摘要
目的开发用于甲型流感病毒快速分型的Taqman低密度芯片(Taqman low density array,TLDA),可以同时开展甲型流感病毒通用、H1-H16及N1-N9亚型高通量快速鉴定,适用于流感样病例及禽流感标本中甲型流感病毒的快速分型鉴定。方法设计甲型流感病毒通用、H1-16亚型、N1-9亚型的引物探针,并将其预先定制在TLDA反应芯片上;优化TLDA退火温度;用多种亚型的甲型流感病毒核酸进行10倍梯度稀释,结合微滴式数字PCR定量方法评价TLDA检测灵敏度;用乙型流感病毒、腺病毒、呼吸道合胞等其他多种常见的呼吸道病毒验证该TLDA检测特异性,同时选用多种已经明确分型的甲型流感病毒评价该TLDA的特异性和有效性;采集16例流感样病例标本及24例禽流感外环境标本用该芯片开展检测。结果该TLDA的最佳退火温度为60℃;针对甲型流感病毒H1N1、H3N2、H5N6、H7N9和H9N2,其检测灵敏度为1.52~8.00拷贝/μl;能特异性检测甲型流感病毒,与其他常见呼吸道病毒无交叉反应,并能精准鉴定多种甲型流感病毒的亚型;应用该方法检测16例流感样病例标本和24例禽流感外环境监测标本,均可分型。结论基于TLDA技术建立的甲型流感病毒高通量快速分型检测方法具有较好的灵敏度和特异性,适用于流感样病例和禽流感标本的病原快速检测,特别是对目前用常规商业化试剂尚无法鉴定的新型甲型流感病毒能够进行快速的亚型筛查与鉴定。
Objective Using the Taqman low-density array(TLDA)to develop a detection method which can rapidly type influenza A virus and the subtypes of H1-H16 and N1-N9,which is suitable for rapid typing and identification of influenza A in influenza like cases and avian influenza samples.Methods The primers and probes of influenza A,H1-16 subtypes,N1-9 subtypes were designed and pre-customized on the TLDA reaction chip;Optimize the TLDA annealing temperature;The sensitivity of TLDA was evaluated by 10 fold gradient dilution of influenza A virus nucleic acid of various subtypes and the digital PCR;The specificity of the TLDA was verified by influenza B,adenovirus,respiratory syncytial virus and other respiratory viruses.At the same time,a variety of influenza A viruses that have been clearly typed were selected to evaluate the specificity and effectiveness of TLDA;The TLDA was used to detect 16 samples of influenza like cases and 24 samples of external environment of avian influenza.Results The best annealing temperature of TLDA was 60℃,the detection sensitivity of TLDA was between 1.52 and 8.00 copies/μl for H1N1,H3N2,H5N6,H7N9 and H9N2,there was no specific cross reaction with influenza B,avian influenza,adenovirus,respiratory syncytial virus and other respiratory viruses.A total of 12 influenza positive samples and 24 samples from environmental surveillance of avian influenza were detected by TLDA,all of the result of samples were correct in typing.Conclusions The high-throughput detection method of influenza A virus based on TLDA has good sensitivity and specificity,and the result of clinical samples show that the TLDA is suitable for rapid detection of influenza like cases and avian influenza samples,especially for rapid subtype screening and identification of new influenza A virus that cannot be identified by conventional commercial kits.
作者
楼秀玉
赵翔
王欣莹
陈寅
孙逸
李榛
余昭
茅海燕
Lou Xiuyu;Zhao Xiang;Wang Xinyin;Chen Yin;Sun Yi;Li Zhen;Yu Zhao;Mao Haiyan(Microbiological Laboratory of Zhejiang Provincial Center for Disease Control and Prevention,Hangzhou 310051,China;National Institute of Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2022年第3期311-316,共6页
Chinese Journal of Experimental and Clinical Virology
作者简介
通信作者:茅海燕,Email:hymao@cdc.zj.cn,电话:0571-87115199。
