title: Development of a multiplex real-time PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A.
abstract: 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 (T(m)) discriminations. Three melting peaks generated simultaneously at temperatures 85.2+/-1.0, 81.9+/-0.9 and 78.7+/-0.9 degrees 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.
authors.affiliation: Institute of Biosciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Wee Theng
authors.initials: WT
authors.lastname: Ong
authors.forename: Abdul Rahman
authors.initials: AR
authors.lastname: Omar
authors.forename: Aini
authors.initials: A
authors.lastname: Ideris
authors.forename: Sharifah Syed
authors.initials: SS
authors.lastname: Hassan
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002645:Chickens; D003959:Diamines; D053127:Influenza A Virus, H9N2 Subtype / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis*; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I