Establishment and application of high throughput rapid typing method for influenza A virus based on Taqman low-density array
10.3760/cma.j.cn112866-20211201-00205
- VernacularTitle:基于TLDA技术的甲型流感病毒高通量快速分型方法的建立及应用
- Author:
Xiuyu LOU
1
;
Xiang ZHAO
;
Xinyin WANG
;
Yin CHEN
;
Yi SUN
;
Zhen LI
;
Zhao YU
;
Haiyan MAO
Author Information
1. 浙江省疾病预防控制中心微生物检验所，杭州 310051
- Keywords:
Taqman low-density array;
Influenza A virus;
High-throughput detection
- From:
Chinese Journal of Experimental and Clinical Virology
2022;36(3):311-316
- CountryChina
- Language:Chinese
-
Abstract:
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.
