TY - JOUR
T1 - Development of a multiplex real-time PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A
AU - Ong, Wee Theng
AU - Omar, Abdul Rahman
AU - Ideris, Aini
AU - Hassan, Sharifah Syed
N1 - Funding Information:
This research was supported by Grant number 01-02-04-009 BTK/ER/38 from Ministry of Science, Technology and Innovation, Government of Malaysia. We thank Mohd. Rajik from Virology Laboratory, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia for kindly providing H9N2 antisera. We would also like to thank Prof. Dr. Malik Peiris from Department of Microbiology, University of Hong Kong, China for generously providing five H9N2 isolates of different lineages from Hong Kong. Lastly, we thank L.L. Kong and S.W. Tan for their technical assistances in optimizing the real-time PCR procedures.
Copyright:
Copyright 2009 Elsevier B.V., All rights reserved.
PY - 2007/9
Y1 - 2007/9
N2 - Avian influenza viruses are pathogens of economical and public health concerns. However, infections caused by low pathogenic avian influenza particularly H9N2 subtype are not associated with clear clinical features. Hence, rapid detection and subtyping of the virus will enable immediate measures to be implemented for preventing widespread transmission. This study highlights the development of a multiplex real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) assay using SYBR Green 1 chemistry for universal detection of avian influenza viruses and specific subtyping of H9N2 isolates based on melting temperatures (Tm) discriminations. Three melting peaks generated simultaneously at temperatures 85.2 ± 1.0, 81.9 ± 0.9 and 78.7 ± 0.9 °C represent NP, H9 and N2 gene products, respectively. The RRT-PCR assay was about 10-100-fold more sensitive when compared to the conventional RT-PCR method using reference H9N2 isolate. In addition, the RRT-PCR assay was 100% sensitive as well as 92% specific according to the standard virus isolation method in detecting experimentally infected specific-pathogen-free (SPF) chickens.
AB - Avian influenza viruses are pathogens of economical and public health concerns. However, infections caused by low pathogenic avian influenza particularly H9N2 subtype are not associated with clear clinical features. Hence, rapid detection and subtyping of the virus will enable immediate measures to be implemented for preventing widespread transmission. This study highlights the development of a multiplex real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) assay using SYBR Green 1 chemistry for universal detection of avian influenza viruses and specific subtyping of H9N2 isolates based on melting temperatures (Tm) discriminations. Three melting peaks generated simultaneously at temperatures 85.2 ± 1.0, 81.9 ± 0.9 and 78.7 ± 0.9 °C represent NP, H9 and N2 gene products, respectively. The RRT-PCR assay was about 10-100-fold more sensitive when compared to the conventional RT-PCR method using reference H9N2 isolate. In addition, the RRT-PCR assay was 100% sensitive as well as 92% specific according to the standard virus isolation method in detecting experimentally infected specific-pathogen-free (SPF) chickens.
KW - Avian influenza virus
KW - Melting temperature (T)
KW - Multiplex
KW - Rapid detection and subtyping
KW - Sensitive
KW - Specific
KW - SYBR Green 1 based real-time reverse-transcriptase polymerase chain reaction (RRT-PCR)
UR - http://www.scopus.com/inward/record.url?scp=34547112040&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2007.03.019
DO - 10.1016/j.jviromet.2007.03.019
M3 - Article
C2 - 17512062
AN - SCOPUS:34547112040
VL - 144
SP - 57
EP - 64
JO - Journal of Virological Methods
JF - Journal of Virological Methods
SN - 0166-0934
IS - 1-2
ER -