Pathogen Quantitation in Complex Matrices: A Multi-Operator Comparison of DNA Extraction Methods with a Novel Assessment of PCR Inhibition

Abstract

Background Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.Methodology/Principal FindingsWe report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities.Conclusions M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×105 cells g−1 using Griffiths and 4.25×106 cells g−1 using FastDNA® Spin kit.

Influences of sample interference and interference controls on quantification of enterococci fecal indicator bacteria in surface water samples by the qPCR method

“…This process, however, adds time and expense to the method and does not necessarily ensure the elimination of all inhibitory factors. A recent study demonstrated that a new commercially available PCR reagent formulation, TaqMan Ò Environmental Master Mix, was successful in amplifying and detecting target sequences in purified DNA extracts of a number of fecal samples that were recalcitrant to analysis by Universal Master Mix (Pontiroli et al, 2011). In the present study, none of the undiluted Ohio River water sample extracts failed the Sketa22 SPC assay acceptance criteria in analyses using Environmental Master Mix as compared to an 18% failure rate with Universal Master Mix.…”

Multicentric Evaluation of a New Real-Time PCR Assay for Quantification of Cryptosporidium spp. and Identification of Cryptosporidium parvum and Cryptosporidium hominis

“…The extraction rates for the other methods were significantly better, but each method had distinctive features depending on the principle of purification and the parasite burden. The two kits using silica columns, i.e., QIAamp DNA Mini and NucleoSpin kits, were characterized by higher saturation limits than the methods based on magnetic silica, as already observed for Mycobacterium (32), while NucliSENS easyMAG gave higher extraction yields with fecal samples containing low parasite burdens. Curiously, NucliSENS easyMAG demonstrated a better extraction rate with fecal samples than with saline suspensions of oocysts.…”

The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance

“…A total of 25 soil samples recovered from six different geographical locations (Algeria, United Kingdom, Germany, Denmark, Norway, and Spain) and soil uses (agricultural and nonagricultural) were analyzed, including 10 samples collected in previous studies (17)(18)(19)(20)(21)(22)(23)) (Table 1). Fifteen samples were collected in this study from 0-to 15-cm depth using sterile plastic bags and stored at Ϫ20°C.…”