Dual priming oligonucleotides for broad-range amplification of the bacterial 16S rRNA gene directly from human clinical specimens

Abstract

Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens.

Fig 1

Examples of various cross-reactivities using different primer sets for sample 3 (Table 2). The top chromatogram was obtained using the DPO primer set. It displays no ambiguity and represents the 16S rRNA gene of Kingella kingae only. The middle chromatogram shows amplification using the CLSI forward/Bosshard reverse primer combination. The area shown is identical to that of the top chromatogram. Coamplification of human DNA gives ambiguity up to bp 121 (red arrow). Thereafter, the 16S rRNA gene of K. kingae continues as a pure sequence. The bottom chromatogram shows amplification using the CLSI primer set. Terminal part of the reverse chromatogram is shown, and the length of the chromatogram is 667 bp (red circle), significantly longer than the expected 16S rRNA amplicon length of 500 bp. The chromatogram represents only human DNA, and the 16S rRNA gene of K. kingae could not be detected.