Description
Microbial DNA qPCR Assays are a mix of two PCR primers (10 µM each) and one 5′-hydrolysis probe (5 µM) that enables quantitative real-time PCR for 100 reactions. Microbial DNA qPCR Assays are designed using a proprietary and experimentally verified algorithm, providing uniform PCR efficiency and amplification conditions. Each Microbial DNA qPCR Assay undergoes rigorous experimental verification to ensure high PCR efficiency, and this high efficiency is guaranteed when the assays are used with Microbial qPCR Mastermixes and Microbial DNA-Free Water (sold separately). What is the difference between microbial identification and profiling? Identification is determining the microbe’s presence or absence in your sample which requires you to run a No Template Control during your analysis. Profiling is determining the microbe’s relative expression in two or more experimental conditions and you will need to run a reference sample and a normalizer (provided by QIAGEN).
Lower limit of quantification (LLOQ) The LLOQ is the lowest concentration of template that still falls into the linear range of the standard curve (see figure, Limit of detection versus lower limit of quantification). Across all Microbial DNA qPCR Assays, 93% have an LLOQ of <100 gene copies (see figure, The LLOQ for all Microbial DNA qPCR Assays reveals high sensitivity). 92% of microbial identification assays meet this LLOQ, as do 95% of virulence factor gene assays and 97% of antibiotic resistance gene assays (see figures, The LLOQ for microbial identification Microbial DNA qPCR Assays reveals high sensitivity, The LLOQ for virulence factor genes Microbial DNA qPCR Assays reveals high sensitivity, and The LLOQ for antibiotic resistance genes Microbial DNA qPCR Assays reveals high sensitivity). Specificity Each Microbial DNA qPCR Assay is stringently tested to ensure that it detects only one target species or gene (see figure, Microbial DNA qPCR Assays are highly specific). For assays that do detect more than one target, a list of detected targets and in silico predictions is included on the product sheet. This specificity is maintained even when samples have high species complexity, such as in stool, sputum, and plaque (see figure, Microbial DNA qPCR Assays display high sensitivity even in complex metagenomic samples).
Microbial DNA qPCR Assays are designed to detect bacterial 16S rRNA gene and fungal ribosomal rRNA gene sequences for species identification, as well as detecting virulence factor genes and antibiotic resistance genes using PCR amplification primers and hydrolysis-probe detection.
The procedure for Microbial DNA qPCR Assays is simple, and can be performed in any laboratory with a real-time PCR instrument. DNA is isolated from the sample using the appropriate QIAamp kit, and then PCR reactions are set up using the appropriate Supplemental Microbial qPCR Mastermix for the PCR instrument. Four separate PCR reactions should be prepared for each sample, including a Positive PCR Control, a No Template Control, and the Microbial DNA Positive Control, as well as the Microbial DNA qPCR Assay. Real-time PCR is performed and data is analyzed using web-based data analysis software or Excel templates.
