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Based on the 16S rRNA gene data we constructed quantitative PCR's (Q-PCR) for the seven most dominating bacterial groups.
Eighteen of these nasopharyngeal specimens were randomly selected (double blind) and included in this study and proven positive by specific diagnostic PCR's for either human rhinovirus (HRV), respiratory syncytial virus (RSV), human coronavirus OC43 (HCoV-OC43), HCoV-NL63, Influenzavirus A, Influenzavirus B, parainfluenzavirus 3 (PIV3) or adenovirus.
We compared rRNA-FISH hybridisation results with two broad-range fungal rRNA gene PCR's on FFPE tissue specimens from patients with proven IFI.
However, comprehensive data on sensitivity, specificity and performance of the multiplex PCR compared to the single target PCR's is limited for most published respiratory multiplex real time PCR assays.
Multiplex PCR's with unlabeled primer pairs were conducted using the standard PCR procedure (see above) and were run on the QIAxcel system (see above).
All PCR's exhibited high amplification efficiency (>90%) and the specificity of PCR products was confirmed by sequencing.
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Typically in a single experiment the Tm curves from the same individual are exactly superimposable upon one another The donors used in this study were also used repeatedly throughout testing, with both separate RNA extractions and multiple RT-PCR's from the same individual extract being employed.
Since antiretroviral registrational trials utilize the TLOVR endpoint, RT-PCR's inferior reliability near the LLOQ, especially when using PPTs, may both inflate virologic failure rates and confound efforts to compare failure rates between trials [12] [14].
To account for RT-PCR's slightly lower LLOQ (down to pVL of 50) vs. bDNA (LLOQ down of 75), for analysis purposes, RT-PCR results between 50 and 75 copies/ml were considered undetectable.As secondary endpoints, we compared censored mean pVLs and mean coefficients of variation (CV).
All QRT-PCR's were performed in triplicate.
Long-PCR's ranging from nad4 to 16S were not successful.
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