Typing detection kit for human seasonal influenza viruses and application method thereof
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A technology for seasonal influenza and detection kits, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of poor PCR sensitivity, expensive dependence, unsuitable detection, etc., and achieve easy operation, low cost, High throughput effect
Inactive Publication Date: 2016-01-06
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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Problems solved by technology
Real-timetimePCR method and LAMP method have good specificity and sensitivity, but the former relies on expensive instruments and reagents; while the latter is difficult to achieve multiple detection
In contrast, ordinary PCR is less sensitive and requires agarose electrophoresis to determine the results, which is not suitable for the detection of large-scale specimens
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Embodiment 1
[0016] Embodiment 1: Target gene plasmid construction
[0017] Design specific primers for the M gene of influenza A virus, the HA gene of H1 subtype virus, the HA gene of H3 subtype virus and the NS gene of B virus respectively, and obtain four target gene fragments by PCR amplification. Connect with the T vector to obtain target gene positive plasmids, named as Pa-m, Ph1-ha, ph3-ha and pb-ns respectively.
Embodiment 2
[0018] Embodiment 2: PCR-ELISA method sensitivity
[0019] (1) PCR reaction: Dilute the target gene cloning plasmids Pa-m, Ph1-ha, ph3-ha and pb-ns by 100 times, and then dilute to 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 were: denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 90 s, and after 35 cycles, extension at 72°C for 7 min to obtain biotin-labeled PCR products, and set a blank control group.
[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, denature at room temperature for 10 minutes, and then add 10nmol / L digoxin-labeled specific probe (Dilute with hybridization solution containing 300mmol / L NaCl, 100mmol / LTris-HCl pH6.5, 10mmol / LEDTA, 0.1% Twee-20), incubate at...
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 4 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 biotechnologies, and particularly relates to a multi-PCR-ELISA detection kit for human seasonal influenza viruses and an application method thereof. The detection kit disclosed by the invention is composed of a RT-PCR reaction system, an ELISA detection system, four target-gene (an influenza-A-virus M gene, a H1 subtype virus HA gene, a H3 subtype virus HA gene, and a B type virus NS gene) positive plasmids (Pa-m, Ph1-ha, Ph3-ha and Pb-ns), a negative quality control specimen and four target-gene specific primer probes. Specific amplification is performed by using specific primers labeled by using four sets of biotins through RT-PCR, an amplified product after being denatured is hybridized with a specific probe labeled by using digoxin, a hybridized product is enveloped with a streptavidin-enveloped 96-hole micro-plate, and an anti-digoxin antibody labeled by using horse radish peroxidase is added for carrying out detection through an ELISA method, so that a rapid, sensitive and specific typing detection kit for seasonal influenza viruses such as H1 and H3 subtypes and hepatitis B viruses is established. The invention relates to the application of four sets of specific primer probes in the clinical differential diagnosis of influenza virus infection and the typing authentication of influenza virus isolates.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a human seasonal influenza virus multiplex PCR-ELISA detection kit and a use method thereof. Background technique [0002] Seasonal influenza is an important global respiratory infectious disease caused by influenza virus. It is prevalent in tropical regions all the year round, while it mainly occurs in winter in temperate regions and has certain seasonal epidemic characteristics. Influenza viruses are divided into three influenza genera of the family Orthomyxoviridae: influenza A (A), B (B) and C (C) influenza viruses. The clinical symptoms of influenza C virus are mild, and the public health significance is not significant. Influenza A and B viruses are the main pathogens of seasonal influenza epidemics every year, can infect all people, and can cause severe disease and even death to some special groups (such as pregnant women, the elderly and patients with underlying ...
Claims
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Application Information
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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70
CPCC12Q1/701C12Q1/686C12Q2600/112
Inventor 祁贤宋勇春章倩云汤奋扬鲍昌俊王慎骄邓婓余慧燕
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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