A Multiplex Asymmetric Reverse Transcription-PCR Assay Combined With an Electrochemical DNA Sensor for Simultaneously Detecting and Subtyping Influenza A Viruses
机构：[1]Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Key Discipline of Pediatrics (Capital Medical University), Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China医技科室科研平台职能科室呼吸疾病研究室临床流行病与循证医学中心临床研究中心儿科研究所首都医科大学附属北京儿童医院[2]DAAN Gene Co., Ltd., Sun Yat-sen University, Guangzhou, China[3]The Medicine and Biological Engineering Technology Research Center of the Ministry of Health, Guangzhou, China[4]National Clinical Research Center for Respiratory Diseases, Department of Respiratory, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China临床科室医技科室职能科室呼吸科临床流行病与循证医学中心临床研究中心首都医科大学附属北京儿童医院
The reliable and rapid detection of viral pathogens that cause respiratory infections provide physicians several advantages in treating patients and managing outbreaks. The Luminex respiratory virus panel (RVP) assay has been shown to be comparable to or superior to culture/direct fluorescent-antibody assays (DFAs) and nucleic acid tests that are used to diagnose respiratory viral infections. We developed a multiplex asymmetric reverse transcription (RT)-PCR assay that can simultaneously differentiate all influenza A virus epidemic subtypes. The amplified products were hybridized with an electrochemical DNA sensor, and the results were automatically acquired. The limits of detection (LoDs) of both the Luminex RVP assay and the multiplex RT-PCRelectrochemical DNA sensor were 10(1) TCID50 for H1N1 virus and 10(2) TCID50 for H3N2 virus. The specificity assessment of the multiplex RT-PCR-electrochemical DNA sensor showed no cross-reactivity among different influenza A subtypes or with other non-influenza respiratory viruses. In total, 3098 respiratory tract specimens collected from padiatric patients diagnosed with pneumonia were tested. More than half (43, 53.75%) of the specimens positive for influenza A viruses could not be further subtyped using the Luminex RVP assay. Among the remaining 15 specimens that were not subtyped, not degraded, and in sufficient amounts for the multiplex RT-PCR-electrochemical DNA sensor test, all (100%) were H3N2 positive. Therefore, the sensitivity of the Luminex RVP assay for influenza A virus was 46.25%, whereas the sensitivity of the multiplex RT-PCR-electrochemical DNA sensor for the clinical H1N1 and H3N2 specimens was 100%. The sensitivities of the multiplex RT-PCR-electrochemical DNA sensor for the avian H5N1, H5N6, H9N2, and H10N8 viruses were 100%, whereas that for H7N9 virus was 85.19%. We conclude that the multiplex RT-PCR-electrochemical DNA sensor is a reliable method for the rapid and accurate detection of highly variable influenza A viruses in respiratory infections with greater detection sensitivity than that of the Luminex xTAG assay. The high mutation rate of influenza A viruses, particularly H3N2 during the 2014 to 2016 epidemic seasons, has a strong impact on diagnosis. A study involving more positive specimens from all influenza A virus epidemic subtypes is required to fully assess the performance of the assay.
基金：
National Major S&T Research Projects for the Control and Prevention of Major Infectious Diseases in China [2017ZX10103004-004, 2018ZX10305409-001-004]; Capital Health Research and Development of Special Projects [2016-2-1142]; Beijing Municipal Administration of Hospitals' Youth Program [QML20171201]
第一作者机构：[1]Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Key Discipline of Pediatrics (Capital Medical University), Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China
共同第一作者：
通讯作者：
通讯机构：[1]Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Key Discipline of Pediatrics (Capital Medical University), Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China
推荐引用方式(GB/T 7714)：
Xu Lili,Jiang Xiwen,Zhu Yun,et al.A Multiplex Asymmetric Reverse Transcription-PCR Assay Combined With an Electrochemical DNA Sensor for Simultaneously Detecting and Subtyping Influenza A Viruses[J].FRONTIERS IN MICROBIOLOGY.2018,9(JUN):-.doi:10.3389/fmicb.2018.01405.
APA：
Xu, Lili,Jiang, Xiwen,Zhu, Yun,Duan, Yali,Huang, Taosheng...&Xie, Zhengde.(2018).A Multiplex Asymmetric Reverse Transcription-PCR Assay Combined With an Electrochemical DNA Sensor for Simultaneously Detecting and Subtyping Influenza A Viruses.FRONTIERS IN MICROBIOLOGY,9,(JUN)
MLA：
Xu, Lili,et al."A Multiplex Asymmetric Reverse Transcription-PCR Assay Combined With an Electrochemical DNA Sensor for Simultaneously Detecting and Subtyping Influenza A Viruses".FRONTIERS IN MICROBIOLOGY 9..JUN(2018):-
生物学