| Avian influenza is an acute respiratory disease of avians caused by the avian influenza virus,which can infect a variety of poultry and wild birds.Since the discovery of the avian influenza virus,epidemics have been reported every year,and it is currently prevalent in many countries and regions around the world.Among them,every outbreak of highly pathogenic avian influenza of H5 and H7 subtypes will cause huge economic losses to the poultry industry.Both H5 and H7 subtypes of avian influenza viruses have been reported to infect humans,posing a huge threat to human life and health,and seriously endangering public health safety.Therefore,the establishment of a high-throughput rapid detection and identification method for avian influenza viruses is of great significance for the monitoring and early warning of avian influenza epidemics and the protection of public health safety.In order to detect avian influenza viruses more quickly and accurately,especially the H5 and H7 subtypes of avian influenza viruses,this study is based on the existing triple fluorescent RT-PCR detection methods for avian influenza viruses(universal,H5,H7),according to the production The demand is further optimized to form a complete product of avian influenza virus(universal,H5,H7)triple real-time RT-PCR detection kit products,use the prepared quality control products to conduct quality research on the kit,and use the kit in a large number of clinical trials Sample testing,evaluation of testing results.In order to study the quality of avian influenza virus(universal,H5,H7)triple real-time RT-PCR detection kit,this study selected 14 different subtypes of avian influenza virus strains,1 strain of Newcastle disease virus,and 1 strain of chicken infectivity Bronchitis virus strain,1 avian infectious bursal bursal virus strain,1 avian leukemia virus strain,1 avian reticuloendothelial hyperplasia virus strain and 2 SPF chicken tissue samples were used as quality control materials for the kit quality Control product preparation and identification.First of all,the preparation process of the quality control product is studied to determine the most suitable combination of inactivation agent and lyoprotectant.The results showed that the combination of ?-propiolactone and sucrose skim milk for inactivation and freeze-drying had the best effect.The freeze-dried product was stored at 4? on the 7th,30th,and 90th day,and the Ct value did not change much;All strains were cultured,inactivated,freeze-dried,and identified with published specific fluorescence detection primers;1 strain of H5 subtype avian influenza virus and 1 strain of H7 subtype avian influenza virus were selected as sensitive quality control materials and calibrated,The result of detection with the corresponding primer probe was positive,and no non-specific amplification occurred.The detection sensitivity of the universal detection primer probe for avian influenza virus quality control product H7-2 has a strong positive Ct value of 25.296±0.981,and the H5 subtype avian influenza The strong positive Ct value of the virus was 25.439±1.161,and the strong positive Ct value of H7 subtype avian influenza virus was 27.658±1.020;the remaining strains were used as specific quality control materials,and the corresponding primer probes showed positive results,and did not appear Non-specific amplification;finally,the serum,homogenate supernatant and swabs of 2 SPF chickens were used as negative controls,and the identification results were all negative.The prepared sensitive quality control products,pecific quality control products and negative quality control products can meet the needs of quality research of avian influenza virus(universal,H5,H7)triple real-time RT-PCR detection kits.In order to test whether the quality of the avian influenza virus(Universal,H5,H7)triple real-time RT-PCR detection kit meets the standards,the following studies have been done:(1)Sensitivity study:3 batches of avian influenza virus(general,H5,H7)The triple real-time RT-PCR detection kit is performed in accordance with the kit instructions for sensitive quality control products,specific quality control products,negative control products,45 H5 subtype avian influenza virus positive clinical samples and 45 H7 subtype poultry Influenza virus-positive clinical samples are tested,and parallel tests are performed with OIE recommended or published in the paper.The test conformity rate with the standard method is 100%;the sensitive quality control products H5-2 and H7-2 are performed 10 times with PBS Stepwise dilution,select 101,102,103,104,105,106,107,108 fold dilution samples to extract nucleic acid,and use avian influenza virus(universal,H5,H7)triple real-time RT-PCR detection kit for detection,each Do 20 replicates of the dilution to determine the sample dilution when the positive detection rate in the test result is just greater than or equal to 95%.This dilution is the minimum detection amount of the corresponding subtype,and the corresponding Ct value is the positive criterion.The results showed that the minimum detection limit of the M gene primer probe to the sensitive quality control H7-2 was 107 times dilution,and the Ct value was 37.614±0.250.The minimum detection limit of the H5 gene primer probe to the sensitive quality control H5-2 was 106.Double dilution,Ct value is 37.736±0.210,the lowest detection limit fordetecting H7 gene primer probe to sensitive quality control H7-2 is 107 times dilution,Ct value is 37.833±0.312;(2)Specificity study:use 3 batches respectively The avian influenza virus(universal,H5,H7)triple real-time RT-PCR detection kit detects sensitive quality control materials,specific quality control materials,negative control materials and 60 negative clinical samples,and none of the 3 batches of kits have been tested.Abnormal amplification curve appears;(3)Repeatability study:3 batches of avian influenza virus(universal,H5,H7)triple real-time RT-PCR detection kits are used to detect sensitive quality control products,and sensitive quality control products need to be tested The 10-fold serial dilution was made to the lowest detection limit,and each dilution was made in 3 replicates.The average intra-assay coefficient of variation was 1.31%,the inter-assay average coefficient of variation was 1.84%,and the intra-assay inter-assay coefficient of variation was less than 3%.Avian influenza virus(universal,H5,H7)triple real-time RT-PCR detection kit has good sensitivity,specificity and reproducibilityIn order to evaluate the clinical applicability of the avian influenza virus(universal,H5,H7)fluorescent RT-PCR detection kit under practical conditions,the test kit used 1920 swab samples from live poultry trading markets in 4 provinces to be tested.The results showed that there were 775 positive samples of avian influenza virus,of which 61 were H5 subtype avian influenza virus and 6 were H7 subtype avian influenza virus.This shows that the kit can be used for clinical detection of avian influenza virus,which can not only detect avian influenza virus,but also distinguish H5 and H7 subtypes of highly pathogenic avian influenza viruses,which can meet the clinical testing needs. |
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