Development of multiplex rt-PCR assays for rapid detection and subtyping of influenza type A viruses from clinical specimens.
We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus negative specimens. Furthermore, the assays could detect and subtype up to 105 dilution of each of the reference viruses that had an original infectivity titer of 106 EID50/ml. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.
Chang
Hee Kyoung
HK
College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Korea.
Park
Jeung Hyun
JH
Song
Min-Suk
MS
Oh
Taek-Kyu
TK
Kim
Seok-Young
SY
Kim
Chul-Jung
CJ
Kim
Hyunggee
H
Sung
Moon-Hee
MH
Han
Heon-Seok
HS
Hahn
Youn-Soo
YS
Choi
Young-Ki
YK
D000818:Animals; D001717:Birds / Q000821:virology; D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000821:virology; D007723:Korea; D009439:Neuraminidase / Q000235:genetics*; D009976:Orthomyxoviridae Infections / Q000662:veterinary / Q000821:virology; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000821:virology
D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral; D009439:Neuraminidase