Abstract
The study of the intestinal microflora has been developed mainly using conventional microbiological approaches. These studies have made ​​great progress, but it is imperative that new methods will be applied to facilitate scientific progress. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) has been used to study microbial communities in many environmental samples. So, we must do our best to enhance the quality and accuracy of upstream analyses, such as DNA extraction. In this study, the relative efficacy of four DNA extraction methods (QIAamp DNA stool mini kit method, Q; QIAamp DNA stool mini kit+ Bead beating method, QB; QIAamp DNA stool mini kit+ Frozen thawed method, QF; E.Z.N.A. Stool DNA Kit method, E) were evaluated. Further, PCR-DGGE technique was also assessed in detecting diversity in infant intestinal bacterial fingerprint profiles. The total DNA was extracted from the infant fecal specimens using four different methods, followed by PCR amplification of bacterial 16S rRNA-V3 region, and DGGE separation of the amplifications. The number of extracted DNA of the infant feces using the Q method was larger than the QB, QF and E methods, and the produced bands of its DGGE profiles were more than the other three methods, which was due to its cracking temperature (95°C). We concluded that DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of the infant intestinal microbial community and the QIAamp DNA stool mini kit method was appropriate for infant fecal DNA extraction. PCR-DGGE could be an important tool for DNA studies. Key words: DNA extraction method, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), infant intestinal microflora.
Highlights
The microbial community colonizing the human gastrointestinal (GI) tract is diverse (Eckburg et al, 2005) and plays an important role in digestion, production of essential vitamins, as well as protecting the GI tract from pathogen colonization
The size of the deoxyribonucleic acid (DNA) fragment was about 21kb; it indicated that the large fragment of genomic DNA had been acquired, but the effect of all DNA bands obtained by the four methods was not very good, the brightness was low and fuzzy, which was related to the reason that the sample itself contained fewer microorganisms
In order to evaluate the effect of DNA extraction in quality, we needed to further detect the biodiversity of microorganisms
Summary
The microbial community colonizing the human gastrointestinal (GI) tract is diverse (Eckburg et al, 2005) and plays an important role in digestion, production of essential vitamins, as well as protecting the GI tract from pathogen colonization. The influence of diet on intestinal microflora has been largely studied using conventional microbiological techniques. The DNA extraction is crucial for the molecular technique. The ability to obtain the representtative total DNA will determine the credibility of subsequent intestinal microbial diversity analysis. The extraction efficiency of the direct extracted method is high and the total number of extracted DNA is large, but the content of impurities is higher, it may affect the subsequent PCR amplification; the purity of the indirect extracted DNA is high, but the total number is smaller; it is not suitable for microbial diversity research, so some suitable methods are demanded
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