Evaluation of inhibitor-resistant real-time PCR methods for diagnostics in clinical and environmental samples

Polymerase chain reaction (PCR) is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at low...

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Published in:	PloS one Vol. 8; no. 9; p. e73845
Main Authors:	Trombley Hall, Adrienne, McKay Zovanyi, Ashley, Christensen, Deanna Rose, Koehler, Jeffrey William, Devins Minogue, Timothy
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 09-09-2013 Public Library of Science (PLoS)
Subjects:	Blood Buffers (chemistry) Deoxyribonucleic acid DNA DNA polymerase DNA, Bacterial DNA-directed DNA polymerase Environmental aspects Environmental Microbiology Evaluation Francisella tularensis - genetics Hepatitis Humans Infectious diseases Inhibitors Laboratories Medical research Methods Pathogens Polymerase chain reaction Purification Reagent Kits, Diagnostic Reagents Real time Real-Time Polymerase Chain Reaction - methods Real-Time Polymerase Chain Reaction - standards Reproducibility of Results Sandy soils Sediments Sensitivity and Specificity Soils Sputum Maryland
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Summary:	Polymerase chain reaction (PCR) is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella tularensis in seven different clinical and environmental samples using an established real-time PCR assay to assess ability to overcome PCR inhibition. While several of these reagents were designed for standard PCR, the described inhibitor resistant properties (ex. Omni Klentaq can amplify target DNA samples of up to 20% whole blood or soil) led to our evaluation with real-time PCR. A preliminary limit of detection (LOD) was determined for each chemistry in whole blood and buffer, and LODs (20 replicates) were determined for the top five chemistries in each matrix (buffer, whole blood, sputum, stool, swab, soil, and sand). Not surprisingly, no single chemistry performed the best across all of the different matrices evaluated. For instance, Phusion Blood Direct PCR Kit, Phire Hot Start DNA polymerase, and Phire Hot Start DNA polymerase with STR Boost performed best for direct detection in whole blood while Phire Hot Start DNA polymerase with STR Boost were the only reagents to yield an LOD in the femtogram range for soil. Although not the best performer across all matrices, KAPA Blood PCR kit produced the most consistent results among the various conditions assessed. Overall, while these inhibitor resistant reagents show promise for direct amplification of complex samples by real-time PCR, the amount of template required for detection would not be in a clinically relevant range for most matrices.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Conceived and designed the experiments: JWK TDM. Performed the experiments: ATH AMZ DRC. Analyzed the data: ATH AMZ DRC JWK TDM. Wrote the manuscript: ATH JWK TDM. Competing Interests: The authors have declared that no competing interests exist.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0073845