[One-step multiplex RT-PCR for rapid screening of type A, B and novel A (H1N1) influenza viruses].
OBJECTIVE
To developed a multiplex RT-PCR assay for simultaneous screening of type A, B and novel A (H1N1) influenza viruses.


METHODS
Two pairs of universal primers in were designed for amplifying the M gene and NS gene of type A and B influenza viruses, respectively. A pair of specific primers of HA gene was designed to detect novel A (H1N1) influenza virus. A one-step method was used to establish the multiplex RT-PCR system. A blinded experiment was carried out to validate the accuracy of this assay in comparison with the results of real-time fluorescence RT-PCR. The clinical practicability and efficacy of this assay was also evaluated.


RESULTS
The RT-PCR products were analyzed using agarose gel electrophoresis, which yielded distinct bands of the target fragments without non-specific reactions, suggesting the high efficiency and specificity of the multiplex RT-PCR. Blinded study of 50 samples demonstrated a concordance rate of 100%.


CONCLUSIONS
This multiplex RT-PCR assay allows one-step simultaneous detection of type A, B and novel A (H1N1) influenza viruses rapidly and accurately, and provides a valuable low-cost screening technique for influenza epidemic monitoring and early diagnosis.
Mo
Qiu-hua
QH
Zhuhai Entry-Exit Inspection and Quarantine Bureau, Zhuhai 519020, China.
Yang
Cui-lan
CL
Lin
Ji-can
JC
Tan
Hua
H
Tu
Cheng-ning
CN
Ye
Li-qing
LQ
Liu
Zhi-ming
ZM
DU
Jian
J
Sun
Hong
H
Yang
Ze
Z
D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000302:isolation & purification*; D009981:Influenza B virus / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D014763:Viral Matrix Proteins / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics
D014763:Viral Matrix Proteins; D017361:Viral Nonstructural Proteins