Comparison of an automated DNA extraction and 16S rDNA real time PCR/sequencing diagnostic method using optimized reagents with culture during a 15-month study using specimens from sterile body sites

Abstract

Background: 16S rDNA-PCR for the identification of a bacterial species is an established method. However, the DNA extraction reagents as well as the PCR reagents may contain residual bacterial DNA, which consequently generates false-positive PCR results. Additionally, previously used methods are frequently time-consuming. Here, we describe the results obtained with a new technology that uses DNA-free reagents for automated DNA extraction and subsequent real time PCR using sterile clinical specimens.

Results: In total, we compared 803 clinical specimens using real time PCR and culturing. The clinical specimens were mainly of orthopedic origin received at our diagnostic laboratory. In 595 (74.1%) samples, the results were concordant negative, and in 102 (12.7%) the results were concordant positive. A total of 170 (21.2%) clinical specimens were PCR-positive, of which 62 (36.5% from PCR positive, 7.7% in total) gave an additional benefit to the patient since only the PCR result was positive. Many of these 62 positive specimens were strongly positive based on crossingpoint values (54% < Cp 30), and these 62 positive clinical specimens were diagnosed as medically relevant as well. Thirty-eight (4.2%) clinical specimens were culture-positive (25 of them were only enrichment culture positive) but PCR-negative, mainly for S. epidermidis, S. aureus and C. acnes. The turnaround times for negative specimens were 4 hours (automated DNA extraction and real time PCR) and 1 working day for positive specimens (including Sanger sequencing). Melting-curve analysis of SYBR Green-PCR enables the differentiation of specific and unspecific PCR products. Using Ripseq, even mixed infections of 2 bacterial species could be resolved.

Conclusions: For endocarditis cases, the added benefit of PCR is obvious. The crucial innovations of the technology enable timely reporting of explicit reliable results for adequate treatment of patients. Clinical specimens with truly PCR-positive but culture-negative results represent an additional benefit for patients. Very few results at the detection limit still have to be critically examined.

Keywords: 16S rDNA real time PCR; Additional benefit; DNA-free reagents; Evolution of methods; Mixed sequences.

Fig. 1

Amplification curves (A top) and corresponding melting curve (B bottom). Seven PCR-positive clinical specimens from one patient (numbers correspond to anonymized patient identity) as well as 3 controls (P1, P2 as positive control and negative control) are shown. The yellow curve was clearly PCR-positive but negative by culture. The arrow indicates a positive result as well, the result of which from Sanger sequencing was congruent with other clinical specimens of the same patient. The negative control was flat above 84 °C (marked by black line), which indicates the absence of specific PCR products. Peaks below 84 °C indicate nonspecific amplification (e.g., primer dimers). Different Tm values (above 84 °C) are caused by different sequences (in this case, S. epidermidis has lower Tm, and B. subtilis (present in P1 and P2) has higher Tm

Fig. 2

Example of a mixed chromatogram. RiSeq results show the presence of Citrobacter koseri and coagulase-negative Staphylococcus (Case 2,111,805,855). Culture confirmed only Citrobacter koseri. The material was a biopsy of the malleolus, and the patient was treated with augmentin. Because Sanger sequencing mainly detects concentration differences of approximately less than 1:10 [13], this must be a relevant mixed infection due to a Cp of 29.9 of the amplification. In addition, the same patient had a second clinical specimen with the same mixed sequence. Chromatogram shows stretches of sequence diversity and sequence identity of the two strains. Example is after the 15 months study period