Subject(s)
Heart Neoplasms , Lymphoma, Large B-Cell, Diffuse , Abdominal Neoplasms/diagnostic imaging , Abdominal Neoplasms/pathology , Abdominal Neoplasms/therapy , Adult , Chest Pain/etiology , Dyspnea/etiology , Female , Heart Neoplasms/complications , Heart Neoplasms/diagnostic imaging , Heart Neoplasms/pathology , Heart Neoplasms/therapy , Humans , Kidney Neoplasms/diagnostic imaging , Kidney Neoplasms/pathology , Kidney Neoplasms/therapy , Lymphoma, Large B-Cell, Diffuse/complications , Lymphoma, Large B-Cell, Diffuse/diagnostic imaging , Lymphoma, Large B-Cell, Diffuse/pathology , Lymphoma, Large B-Cell, Diffuse/therapy , Magnetic Resonance Imaging , Pelvic Neoplasms/diagnostic imaging , Pelvic Neoplasms/pathology , Pelvic Neoplasms/therapy , Tomography, X-Ray ComputedABSTRACT
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.