Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Cascade Invasive Reaction and Nanoparticle Hybridization for Subtyping of Influenza A Virus.
Considering the fatal human victims and economic loss caused by influenza virus infection every year, methodologies for rapid and on-site detection of influenza viruses are urgently needed. LAMP is the most commonly used nucleic acid isothermal amplification technology suitable for on-site use. However, for multiplex LAMP, differentiation of the amplicons derived from multiple targets is still challengeable currently. Here we developed a multiplex RT-LAMP assay for simultaneous amplification of three prominent subtypes of influenza viruses (A/H5, A/H7 and 2009A/H1). The amplicons were further identified by cascade invasive reaction and nanoparticle hybridization in separate target-specific detection tubes (referred to as mRT-LAMP-IRNH). The analytic sensitivities of the assay are 10 copies of RNA for all the three HA subtypes, and the specificity reached 100%. Clinical specimen analysis showed this assay had a combined sensitivity and specificity of 98.1% and 100%, respectively. Overall, the mRT-LAMP-IRNH assay can be used as a cost-saving method that utilizes a simple instrument to detect A/H5, A/H7, and 2009A/H1 influenza viruses, especially in resource-limited settings.
Department of Pathogen Biology, Key Laboratory of Pathogen Biology of Jiangsu Province, Nanjing Medical University, Nanjing, China.|Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
Ying
Y
Chi
Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
Yiyue
Y
Ge
Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
Kangchen
K
Zhao
Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing, China.
Bingjie
B
Zou
Department of Biomedical Engineering, Nanjing medical university, Nanjing, China.
Bin
B
Liu
Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
Xian
X
Qi
Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
Qian
Q
Bian
Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
Zhiyang
Z
Shi
Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
Fengcai
F
Zhu
Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
Minghao
M
Zhou
Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
Lunbiao
L
Cui
Department of Pathogen Biology, Key Laboratory of Pathogen Biology of Jiangsu Province, Nanjing Medical University, Nanjing, China.
Chuan
C
Su
D006046:Gold; D006801:Humans; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics*; D007251:Influenza, Human / Q000175:diagnosis / Q000821:virology*; D053758:Nanoparticles; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral; D015203:Reproducibility of Results; D048348:Reverse Transcription; D012680:Sensitivity and Specificity
D012367:RNA, Viral; D006046:Gold