Description
Microbial DNA qPCR Arrays are collections of assays to identify and profile microbial species, virulence factor genes or antibiotic resistance genes from a variety of sample types. Species identification assays target bacterial 16S rRNA genes and fungal ribosomal RNA genes, and each array includes controls for host DNA, presence of bacterial DNA and success of the PCR reaction. Microbial qPCR Mastermix is also included in the array kit. The simplicity of the product format and operating procedure allow routine, reliable identification or profiling of microbial species and genes from samples in any research laboratory with access to a real-time PCR instrument. What is the difference between microbial identification and profiling? Identification is determining the microbe’s presence or absence in your sample which requires running a No Template Control during your analysis. Profiling is determining the microbe’s relative expression in two or more experimental conditions for which you will need to run a reference sample and a normalizer (provided by QIAGEN).
Linearity and dynamic range Microbial DNA qPCR Assays display linear amplification across a range from 10 to 10 6 copies of DNA template (see figure, Linearity and sensitivity of Microbial DNA qPCR Assays). 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 gene detection Microbial DNA qPCR Assays reveals high sensitivity, and The LLOQ for antibiotic resistance gene detection 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), and is verifiable by sequencing methods (see figure, Specificity of the Antibiotic Resistance Genes Microbial DNA qPCR Array is confirmed by pyrosequencing). Reproducibility Microbial DNA qPCR Assays are highly reproducible, both in intra- and inter-individual variability tests (see figure, Microbial DNA qPCR Arrays generate reliably reproducible results).
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. Microbial DNA qPCR Arrays are 96- or 384-well plates comprising related assays; for example, microbial species associated with respiratory infections, or microbes that are studied for biodefense. Controls are integrated on each plate to test for the presence of fungal DNA, bacterial DNA, host genomic DNA, and success of the PCR reaction, ensuring confidence in the results.
The Microbial DNA qPCR Array procedure is simple and can be carried out in any laboratory with a real-time PCR instrument. DNA is isolated using the QIAamp kit that is suited for the sample type, and then mixed with the appropriate Microbial qPCR Mastermix. The mixture is aliquotted across the array plate, and real-time PCR is performed to obtain the raw C T values for each assay. The complimentary data analysis software is then used to profile genes or species in the sample.
