Abstract
Molecular microbiological methods, such as competetive PCR, real-time PCR, denaturing gradient gel electrophoresis (DGGE) and large-scale parallel-pyrosequencing, require the extraction of sufficient quantity of high quality DNA from microbiologically and chemically complex matrices. Due to difficulties in the field to standardize/select the optimum DNA preservation-extraction methods in view of laboratories differences, this article attempts to present a straight-forward mathematical framework for comparing some of the most commonly used methods. To this end, as a case study, the problem of selecting an optimum sample preservation-DNA extraction strategy for obtaining total bacterial DNA from swine feces was considered. Two sample preservation methods (liquid nitrogen and RNAlater?) and seven extraction techniques were paired and compared under six quantitative DNA analysis criteria: yield of extraction, purity of extracted DNA (A260/280 and A 260/230 ratios), duration of extraction, degradation degree of DNA, and cost. From a practical point of view, it is unlikely that a single sample preservation-DNA extraction strategy can be optimum for all selected criteria. Hence, a systematic multi-criteria decision-making (MCDM) approach was used to compare the methods. As a result, the ZR Fecal DNA MiniPrepTM DNA extraction kit for samples preserved either with liquid nitrogen or RNAlater? were identified as potential optimum solutions for obtaining total bacterial DNA from swine feces. Considering the need for practicality for in situ applications, we would recommend liquid nitrogen as sample preservation method, along with the ZR Fecal DNA MiniPrepTM kit. Total bacterial DNA obtained by this strategy can be suitable for downstream PCR-based DNA analyses of swine feces.
Highlights
The intestinal tract of animals is a complex ecosystem composed of at least 400 - 500 different microbial species which have critical roles in the nutrition and health of their hosts, including the fermentation of potential energy sources, the production of short chain fatty acids, and the activation or deactivation of carcinogens [1]
Samples preserved by liquid nitrogen (L) offered identical (FTE, M, E) or higher (PC, Q, Z, FSP) yields of extracted DNA than samples preserved by RNAlater® (R) (Table 4)
Samples preserved by RNAlater® showed similar (M, Z, E) or less DNA degradation (PC, Q, FSP) than samples preserved by liquid nitrogen (Figure 1 and Table 4)
Summary
The intestinal tract of animals is a complex ecosystem composed of at least 400 - 500 different microbial species which have critical roles in the nutrition and health of their hosts, including the fermentation of potential energy sources, the production of short chain fatty acids, and the activation or deactivation of carcinogens [1]. Classical microbiological methods for identifying and characterizing bacteria in the gut microbiota are labor-intensive, time-consuming and limited to the study of microorganisms which are active and can be grown under laboratory conditions [4,5,6,7,8] These restrictions can be overcome using culture-independent molecular microbiological methods such as competitive PCR, real-time PCR, denaturing gradient gel electrophoresis (DGGE) and large-scale parallel-pyrosequencing, all of which require proper sample preservation and extraction of sufficient quantity of high quality DNA (i.e., free from contaminants and representative of the microbial phylogenetic and/or functional biodiversity of the intestinal microbiota) from samples. Feces contain inhibitors such as bile salts, haemoglobin degradation products
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