Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial Populations in human Feces

被引:181
作者
Nagashima, K
Hisada, T
Sato, M
Mochizuki, J
机构
[1] Hokkaido Food Proc Res Ctr, Ebetsu, Hokkaido 0690836, Japan
[2] NCIMB Japan Co Ltd, Res Ctr, Shimizu, Shizuoka 4248610, Japan
关键词
D O I
10.1128/AEM.69.2.1251-1262.2003
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.
引用
收藏
页码:1251 / 1262
页数:12
相关论文
共 22 条
[1]   Community structure of denitrifiers, Bacteria, and Archaea along redox gradients in pacific northwest marine sediments by terminal restriction fragment length polymorphism analysis of amplified nitrite reductase (nirS) and 16S rRNA genes [J].
Braker, G ;
Ayala-del-Río, HL ;
Devol, AH ;
Fesefeldt, A ;
Tiedje, JM .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2001, 67 (04) :1893-1901
[2]   DETECTION OF STAPHYLOCOCCUS-AUREUS BY POLYMERASE CHAIN-REACTION AMPLIFICATION OF THE NUC GENE [J].
BRAKSTAD, OG ;
AASBAKK, K ;
MAELAND, JA .
JOURNAL OF CLINICAL MICROBIOLOGY, 1992, 30 (07) :1654-1660
[3]   Molecular monitoring of succession of bacterial communities in human neonates [J].
Favier, CF ;
Vaughan, EE ;
De Vos, WM ;
Akkermans, ADL .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2002, 68 (01) :219-226
[4]  
Franks AH, 1998, APPL ENVIRON MICROB, V64, P3336
[5]  
Gong JH, 2002, FEMS MICROBIOL LETT, V208, P1, DOI 10.1111/j.1574-6968.2002.tb11051.x
[6]   Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods [J].
Harmsen, HJM ;
Wildeboer-Veloo, ACM ;
Raangs, GC ;
Wagendorp, AA ;
Klijn, N ;
Bindels, JG ;
Welling, GW .
JOURNAL OF PEDIATRIC GASTROENTEROLOGY AND NUTRITION, 2000, 30 (01) :61-67
[7]  
Harmsen HJM, 2000, FEMS MICROBIOL LETT, V183, P125, DOI 10.1111/j.1574-6968.2000.tb08945.x
[8]   Terminal restriction pattern analysis of 16S rRNA genes for the characterization of bacterial communities of activated sludge [J].
Hiraishi, A ;
Iwasaki, M ;
Shinjo, H .
JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 2000, 90 (02) :148-156
[9]   QUANTITATIVE FLUORESCENCE IN-SITU HYBRIDIZATION OF BIFIDOBACTERIUM SPP WITH GENUS-SPECIFIC 16S RIBOSOMAL-RNA-TARGETED PROBES AND ITS APPLICATION IN FECAL SAMPLES [J].
LANGENDIJK, PS ;
SCHUT, F ;
JANSEN, GJ ;
RAANGS, GC ;
KAMPHUIS, GR ;
WILKINSON, MHF ;
WELLING, GW .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1995, 61 (08) :3069-3075
[10]   Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae [J].
Leser, TD ;
Lindecrona, RH ;
Jensen, TK ;
Jensen, BB ;
Moller, K .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2000, 66 (08) :3290-3296