Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: possible consequences for population fingerprinting of complex microbial communities.
The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V3, V3-V5, and V6-V8 hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V3 and V3-V5 primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.
Huys
Geert
G
Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000, Ghent, Belgium. Geert.Huys@UGent.be
Vanhoutte
Tom
T
Joossens
Marie
M
Mahious
Amal S
AS
De Brandt
Evie
E
Vermeire
Severine
S
Swings
Jean
J
D000818:Animals; D001419:Bacteria / Q000737:chemistry / Q000145:classification* / Q000235:genetics; D015373:Bacterial Typing Techniques; D016172:DNA Fingerprinting; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005243:Feces / Q000382:microbiology*; D005399:Fishes / Q000382:microbiology*; D041981:Gastrointestinal Tract / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012337:RNA, Ribosomal, 18S