An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) was established for the precise evaluation of human intestinal microbiota. Group- and species-specific primer sets for Clostridium perfringens, Lactobacillus spp. (six subgroups and three species), Enterococcus spp., and Staphylococcus spp. targeting 16S rRNA gene sequences were newly developed for the ...

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our...

We evaluated a previously described quantitative real-time PCR (qPCR) for quantifying and differentiating Ureaplasma parvum and U. urealyticum. Because of nonspecific reactions with Staphylococcus aureus DNA in the U. parvum PCR, we developed a modified qPCR and designed new primers. These oligonucleotides eradicated cross-reactions, indicating higher specificity. The detection limits of the qP...

BACKGROUND Potentially pathogenic microorganisms can be detected by quantitative real-time polymerase chain reaction (qPCR) in sputum from patients with COPD, although how this technique relates to culture and clinical measures of disease is unclear. We used cross-sectional and longitudinal data to test the hypotheses that qPCR is a more sensitive measure of bacterial presence and is associated...

Quantitative real-time PCR is an important high-throughput method in the biomedical sciences. However, existing software has limitations in handling both relative and absolute quantification. We designed quantitative PCR data analysis and management system (qPCR-DAMS), a database tool based on Access 2003, to deal with such shortcomings by the addition of integrated mathematical procedures. qPC...

Human cytomegalovirus (CMV) has historically been the major infectious cause of morbidity and mortality among patients receiving hematopoietic cell or organ transplant. Standard care in a transplant setting involves frequent monitoring of CMV viral load over weeks to months to determine when antiviral treatment may be required. Quantitative PCR (qPCR) is the standard molecular diagnostic method...

Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily gene...

Background and objective The assessment of human epidermal growth factor receptor 2 (HER2) status has become of great importance in the diagnosis of breast cancer. The aim of this study was to investigate the diagnostic value of quantitative Polymerase Chain Reaction (qPCR) and Chromogenic In Situ Hybridization (CISH) to assess HER2 status of biopsy specimens. Methods To elucidate the status ...

The purpose of this manuscript is to describe a reliable approach to quantitative real-time polymerase chain reaction (qPCR) assay development and project management, which is currently embodied in the Excel 2003-based software program named "PREXCEL-Q" (P-Q) (formerly known as "FocusField2-6Gallup-qPCRSet-upTool-001," "FF2-6-001 qPCR set-up tool" or "Iowa State University Research Foundation [...

Real-time quantitative polymerase chain reaction (qPCR) is one of the most important methods for analyzing the expression patterns of target genes. However, successful qPCR experiments rely heavily on the use of high-quality primers. Various qPCR primer databases have been developed to address this issue, but these databases target only a few important organisms. Here, we developed the qPrimerD...