ArticlePloS one2013
The critical role of DNA extraction for detection of mycobacteria in tissues.
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 22 papers.
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Who cites it
22 citing papers in PubMed, 38 citations in OpenAlex.
- Quantitative metagenomics using a portable protocol.Applied and environmental microbiology · 2026Article
- Shaken, not stirred: magnetic bead DNA extraction as a rapid and effective method for the scaling up of bovine tuberculosis diagnosis.BMC veterinary research · 2024Article
- Non-Microbiological Mycobacterial Detection Techniques for Quality Control of Biological Products: A Comprehensive Review.Microorganisms · 2024Review
- Optimization of real-time PCR protocols from lymph node bovine tissue for direct detection ofMicrobiology spectrum · 2023Article
- Rapid Diagnostic ofMicroorganisms · 2023Article
- What about Current Diversity of Mycolactone-Producing Mycobacteria? Implication for the Diagnosis and Treatment of Buruli Ulcer.International journal of molecular sciences · 2023Review
- Mycobacterium Tuberculosis and Avium Complex Investigation among Malaysian Free-Ranging Wild Boar and Wild Macaques at Wildlife-Livestock-Human Interface.Animals : an open access journal from MDPI · 2021Article
- Direct PCR on Tissue Samples To Detect Mycobacterium tuberculosis Complex: an Alternative to the Bacteriological Culture.Journal of clinical microbiology · 2021Article
- Ultrasensitive hybridization capture: Reliable detection of <1 copy/mL short cell-free DNA from large-volume urine samples.PloS one · 2021Article
- The Paratuberculosis Paradigm Examined: A Review of Host Genetic Resistance and Innate Immune Fitness inFrontiers in veterinary science · 2021Review
- Liver Bacterial Dysbiosis With Non-Tuberculosis Mycobacteria Occurs in SIV-Infected Macaques and Persists During Antiretroviral Therapy.Frontiers in immunology · 2021Article
- Validation of real-time PCR technique for detection of Mycobacterium bovis and Brucella abortus in bovine raw milk.Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] · 2020Article
- A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows' milk.Applied microbiology and biotechnology · 2020Article
- Potential Value of Qiagen and PrepIT•MAX Kits in Extraction of Mycobacterial DNA From Presumptive Tuberculosis Archived Formalin-Fixed Paraffin-Embedded Tissues.The East African health research journal · 2018Article
- PCR Inhibition of a Quantitative PCR for Detection ofFrontiers in microbiology · 2017Article
- Culture-Independent Identification ofFrontiers in veterinary science · 2017Article
- Two alternative DNA extraction methods to improve the detection of Mycobacterium-tuberculosis-complex members in cattle and red deer tissue samples.BMC microbiology · 2016Article
- Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples.PloS one · 2016Article
- 16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice.Microbiome · 2015Article
- Disruption of Mycobacterium avium subsp. paratuberculosis-specific genes impairs in vivo fitness.BMC genomics · 2014Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
backgroundNucleic acid-based methods offer promise for both targeted and exploratory investigations of microbes in tissue samples. As the starting material for such studies is a mixture of host and microbial DNA, we have critically evaluated the DNA extraction step to determine the quantitative and qualitative parameters that permit faithful molecular detection of mycobacteria in infected tissue. Specifically, we assessed: 1) tissue disruption procedures; 2) DNA extraction protocols; and 3) inhibition of bacterial PCR by host DNA. PRINCIPAL
findingsRegarding DNA extraction, we found that 1) grinding was not necessary if bead-beating is done, 2) the reference mycobacterial DNA extraction method recovered more pure DNA than commercial spin column kits, 3) lysozyme digestion of 1 hour was sufficient, and 4) repeated steps of phenol:chloroform:isoamyl alcohol offered minimal gain in DNA quality. By artificially mixing mycobacterial DNA with DNA extracted from uninfected mice, we found that bacterial real-time quantitative PCR was only reliable when the quantity of host DNA was < 3 µg in a final volume of 25 µl and the quality was high (260/280 nm ratio = 1.89 ± 0.08). Findings from spiked DNA studies were confirmed using DNA extracted from mice infected with different intracellular pathogens (M. tuberculosis, M. avium subsp. paratuberculosis).
conclusionsOur findings point to the most appropriate methods for extracting DNA from tissue samples for the purpose of detecting and quantifying mycobacteria. These data also inform on the limits of detection for two mycobacterial species and indicate that increasing the sample mass to improve analytic sensitivity comes at the cost of inhibition of PCR by host DNA.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.