Evaluation of Single-Nucleotide Primer Extension for Detection and Typing of Phylogenetic Markers Used for Investigation of Microbial Communities

Abstract

Single-nucleotide primer extension (SNuPE) is an emerging tool for parallel detection of DNA sequences of different target microorganisms. The specificity and sensitivity of the SNuPE method were assessed by performing single and multiplex reactions using defined template mixtures of 16S rRNA gene PCR products obtained from pure bacterial cultures. The mismatch discrimination potential of primer extension was investigated by introducing different single and multiple primer-target mismatches. The type and position of the mismatch had significant effects on the specificity of the assay. While a 3-terminal mismatch has a considerable effect on the fidelity of the extension reaction, the internal mismatches influenced hybridization mostly by destabilizing the hybrid duplex. Thus, carefully choosing primer-mismatch positions should result in a high signal-to-noise ratio and prevent any nonspecific extension. Cyclic fluorescent labeling of the hybridized primers via extension also resulted in a significant increase in the detection sensitivity of the PCR. In multiplex reactions, the signal ratios detected after specific primer extension correlated with the original template ratios. In addition, reverse-transcribed 16S rRNA was successfully used as a nonamplified template to prove the applicability of SNuPE in a PCR-independent manner. In conclusion, this study demonstrates the great potential of SNuPE for simultaneous detection and typing of various nucleic acid sequences from both environmental and engineered samples.

Borrelia burgdorferi Not Confirmed in Human-Biting Amblyomma americanum Ticks from the Southeastern United States

“…However, a low level of polymerase readthrough can still occur (37), and because PCR results in exponential amplification of a template, even a low level of readthrough can become significant after sufficient PCR cycles. The ability of the Clark primers to amplify B. andersonii and B. americana has already been experimentally identified (26,28), but the B. lonestari result is unexpected.…”

Tetrachloroethene conversion to ethene by aDehalococcoides-containing enrichment culture from Bitterfeld

“…sequences obtained from chloroethene‐contaminated groundwater samples. Our recent study demonstrated the excellent specificity of the incorporated labeled nucleotide analogues in SNuPE (Nikolausz et al , 2009b), which is associated with a good dynamic range and the detection of a minority template (Nikolausz et al , 2008).…”

Quantitative analysis of classical and new putative periodontal pathogens in subgingival biofilm: a case–control study

“…Blasting of primer pairs for the five tested species against NCBI returned no hits other than the intended targets, meaning that at least one primer of each pair had at least two 3′ or six mismatches to unintended targets. For oral Synergistetes and TM7s, blasting returned unintended targets from some phyla; however, at least either primer had at least one middle and one 3′ mismatch or 3′ nt 1 mismatch to ensure no or, at least, much lower amplification efficiency . In addition, probes also had enough mismatches to compromise detection of non‐specific amplification (Table S1).…”