The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues

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.

Fecal culture and two fecal-PCR methods for the diagnosis of Mycobacterium avium subsp. paratuberculosis in a seropositive herd

“…On the other hand, F57, a single copy-segment, has demonstrated high specificity for the detection of MAP (Coetsier et al, 2000;Ellingson et al, 2000;Harris and Barletta, 2001;Strommenger et al, 2001;Vansnick et al, 2004;Rajeev et al, 2005). The nested IS900 assay can detect 0.01 pg of DNA (corresponding to 10 genomes) when extracted from a pure culture, while the F57 assay can detect 0.1 pg of DNA (corresponding to 100 genomes; Radomski et al, 2013). Vansnicka et al (2004), Tasara and Stephan (2005), and Schönenbrücher et al (2008) recommend including the F57-PCR assay to confirm the presence of MAP after a positive IS900-PCR.…”

Comparison of DNA extraction protocols to detect Mycobacterium bovis in bovine tissue by PCR

“…In addition, PCR sensitivity can be affected by the inhibitors present in the clinical samples, those from the reagents used in the extraction method, or the amount of extracted DNA (CARDOSO et al, 2009;RADOMSKI et al, 2013). Cardoso et al (2009) demonstrated that the potential effect of PCR inhibitors can be reduced by diluting (1:2) or increasing the amount of DNA in the samples.…”

“…The application of molecular methods on clinical specimens encounters a number of problems, including a complex mycobacterial cell wall, the presence of the bacterial cell within a phagosome, a host cell and a granuloma, and the low amount of bacterial cells or DNA (KUMAR et al, 2010;RADOMSKI et al, 2013). Additionally, inhibitory substances present in the samples or the reagents used during DNA extraction, and an excessive amount of host DNA compared to the target DNA of the pathogen, also make accurate pathogen detection in clinical specimens difficult (PARK et al, 2014).…”

Standing of nucleic acid testing strategies in veterinary diagnosis laboratories to uncover Mycobacterium tuberculosis complex members

“…Indeed, most of the amplification-based assays described for detecting MTC nucleic acids directly in fresh or formalin-fixed paraffin-embedded animal tissues only yield a moderate sensitivity, usually up to 75% when compared to the reference bacteriological culture, particularly when testing tissues without the characteristic lesions (Liébana et al, 1995 ; Coetsier et al, 2000 ; Roring et al, 2000 ; Taylor et al, 2001 , 2007 ; Parra et al, 2008 ; Thacker et al, 2011 ). This limitation is mostly related to the increased complexity for disrupting and recovering genomic DNA from the tough mycobacterial cells, which harbor complex walls, and to the inefficiency of the extraction procedures from affected animal tissues, especially those exhibiting strong fibrosis, calcification, and with no histological evidence of acid-fast bacteria (Liébana et al, 1995 ; Taylor et al, 2007 ; Amaro et al, 2008 ; Parra et al, 2008 ; Costa et al, 2013a ; Radomski et al, 2013 ). The presence of amplification inhibitors in crude tissue extracts, and of large amounts of co-extracted host eukaryotic DNA, often represent an additional problem.…”