ArticlePloS one2013
Evaluation of inhibitor-resistant real-time PCR methods for diagnostics in clinical and environmental samples.
Article in PloS one, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 42 papers.
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Who cites it
42 citing papers in PubMed, 80 citations in OpenAlex.
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- Development of a PCR Assay for the Detection ofInfectious disease reports · 2025Article
- Development of a Real-Time Quantitative PCR Assay for Direct Detection and Quantification of the Root-Lesion NematodeInternational journal of molecular sciences · 2025Article
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- A simple and cost-effective extraction for piscine environmental DNA metabarcoding using guanidine hydrochloride method.MethodsX · 2024Article
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- Direct Real-Time PCR for the Detection and Serotyping of Haemophilus influenzae without DNA Extraction.Journal of clinical microbiology · 2022Article
- Inhibition monitoring in veterinary molecular testing.Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc · 2020Article
- Degradation of nuclear and mitochondrial DNA after γ-irradiation and its effect on forensic genotyping.Forensic science, medicine, and pathology · 2020Article
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- Advances in Directly Amplifying Nucleic Acids from Complex Samples.Biosensors · 2019Review
- Detection of Single-Nucleotide Polymorphism Markers of Antimalarial Drug Resistance Directly from Whole Blood.The Journal of molecular diagnostics : JMD · 2019Article
- A diffusion-based microfluidic device for single-cell RNA-seq.Lab on a chip · 2019Article
Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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