What is Direct PCR?
Direct PCR (dPCR) is a technique of DNA amplification instantly from an animal or plant tissue pattern with out performing DNA isolation and purification steps. This approach vastly reduces experimental time, and price in genotyping and high-volume initiatives. It additionally offers a greater possibility when confronted with the challenges of amplifying a really small amount of pattern the place the purification step might doubtlessly result in pattern loss.
In some ways, dPCR works like typical PCR. Both methods rely upon template DNA, primers and a grasp combine or PCR reagents, and each use a thermal cycler for amplification. The major distinction between dPCR and typical PCR is the tailor-made buffers utilized in dPCR. The modified parts of dPCR allow strong amplification regardless of the presence of PCR inhibitors typically present in crude samples.
ALS SARS-CoV-2 RT-PCR 4G has been developed and produced fully at ALS in Portugal and is designed primarily for export. This new equipment is ready to detect all identified SARS-CoV-2 variants, together with British, Brazilian and South African variants, with out cross-reacting with different identified associated viruses or frequent respiratory pathogens. The alternative of the genomic areas used as targets and the sequences of the primers and hydrolysis probes had been primarily based on WHO, CDC and DGS suggestions.
The ALS SARS-CoV-2 RT-PCR 4G equipment is an in vitro diagnostic medical gadget (CE-IVD) that makes use of real-time PCR nucleic acid amplification know-how for the detection of SARS-CoV-2 in medical samples from the higher respiratory tract of people who might or is probably not suspected of getting COVID-19.
The equipment makes use of the RT-PCR approach, combining amplification of sure areas of the viral genome with its detection by particular hydrolysis probes. The SARS-CoV-2 genome goal areas are extremely conserved and correspond to 2 particular genes: ORF polyprotein (ORF1ab gene) and nucleocapsid phosphoprotein (N gene). The equipment may detect the gene that codifies the structural protein of the envelope (gene E) particular to the Sarbecovirus subgenus.
Direct PCR Reagent
Advantages
- Rapid amplification: The amplification price of the polymerase is 6kb/min. 1kb fragment might be amplified inside 25min.
- Long fragment amplification: For plasmid, λ DNA and different straightforward templates, the polymerase can successfully amplify > 20kb. For genome, the polymerase can successfully amplify > 8kb. And for cDNA, the polymerase can successfully amplify > 8kb.
- High specificity: With sizzling begin know-how, the polymerase is 100% inactive under 50°C, and can solely be restored by heating at 95°C for 5min.
- High tolerance to impurities: Samples of entire blood, serum, cultured cells and urine might be instantly amplified with out prior DNA purification.
Unit Definition
One unit is outlined as the quantity of enzyme required to catalyze the incorporation of 10 nmol dNTP into an acid insoluble substance inside 30 min at 74°C.
| Description | Code | Specification | Category | Technology |
|---|---|---|---|---|
| ALS SARS-CoV-2 RT-PCR Test for surfaces | ALS-B01-Adv | RUO | Food /Water Virology Detection | Real-Time PCR |
| ALS SARS-CoV-2 RT-PCR Test for Wastewater | ALS-B02-Adv | RUO | Food /Water Virology Detection | Real-Time PCR |
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