Contamination of Commercial PCR Master Mix with DNA from Coxiella burnetii

Abstract

Contamination of an in-house diagnostic real-time PCR for Q fever was traced back to a commercially obtained PCR Master Mix. It was established that this Master Mix contained DNA from Coxiella burnetii, probably as a result of the use of compounds of animal origin such as bovine serum albumin.As of 2007 and up to the present time, there has been a large ongoing outbreak of Q fever in Netherlands that is unprecedented both in the number of affected individuals and in its duration (5). Real-time PCR has rapidly been implemented in many diagnostic laboratories in Netherlands as a first-line diagnostic test to identify patients infected with Coxiella burnetii in the acute phase of the disease. In a recent interlaboratory study, it has been shown that multiple PCR approaches based on different combinations of extraction procedures, amplification primers and probes, PCR Master Mixes, and real-time PCR platforms offer equivalent solutions to screening for Q fever (6). In the spring of 2009, the medical microbiology laboratory of the Canisius Wilhelmina Hospital (CWZ) was suddenly confronted with positive PCR results in a no-template control (NTC), indicating contamination of the diagnostic pathway. Analysis of multiple NTCs showed that 10 to 30% yielded a positive PCR result with threshold cycle values such as those found in many clinical samples. The laboratory enforces strict precautionary measures to avoid amplicon or DNA carryover during the entire diagnostic process. Since the contamination coincided with the first use of a new batch of PCR primers and probe, it was suspected that one of these was contaminated off synthesis. A newly ordered batch of primers and probe did not alleviate the problem. Around the same time, the medical microbiology laboratory of the Radboud University Nijmegen Medical Center (RUNMC) experienced similar problems, with important clinical consequences. A patient with a history of a Ross procedure was admitted with fever and probable endocarditis with gradual degradation of the aortic homograft and deterioration of cardiac function despite broad-spectrum antibiotics. All blood cultures were negative, and additional serological tests for culture-negative endocarditis (including Q fever) were performed. During this time, hemodynamic problems occurred and a second cardiac surgery was necessary. The day before surgery, the Q-fever PCR was positive but serological tests (complement fixation test and IgM antibodies) were negative, making chronic Q fever unlikely. It was decided to postpone the cardiac surgery and to repeat the PCR in another laboratory. This PCR was negative, confirming the suspicion of a false-positive PCR result. Fortunately, no serious hemodynamic problems occurred during the time while the operation was deferred. According to protocol and good laboratory practice, numerous measures were undertaken to eliminate any potential source of contamination, but without success.

Quantifying and understanding well-to-well contamination in microbiome research

“…Contamination, or the observation of sequence reads in a sample coming from microbes that weren’t originally part of that sample, remains one of the most pernicious types of experimental noise. Microbial rRNA gene copies can be found even in ‘sterile’ reagents, leading to that presence of background signal derived from DNA extraction kits [18], PCR mastermix [19], and other consumables [20]. It is now widely understood that such contaminants must be considered in microbiome analyses, especially when dealing with low-biomass samples where contaminant rRNA gene copies make up a larger fraction of the community [7,21–25].…”

Combinational approach using in situ hybridization targeting 23S ribosomal RNA genes and blood cultures for bacterial identification in patients with neutropenia and fever

“…However, particularly for PCR, there are disadvantages such as contamination of bacterial DNA (e.g. Taq-polymerases or reagents for DNA extraction procedures) [10][11][12][13][14], indicating that these methods require further standardization. In addition, serum biomarkers including PCT and IL-6 might not be useful in patients with FN, compared to those in non-neutropenic sepsis patients, because the levels of these markers could trend lower in neutropenic patients [27], probably due to chemotherapy-induced cytopenia.…”

“…procalcitonin (PCT), interleukin (IL)-6, and IL-8) [3][4][5], polymerase chain reaction (PCR) [6,7], or mass spectrometry [8,9] could improve the management of FN. Although PCR analysis and mass spectrometry have the advantage of obtaining direct evidence of bacteria, the former could be susceptible to contamination with bacterial DNA [10][11][12][13][14], whereas the latter demonstrates insufficient diagnostic accuracy. However, both methods might be useful for identifying causal bacteria from positive blood culture samples [7][8][9].…”

Update Review Article: Spontaneous Bacterial Peritonitis

“…Taq-polymerases may be contaminated with bacterial DNA 65,66 . Moreover, the reagents used for DNA extraction procedures carry a risk of exposing the clinical samples to exogenous bacterial DNA 67,68 . Although PCR is a very sensitive method for detecting DNA, PCR-based methods display discrepant and controversial findings with respect to diagnostic performance in detecting the causative pathogen(s) in SBP patients with ascites, perhaps, or at least in part, due to the problems described above.…”