Evaluation of Inhibitor-Resistant Real-Time PCR Methods for Diagnostics in Clinical and Environmental Samples

Abstract

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.

Cross-Institute Evaluations of Inhibitor-Resistant PCR Reagents for Direct Testing of Aerosol and Blood Samples Containing Biological Warfare Agent DNA

“…The United Kingdom group evaluated reagents specifically designed to support real-time (TaqMan) PCR, while the U.S. group evaluated reagents selected from a previous study (17), which were not specifically designed to support TaqMan PCR chemistry. For reagents that did not support TaqMan chemistry, Platinum Taq was included in the reaction mixture to allow for probe hydrolysis and detection.…”

“…Five different inhibitor-resistant PCR chemistries were selected from a previous study (17). These included three separate chemistries: Phire Hot Start DNA polymerase (New England BioLabs, Ipswich, MA), the Phusion Blood Direct PCR kit (New England BioLabs), and the KAPA Blood PCR kit (KAPA Biosystems, Woburn, MA).…”

“…In addition, two buffers (Ampdirect buffer [Rockland Immunochemicals, Gilbertsville, PA] and STRboost buffer [Biomatrica, San Diego, CA]) were run with the Phire Hot Start polymerase (Phire). As noted in previous studies, these polymerases were not designed for real-time (TaqMan) PCR detection, specifically 5=-to-3= digestion of the labeled PCR probe (17). Therefore, 0.8 U of Platinum Taq (Life Technologies) was added to each reaction mixture to ensure adequate probe hydrolysis and fluorophore detection.…”

“…In the context of BWA detection, the potential for false-negative results becomes increasingly important, since minimal overlap exists between the diagnostic and therapeutic windows for these highly pathogenic viruses and bacteria. To facilitate a faster time-to-answer and to minimize the operative and logistical burdens, PCR reagents with reported resistance to various PCR inhibitors have been developed or sold commercially (15)(16)(17). Modifications include N-terminal DNA polymerase truncation (15), the addition of betaine or protease inhibitors (16), or the addition of bovine serum albumin (BSA) (16,18).…”

Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System

“…For example, the FilmArray platform is reported to have a 250-genome equivalent (GE) limit of detection (22), which is unlikely to be sufficiently sensitive for use as a direct blood test. Furthermore, most other existing F. tularensis detection technologies require manual DNA extraction methods that are labor-intensive and can further compromise assay sensitivity (23,24). A more sensitive NAA assay that also included integrated sample processing and target detection would permit more rapid and potentially lifesaving diagnosis of this disease, while at the same time help to address biosafety concerns by decreasing the potential for laboratory-acquired infections.…”