Evaluation of QIAamp DNA Stool Mini Kit for ecological studies of gut microbiota.
Cell lysis efficiency and the quality of DNA extracts from complex bacterial ecosystems are two major concerns in molecular ecological studies of gut microbiota. In this study, we use PCR-denaturing gradient gel electrophoresis (DGGE) DNA profiling, random cloning and sequence analysis of 16S rRNA genes to compare the QIAamp DNA Stool Mini Kit with the bead beating technique in the preparation of DNA extracts from gut microbiota of pigs. We also developed a washing procedure that can release more than 93% of bacterial cells attached to the gut mucosa. Both the QIAamp kit and bead beating method lysed approximately 95% of bacterial cells. PCR-DGGE DNA profiles of ileal and cecal microbiota from both digesta and mucosa that were generated from the DNA extracts using the two methods were nearly identical. Random cloning and sequence analysis also demonstrated the high quality of DNA extracts using the two methods. Two random clone sets of 16S rRNA genes generated from the DNA extracts had a similar degree of bacterial diversity. Different preparations of DNA extract from a single sample using the QIAamp kit consistently produced similar PCR-DGGE DNA profiles with similarity indexes higher than 99%. Our data suggest the appropriateness of the QIAamp DNA Stool Mini Kit for the studies of gut microbial ecology and the effectiveness of the QIAamp kit in processing multiple samples for cell lysis and DNA extraction.
Food Research Program, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada N1G 5C9.
Mei
M
Li
Jianhua
J
Gong
Michael
M
Cottrill
Hai
H
Yu
Cornelis
C
de Lange
Jeremy
J
Burton
Edward
E
Topp
D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000032:analysis; D017753:Ecosystem; D004586:Electrophoresis / Q000379:methods; D005243:Feces / Q000382:microbiology*; D007413:Intestinal Mucosa / Q000737:chemistry / Q000382:microbiology; D007422:Intestines / Q000382:microbiology*; D008863:Microspheres; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA; D013552:Swine
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D011933:Reagent Kits, Diagnostic