Library of prefabricated locked nucleic acid hydrolysis probes facilitates rapid development of reverse-transcription quantitative real-time PCR assays for detection of novel influenza A/H1N1/09 virus.

BACKGROUND: The emergence of a novel pandemic human strain of influenza A (H1N1/09) has clearly demonstrated the need for flexible tools enabling the rapid development of new diagnostic methods. METHODS: We designed a set of reverse-transcription quantitative real-time PCR (RT-qPCR) assays based on the Universal ProbeLibrary (UPL)--a collection of 165 presynthesized, fluorescence-labeled locked nucleic acid (LNA) hydrolysis probes--specifically to detect the novel influenza A virus. We evaluated candidate primer/UPL-probe pairs with 28 novel influenza A/H1N1/09 patient samples of European and Mexican origin. RESULTS: Of 14 assays in the hemagglutinin (HA) and neuraminidase (NA) genes, 12 detected viral nucleic acids from diluted patient samples without need for further optimization. We characterized the diagnostic specificity of the 2 best-performing assays with a set of samples comprising various influenza virus strains of human and animal origin that showed no cross-reactivity. The diagnostic sensitivity of these 2 primer/probe combinations was in the range of 100-1000 genomic copies/mL. In comparison to a reference assay recommended by the German health authorities, the analytical sensitivities and specificities of the assays were equivalent. CONCLUSIONS: Facing the emergence of novel influenza A/H1N1/09, we were able to develop, within 2 days, a set of sensitive and specific RT-qPCR assays for the laboratory diagnosis of suspected cases. H1N1/09 served as a model to show the feasibility of the UPL approach for the expedited development of new diagnostic assays to detect emerging pathogens.

Comparison of an internally controlled, large-volume LightCycler assay for detection of Mycobacterium tuberculosis in clinical samples with the COBAS AMPLICOR assay.

We present a sensitive and specific assay for reliable and flexible detection of members of the Mycobacterium tuberculosis complex (MTBC) in clinical samples. This real-time PCR assay, which uses the LightCycler 2.0 instrument and 100-mul glass capillaries, can provide a result within 1 h after DNA extraction. The primers amplify a 206-bp fragment of the MTBC 16S rRNA gene. The sensor hybridization probe targets a region highly specific to members of the MTBC. The assay also includes a novel type of internal control that monitors the function of the reaction components and can detect potential inhibitors. Template DNA was extracted by the same procedure used for the COBAS AMPLICOR M. tuberculosis assay, so the LightCycler assay could be directly compared to the COBAS AMPLICOR assay. The LightCycler assay was evaluated with 146 clinical samples of various types. Very good agreement (100% sensitivity, 98.6% specificity) could be shown between the LightCycler and COBAS AMPLICOR assays. Specificity was checked with a panel of nontuberculous mycobacteria, as well as a large panel of bacterial and fungal organisms.