Evaluation of novel H1N1-specific primer-probe sets using commercial RT-PCR mixtures and a premixed reaction stored in a lyophilized format.

Timothy K W Cheung 1 Alex W H Chin 1 Kwok H Chan 1 Michael Schumaker 2 Polly W Y Mak 1 Horasis S Y Leung 1 Ada Wong 2 J S Malik Peiris 1, 3 Olga V Petrauskene 2 Leo L M Poon 1, *
* Corresponding author
1 Department of Microbiology [HKU]
2 R&D, Applied Biosystems
3 Centre de recherche Université de Hong-Kong-Pasteur
Abstract : 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.
Document type :
Journal articles
Complete list of metadatas

Cited literature [9 references]  Display  Hide  Download
https://hal-riip.archives-ouvertes.fr/pasteur-00588911
Contributor : Francois Kien <>
Submitted on : Wednesday, April 27, 2011 - 6:22:27 AM
Last modification on : Monday, October 8, 2018 - 5:44:06 PM
Long-term archiving on : Thursday, July 28, 2011 - 2:33:25 AM

File

Cheung_et_al_JVirolMethods_201...
Explicit agreement for this submission

Identifiers

Collections

RIIP | RIIP_HONGKONG

Citation

Timothy K W Cheung, Alex W H Chin, Kwok H Chan, Michael Schumaker, Polly W Y Mak, et al.. Evaluation of novel H1N1-specific primer-probe sets using commercial RT-PCR mixtures and a premixed reaction stored in a lyophilized format.. Journal of Virological Methods, Elsevier, 2010, 165 (2), pp.302-4. ⟨10.1016/j.jviromet.2010.01.024⟩. ⟨pasteur-00588911⟩

Export

BibTeX TEI DC DCterms EndNote

Share

Metrics

Record views

167

Files downloads

450

Contact

CCSD