Evaluation of novel H1N1-specific primer-probe sets using commercial RT-PCR mixtures and a premixed reaction stored in a lyophilized format.
The recent emergence of a novel H1N1 influenza A virus in humans caused the first influenza pandemic of this century. Many clinical diagnostic laboratories are overwhelmed by the testing demands related to the infection. Three novel H1N1-specific primer-probe sets reported during the early phase of the pandemic were tested in three commercial real-time RT-PCR mixtures. The amplification efficiencies and detection limits of these assays were determined. A ready-to-use premixed RT-PCR stored in a lyophilized format was developed. The detection limits of the studied assays were highly variable, ranging from 1.68E-01 to 1.68E-05 TCID(50) per reaction. The detection limit of the lyophilized reaction mixture was found to be 1.68E-05 TCID(50) per reaction, but the amplification efficiency of the assay was lower than those deduced from the other assays. All respiratory samples from infected patients and all control nasopharyngeal aspirates were positive and negative, respectively, in the newly developed assay. The results highlighted that, to enhance the sensitivity of an assay, it is essential to evaluate a primer-probe set with different commercial RT-PCR assays. This study also demonstrated the feasibility of using lyophilized reaction mixtures for the molecular diagnosis of novel H1N1.
Cheung
Timothy K W
TK
Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China.
Chin
Alex W H
AW
Chan
Kwok H
KH
Schumaker
Michael
M
Mak
Polly W Y
PW
Leung
Horasis S Y
HS
Wong
Ada
A
Peiris
J S Malik
JS
Petrauskene
Olga V
OV
Poon
Leo L M
LL
D017931:DNA Primers; D005612:Freeze Drying; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis*; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D011933:Reagent Kits, Diagnostic