Direct PCR is a reaction that directly uses animal or plant tissues for amplification without nucleic acid extraction. In many ways, direct PCR works like regular PCR

The main difference is the custom buffer used in direct PCR, the sample can be directly subjected to the PCR reaction without nucleic acid extraction, but there are corresponding requirements for the tolerance of the enzymes and the compatibility of the buffer involved in the direct PCR reaction.

Although there are more or less PCR inhibitors in common samples, direct PCR can still achieve reliable amplification under the action of enzymes and buffers. The traditional PCR reaction requires high-quality nucleic acid as a template, which can inhibit the smooth progress of PCR reaction if the template contains proteins and other impurities. Direct PCR is currently one of the more popular technologies in the field of molecular diagnostics.

01 Direct PCR was originally used for animal and plant

The earliest application of direct PCR is in the field of animals and plants, such as the blood, tissue and hair of the rat, cat, chicken, rabbit, sheep, cattle, etc., plant leaves and seeds, etc., used to study genotyping, transgenic, plasmid detection, Gene knockout analysis, DNA source identification, species identification, SNP analysis and other fields.

These fields have some common features, that is, the target gene content is relatively high and nucleic acid extraction is troublesome, so direct PCR can not only save time and have a small impact on the results, but also save cost.

Direct PCR used for pathogen detection is a matter of recent years, some PCR reagent manufacturers have made a lot of efforts in this direction when making innovation. Especially in this COVID-19 epidemic, many such detection products have appeared on the market, such as SARS-CoV-2 Nucleic Acid Detection Kit (Multiplex PCR Fluorescent Probe Method) researched and developed by Foregene, which uses Real-time RT PCR technology (rRT-PCR) for qualitative detection of SARS-CoV-2 nucleic acids in human nasopharyngeal or oropharyngeal swab samples. 

Foregene is one of the companies using Direct PCR technology, for the detection of normal ORF1ab, N, E, and variant lineages nucleic acids in human nasopharyngeal or oropharyngeal swab samples such as SARS-CoV-2 B.1.1.7 lineage (UK), B.1.351 lineage (ZA), B.1.617 lineage (IND) and P.1 lineage (BR).

02  Reagents needed for direct PCR

Sample Lysate

The sample lysate can be configured by yourself or purchased. The difference in the composition of different brands of lysate will make the lysing ability different, and then the lysing time will be slightly different. For example, for the preparation of animal tissue samples, 30 minutes or overnight lysis is generally recommended, and the lysis solution for viruses ranges from 3-10 minutes.

PCR master mix

It is recommended to use hot-start DNA polymerase to enhance specific amplification and increase the amplification ability. The core of direct PCR is a highly tolerant polymerase.

Eliminate or inhibit components in the sample that affect DNA amplification

After the sample is processed with the lysate, proteins, lipids and other cell debris will be released, these substances will inhibit the PCR reaction. Therefore, direct PCR requires the addition of corresponding removal or inhibitors to reduce the influence of these factors.

03  A collection of five knowledge points of direct PCR

First, Direct PCR technology is a direct PCR technology for various biological samples. Under this technical condition, there is no need to separate and extract the nucleic acid, directly use the tissue sample as the object, and add the target gene primers are to perform the PCR reaction.

Second, Direct PCR technology is not only a traditional DNA template amplification technology, but also includes RNA template reverse transcription PCR.

Third, Direct PCR technology not only directly performs routine qualitative PCR reactions on tissue samples, but also includes Real-Time qPCR reactions, which requires the reaction system to have strong anti-background fluorescence interference capability and endogenous fluorescence quenchers the antagonistic ability.

Fourth, the samples targeted by the Direct PCR technology only need the release of nucleic acid templates, and do not remove proteins, polysaccharides, salt ions, etc. that interfere with the PCR reaction. Which requires the nucleic acid polymerase and PCR Mix in the reaction system to have excellent resistance and adaptability to ensure enzyme activity and replication accuracy under complex conditions.

Fifth, the tissue sample targeted by Direct PCR technology without any nucleic acid enrichment treatment and the amount of template is very small, which requires the reaction system to have extremely high sensitivity and amplification efficiency.