Current methods of estimating the concentrations of microorganisms are based on the most probable number (MPN) methods. The culture based MPN method is a slow, and laborious that requires two or more days for completion. Commercially available molecular methods generates presence and absence results for the detection assays. Target bacterial concentration in the test samples can be estimated by these detection assays can be extrapolated using external standard curve. As each sample varies in composition, microbial load, and presence of natural inhibitor (i.e., sample matrix effect for each sample type), a separate standard curve is needed for each food product.
Pathogen detection assays rely on the enrichment of samples for increasing the number of target pathogens in the biological sample (i.e., food, meat, blood, urine, tissue, swabs). DNA isolated from these enrichments is used for amplification of specific DNA sequences in a PCR reaction. These commercially available PCR assays generate only the presence or absence of results and are not geared for estimating initial pathogen levels in the test samples. Knowing initial target organism concentration in the biological sample is critical for food industries and clinicians.
What is needed is a method which not only specifically detects the presence of a target organism, but assay can estimate the initial number of organisms present in the test sample.
This invention describes a novel detection approach which specifically detects the target and estimates the initial target load in the test samples. The method incorporates a novel PCR amplification approach in which each DNA samples is amplified using a combination of multiple primer set with varying amplification efficiencies. A high assay specificity is achieved by the incorporation of a highly specific dual-labeled probe. The primer-pair with higher amplification efficiency detects samples with lower concentration. Whereas the primer-pair with lower amplification efficiency only detects samples with higher concentration. In this way, by knowing which primer-pair applied and which did not, target pathogen concentration in the test sample can be estimated.
