TY - JOUR
T1 - Nanopore sequencing reveals novel targets for detection and surveillance of human and avian influenza a viruses
AU - Yip, Cyril Chik Yan
AU - Chan, Wan Mui
AU - Ip, Jonathan Daniel
AU - Seng, Claudia Win May
AU - Leung, Kit Hang
AU - Poon, Rosana Wing Shan
AU - Ng, Anthony Chin Ki
AU - Wu, Wai Lan
AU - Zhao, Hanjun
AU - Chan, Kwok Hung
AU - Siu, Gilman Kit Hang
AU - Ng, Timothy Ting Leung
AU - Cheng, Vincent Chi Chung
AU - Kok, Kin Hang
AU - Yuen, Kwok Yung
AU - To, Kelvin Kai Wang
PY - 2020/4/23
Y1 - 2020/4/23
N2 - Accurate detection of influenza A virus (IAV) is crucial for patient management, infection control, and epidemiological surveillance. The World Health Organization and the Centers for Disease Control and Prevention have recommended using the M gene as the diagnostic gene target for reverse-transcription-PCR (RT-PCR). However, M gene RT-PCR has reduced sensitivity for recent IAV due to novel gene mutations. Here, we sought to identify novel diagnostic targets for the molecular detection of IAV using long-read third-generation sequencing. Direct nanopore sequencing from 18 nasopharyngeal specimens and one saliva specimen showed that the 5= and 3= ends of the PB2 gene and the entire NS gene were highly abundant. Primers selected for PB2 and NS genes were well matched with seasonal or avian IAV gene sequences. Our novel PB2 and NS gene real-time RT-PCR assays showed limits of detection similar to or lower than that of M gene RT-PCR and achieved 100% sensitivity and specificity in the detection of A(H1N1), A(H3N2), and A(H7N9) in nasopharyngeal and saliva specimens. For 10 patients with IAV detected by M gene RT-PCR conversion in sequentially collected specimens, NS and/or PB2 gene RT-PCR was positive in 2 (20%) of the initial specimens that were missed by M gene RT-PCR. In conclusion, we have shown that PB2 or NS gene RT-PCRs are suitable alternatives to the recommended M gene RT-PCR for diagnosis of IAV. Long-read nanopore sequencing facilitates the identification of novel diagnostic targets.
AB - Accurate detection of influenza A virus (IAV) is crucial for patient management, infection control, and epidemiological surveillance. The World Health Organization and the Centers for Disease Control and Prevention have recommended using the M gene as the diagnostic gene target for reverse-transcription-PCR (RT-PCR). However, M gene RT-PCR has reduced sensitivity for recent IAV due to novel gene mutations. Here, we sought to identify novel diagnostic targets for the molecular detection of IAV using long-read third-generation sequencing. Direct nanopore sequencing from 18 nasopharyngeal specimens and one saliva specimen showed that the 5= and 3= ends of the PB2 gene and the entire NS gene were highly abundant. Primers selected for PB2 and NS genes were well matched with seasonal or avian IAV gene sequences. Our novel PB2 and NS gene real-time RT-PCR assays showed limits of detection similar to or lower than that of M gene RT-PCR and achieved 100% sensitivity and specificity in the detection of A(H1N1), A(H3N2), and A(H7N9) in nasopharyngeal and saliva specimens. For 10 patients with IAV detected by M gene RT-PCR conversion in sequentially collected specimens, NS and/or PB2 gene RT-PCR was positive in 2 (20%) of the initial specimens that were missed by M gene RT-PCR. In conclusion, we have shown that PB2 or NS gene RT-PCRs are suitable alternatives to the recommended M gene RT-PCR for diagnosis of IAV. Long-read nanopore sequencing facilitates the identification of novel diagnostic targets.
KW - Diagnostic assay
KW - Influenza
KW - Next-generation sequencing
UR - http://www.scopus.com/inward/record.url?scp=85084025483&partnerID=8YFLogxK
U2 - 10.1128/JCM.02127-19
DO - 10.1128/JCM.02127-19
M3 - Journal article
C2 - 32132187
AN - SCOPUS:85084025483
SN - 0095-1137
VL - 58
JO - Journal of Clinical Microbiology
JF - Journal of Clinical Microbiology
IS - 5
M1 - e02127-19
ER -