive, and it is difficult for general laboratories to carry out
Real-timetimePCR method and LAMP method have good specificity and sensitivity, but the former also relies on expensive instruments and reagents; while the latter is difficult to achieve multiple detection

Method used

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  • H7N9 subtype avian influenza virus detection kit, and application method thereof
  • H7N9 subtype avian influenza virus detection kit, and application method thereof
  • H7N9 subtype avian influenza virus detection kit, and application method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Embodiment 1: Target gene plasmid construction

[0017] Design specific primers for the M gene of the influenza A virus, the HA gene of the H7 subtype virus and the NA gene of the N9 subtype virus, and obtain 3 target gene fragments by RT-PCR method amplification, and recover and purify the respective T vectors Connect to obtain target gene positive plasmids, named pMD-M, Pmd-H7 and Pmd-N9 respectively.

Embodiment 2

[0018] Embodiment 2: PCR-ELISA method sensitivity

[0019] (1) PCR reaction: Dilute the target gene cloning plasmids pMD-M, Pmd-H7 and Pmd-N9 by 100 times respectively, and then dilute them into 10 times by 10 times. -2 -10 -9 A total of eight concentrations were used as templates, and the above biotin-labeled primers were used to perform PCR amplification reactions using the one-step kit from TaKaRa Company. The reaction conditions are: pre-denaturation at 94°C for 5 minutes, thermal cycle parameters are denaturation at 94°C for 30s, annealing at 56°C for 30s, extension at 72°C for 90s, and after 35 cycles, extension at 72°C for 7 minutes to obtain a digoxin-labeled PCR product. Blank control group. The obtained digoxin-labeled PCR products were determined by ELISA and 1.5% agarose gel electrophoresis to determine their respective sensitivities.

[0020] (2) ELISA detection: Take 5 μL of the amplified biotin-labeled PCR product, add it to 10 μL of 0.1mol / L NaOH solution, d...

Embodiment 3

[0024] Embodiment 3: PCR-ELISA method specificity

[0025] Using enterovirus, respiratory coronavirus, rhinovirus, respiratory syncytial virus, influenza A virus H1, H3, H5, H7 and H9 subtypes and influenza B nucleic acid as templates, PCR-ELISA experiments were performed with the above three sets of primers . Results The 4 sets of primers could not amplify the nucleic acid of enterovirus, respiratory coronavirus, rhinovirus, and respiratory syncytial virus, and there was no cross-reaction between different subtypes of influenza viruses, indicating that the established PCR-ELISA method had good specificity. sex. .

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Abstract

The invention belongs to the field of biotechnology, and relates to a H7N9 subtype avian influenza virus PCR-ELISA detection kit, and an application method thereof. The H7N9 subtype avian influenza virus PCR-ELISA detection kit is composed of a RT-PCR reaction system, an ELISA detection system, three target genes (influenza A virus M gene, H7N9 subtype virus HA gene, and H7N9 subtype virus NA) positive plasmids (pMD-M, Pmd-H7, and Pmd-N9), a negative control, and the specific primer probes of the three target genes. The three biotin-marked specific primers are subjected to specific amplification using RT-PCR, amplification products are denatured, hybridization of the denatured amplification products with digoxin-marked specific probes is carried out, an obtained hybridization product is delivered into a streptavidin-coated 96-well plate, and horse radish peroxidase-marked anti-digoxin antibody is added for ELISA detection. The invention also relates to applications of the three specific primer probes in environmental sample differential diagnosis and H7N9 subtype avian influenza virus isolated strain classification identification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an RT-PCR-ELISA kit for typing and detecting H7N9 subtype avian influenza virus nucleic acid and a use method thereof. Background technique [0002] Influenza A virus belongs to Orthomyxoviridae in classification, and its genome consists of 8 single-stranded negative-sense RNA segments. Influenza A virus has a wide range of hosts and can infect humans, birds and other mammals. According to the antigenicity of the two glycoproteins hemagglutinin (HA) and neuraminidase (NA) on the surface of the virus, influenza A viruses can be divided into different serotypes. So far, 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9) have been found in wild waterfowl, the natural storage hosts of influenza A virus. Under normal circumstances, bird flu viruses do not break through the species barrier to infect humans. However, since the H5N1 avian influenza virus infection incident in H...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70G01N33/53
CPCC12Q1/701C12Q2600/112C12Q2600/16G01N33/56983G01N2333/11
Inventor 祁贤章倩云宋勇春汤奋扬王慎骄余慧燕邓婓
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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