Direct PCR: What sample to use and how to set up reactions?

A practical guide to running the DBdirect^™ PCR & qPCR mixes straight from crude sample

DBdirect™ PCR mixes combine an inhibitor-resistant DB SuperSens Taq DNA Polymerase (DB-1328) with an enhancer-stabilized, single-component formulation, so DNA is amplified directly from cells, viruses and biological matrices without a separate extraction or purification step. Two handling routes cover the various range of sample types: add the sample directly to the mix or perform a fast pre-treatment using Lysis Buffer A (DB-1281). For many sample types both routes work well - the better choice often depends on the number of cells in the reaction, because high cell loads can inhibit the PCR. This application note summarizes each input type to the recommended route and gives the basic reaction setup.

Download this application note in PDF.

Rules of thumb

● Start around 1,000 cells/particles per reaction to check whether an organism is direct-PCR compatible.

● What counts is the final number of cells/particles in the reaction, not the sample volume; high loads can inhibit the reaction.

● Default concentration of primers 400 nM, probe 200 nM - if not, optimizing.

● We recommend to validate for your organism with a dilution series (e.g. 100 - 1,000 - 10,000 - 100,000 per reaction).

Workflow - Three steps, no extraction

Step 1️⃣: Prepare Sample

Suspend cells or virus, or take crude matrix (serum, saliva, media). No pre-treatment needed for the direct route.

Step 2️⃣: Lysis Buffer A (Optional)

For yeast, tough Gram-positives, human cells, enveloped viruses and any high cell load: 20 min at 95 °C in DB Lysis Buffer A (DB-1281), then add up to 5 µL lysate. Skip for Gram-negatives, non-enveloped viruses and low cell numbers.

Step 3️⃣: Add Mix and Run PCR

Combine with the DBdirect™ mix and cycle. The UDG (optional) + 95 °C start also lyses many inputs and prevents carry-over contamination.

Features of DBdirect^™ PCR Mixes

● Inhibitor resistance - DB SuperSens Taq tolerates crude-sample and matrix inhibitors that stall standard polymerases.

● Ultrasensitive - detection down to ~1 copy of DNA; single-cell detection possible.

● Built-in lysis - the 95 °C hot-start releases DNA from many inputs’ matrices without a separate step.

● One tube - single-component 2X mix; just add primers/probe(s) and sample.

Conclusion

For cells with thick cell walls (e.g. yeast, Gram-positive bacteria), human cells, and enveloped viruses, pre-incubation with (DB 1281) DB Lysis Buffer A is recommended.

For Gram-negative bacteria (e.g. E. coli) and non-enveloped viruses (e.g. ADV), direct addition to the PCR mix is effective across a wide range of sample concentrations.

The PCR mix method is in principle compatible with all tested cell types and viruses; however, inhibition was observed for some microorganisms at higher concentrations.