Avoiding false positives when using universal primers for bacterial identification
In PCR experiments, amplification in the “no template control” (NTC) before the ~38th cycle with probe-based assays (or ~34th cycle when using intercalating dyes) is a sign of false positives and/or contamination. This sidebar specifically addresses false positives that occur during bacterial research when primers and probes are designed to detect common sequences, such as ribosomal RNA (rRNA).
Am I amplifying DNA from my reagents or consumables?
The exponential PCR process can amplify a single copy of DNA to detectable levels. Thus, it is important to consider the pervasiveness of the chosen primer or probe sequences. There are some DNA sequences, such as bacterial genes for 16S or 23S rRNA, that can be found almost anywhere. While not commonly used for RT-PCR or gene expression studies, rRNA sequences are often used for characterizing environmental species diversity, such as bacterial strains within the intestine or in salt water marshes. For such applications, genomic DNA, rather than RNA or cDNA, is used as the sample.
Choose a well-conserved, species-specific gene or novel sequence
In 16S rRNA experiments, a better approach may be to choose a unique sequence from the hypervariable region of 16S rRNA. Alternatively, a conserved, species-specific gene may be used. Blocking oligos and/or clamps can also be used to block amplification of common sequences and enhance the amplification of a rare sequence.
As new sequences are deposited into the NCBI database, it is essential to perform a BLAST search of every primer and probe sequence used in PCR to check for specificity and cross reactivity.
Bacterial ribosomal sequences can be amplified readily from virtually any bacterial source, including bacteria-derived Taq polymerases used to amplify them, as well as nonsterile tubes and pipette tips. Hence, if a positive NTC is observed, testing with different master mixes will help rule out the master mix as the cause of contamination.