PMID- 11822677
OWN - NLM
STAT- MEDLINE
DCOM- 20020712
LR  - 20041117
IS  - 0723-2020 (Print)
IS  - 0723-2020 (Linking)
VI  - 24
IP  - 3
DP  - 2001 Nov
TI  - DNA isolation protocols affect the detection limit of PCR approaches of bacteria 
      in samples from the human gastrointestinal tract.
PG  - 405-10
AB  - A major concern in molecular ecological studies is the lysis efficiency of 
      different bacteria in a complex ecosystem. We used a PCR-based 16S rDNA approach 
      to determine the effect of two DNA isolation protocols (i.e. the bead beating and 
      Triton-X100 method) on the detection limit of seven feces-associated bacterial 
      species of different genera. Glycogen was used in these protocols to improve the 
      precipitation of small concentrations of DNA in ethanol without affecting the 
      sequential procedures. The PCR detection limit of 16S rDNA amplicons on agarose 
      gel from the seven strains tested varied between 8.0 (+/- 1.3) x 10(4) and 4.3 
      (+/- 1.6) x 10(6) cells for the bead beating method, and between 8.0 (+/- 1.3) x 
      10(4) and 5.4 (+/- 0.7) x 10(8) cells for the Triton X-100 method. These large 
      differences are most like due to the difference in cell lysis efficiency, since a 
      competitive PCR experiment did not indicate any preference for gram negative, low 
      G+C gram positive or high G+C gram positive bacteria. Denaturing gradient gel 
      electrophoresis (DGGE) analysis was performed to investigate the effect of both 
      DNA isolation protocols on the lysis efficiency of bacteria in fecal samples. A 
      higher diversity in fecal samples was observed with the bead beating method than 
      with the Triton-X100 method. Bands in the bead beating method-derived DGGE 
      profiles corresponding to bands of cloned sequences of the Clostridium 
      coccoides-Eubacterium rectale group and uncultured Fusobacterium prausnitzii were 
      absent or had low intensity in the Triton X-100 method-derived profiles. The 
      applicability of the bead beating method was further investigated by analyzing 
      biopsy samples from the human colon which contain approximately 10(6) cells.
FAU - Zoetendal, E G
AU  - Zoetendal EG
AD  - Wageningen Centre for Food Sciences, The Netherlands. 
      erwin.zoetendal@algemeen.micr.wau.nl
FAU - Ben-Amor, K
AU  - Ben-Amor K
FAU - Akkermans, A D
AU  - Akkermans AD
FAU - Abee, T
AU  - Abee T
FAU - de Vos, W M
AU  - de Vos WM
LA  - eng
PT  - Journal Article
PL  - Germany
TA  - Syst Appl Microbiol
JT  - Systematic and applied microbiology
JID - 8306133
RN  - 0 (DNA, Bacterial)
RN  - 9005-79-2 (Glycogen)
SB  - IM
MH  - Biopsy
MH  - DNA, Bacterial/*isolation & purification
MH  - Digestive System/*microbiology
MH  - Feces/microbiology
MH  - Glycogen/pharmacology
MH  - Humans
MH  - Polymerase Chain Reaction/*methods
EDAT- 2002/02/02 10:00
MHDA- 2002/07/13 10:01
CRDT- 2002/02/02 10:00
PHST- 2002/02/02 10:00 [pubmed]
PHST- 2002/07/13 10:01 [medline]
PHST- 2002/02/02 10:00 [entrez]
AID - S0723-2020(04)70051-X [pii]
AID - 10.1078/0723-2020-00060 [doi]
PST - ppublish
SO  - Syst Appl Microbiol. 2001 Nov;24(3):405-10. doi: 10.1078/0723-2020-00060.