1-Step vs 2-Step PCR for Pathogen
DNA Detection from Whole Blood
Pathogen detection by PCR analysis has become an area of significant interest and product development. Commercial systems exist that can detect panels of pathogens using both one-step and two-step PCR approaches but utilize enclosed systems (a sealed consumable). While enclosed systems are ideal for point-of-care applications, they are typically expensive, inflexible, and do not utilize existing laboratory equipment. Reagent kits exist that are compatible with standard laboratory equipment, but all use single-step PCR to avoid the added steps and contamination risks associated with two-step PCR. Since the Alluvia system can enable the development of two-step PCR pathogen detection reagent kits for standard laboratory equipment, we investigated if the added PCR step will significantly improve PCR results....
1-Step vs 2-Step PCR for
DNA Methylation Detection
DNA methylation is a form of epigenetics that is widely studied in cancer research and developmental biology. The predominant form of DNA methylation is a methyl group added to the 5 position of the Cytosine base in CpG dinucleotides, which are often clustered in transcriptional regulatory regions....Previous reports have shown that utilizing two steps of PCR including a preamplification with outer non-MSP primers followed by a second PCR step using nested MSP primers can be more robust than a single MSP step (Palmisano et al., Cancer Res. 60:5954, 2000), particularly when the samples derive from FFPE tissue samples (Draht et al., Clinical Epigenetics 8:44, 2016). In this study we compared the sensitivity and specificity of 1-step MSP vs. 2-step MSP....
Alluvia System Fluidic and
The Alluvia system facilitates 2-step nucleic acid amplification (i.e. multiplex-nested PCR) while eliminating the risk of amplification product contamination. The advantages of 2-step amplifications are improved sensitivity (as the sample can be preamplified in a multiplex format thereby reducing total sample usage) and specificity (the 2nd step PCR primers can be "nested" relative to the 1st step primers yielding 4 primer specificity). A major problem with 2-step amplification is the necessary handling of the 1st step products, which can lead to contamination of the laboratory environment and false positive results in subsequent reactions. The Alluvia System eliminates this contamination risk by continuously containing the amplification products in plastic consumables. The attractive design and intuitive operation make the system easy to use while reducing user error compared to the pipetting steps it replaces.....