Which are the most probable reasons for a false positive result and how to address the issue?
If you are using highly conserved 16S rRNA genes (one of the most widely used PCR targets for detection of a broad range of organisms) as a target for DNA amplification by PCR, you probably encountered an unexpected PCR product in the non-template control amplified using your primers.
The most probable reason for false positive result is contamination of reagents, especially Taq DNA polymerase, with DNA which may be derived from a bacterial source. In bacterial cultures, nucleic acids (including ribosomal DNA sequences) are co-purified during enzyme production. Therefore, an enzyme may contain a source of contaminating DNA as a result of its manufacture and incomplete purification.
Reagent contamination with bacterial-derived DNA is normally minor. However, it represents a great risk for false positive signals due to 106- to 107-fold amplification of a single copy of template DNA in PCR reaction. Even though the amplicon sizes of contaminating DNA are normally different from the expected target, and thus non-specific amplification can be suspected, using qPCR no information about the amplicon size is given and may lead to incorrect interpretation of results.
In order to diminish the amount of contaminating DNA and thus overcome the problem of false positive signals of non-template controls or negative samples, several strategies for regent decontamination based on physical, chemical, and enzymatic means have been proposed. DNA decontamination is usually applied directly to DNA polymerase enzyme or PCR mix and includes treatments with ultrafiltration, UV irradiation or enzymes such as DNase I, restriction enzyme Sau 3A1 and Exonuclease III (Exo III). However, most of these approaches are not entirely effective and may result in inhibition of the PCR reaction compromising PCR efficiency or detection limits.
As these approaches can be time-consuming and can lead to introduction of other sources of contamination, maybe a better choice for your laboratory is to buy commercially available DNA-free PCR reagents such as MTP Taq DNA Polymerase (SIGMA-ALDRICH), MolTaq 16S and Mastermix 16S (MOLZYM). Approaches which eliminate the need for DNA decontamination are also known.
By Urska Cepin, PhD, Research and Development Assistant, BioSistemika LLC