Comparison of standard, quantitative and digital PCR in the detection of enterotoxigenic Bacteroides fragilis.
Gut colonization with enterotoxigenic Bacteroides fragilis (ETBF) appears to be associated with the development of colorectal cancer. However, differences in carriage rates are seen with various testing methods and sampling sites. We compared standard PCR, SYBR green and TaqMan quantitative PCR (qPCR) and digital PCR (dPCR) in detecting the B. fragilis toxin (bft) gene from cultured ETBF, and from matched luminal and faecal stool samples from 19 colorectal cancer patients. Bland-Altman analysis found that all three quantitative methods performed comparably in detecting bft from purified bacterial DNA, with the same limits of detection (<1 copy/μl). However, SYBR qPCR under-performed compared to TaqMan qPCR and dPCR in detecting bft in clinical stool samples; 13/38 samples were reported positive by SYBR, compared to 35 and 36 samples by TaqMan and dPCR, respectively. TaqMan qPCR and dPCR gave bft copy numbers that were 48-fold and 75-fold higher for the same samples than SYBR qPCR, respectively (p < 0.001). For samples that were bft-positive in both fecal and luminal stools, there was no difference in relative abundance between the sites, by any method tested. From our findings, we recommend the use of TaqMan qPCR as the preferred method to detect ETBF from clinical stool samples.
Department of Surgery, University of Otago, 2 Riccarton Avenue, PO Box 4345, Christchurch 8140, New Zealand.
Rachel V
RV
Purcell
Department of Population Health,University of Otago, 2 Riccarton Avenue,PO Box 4345,Christchurch 8140, New Zealand.
John
J
Pearson
Department of Surgery, University of Otago, 2 Riccarton Avenue, PO Box 4345, Christchurch 8140, New Zealand.
Frank A
FA
Frizelle
Department of Surgery, University of Otago, 2 Riccarton Avenue, PO Box 4345, Christchurch 8140, New Zealand.
Jacqueline I
JI
Keenan
J Bacteriol. 1999 Nov;181(21):6623-33
10542162
Cytometry A. 2003 Feb;51(2):127-8; author reply 129
12541287
Clin Infect Dis. 2015 Jan 15;60(2):208-15
25305284
Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14979-84
9844001
J Clin Microbiol. 2015 Jul;53(7):2008-14
25392365
Eur J Clin Microbiol Infect Dis. 2015 Sep;34(9):1871-7
26173688
PLoS One. 2015 Mar 09;10(3):e0119462
25751261
Lancet. 1986 Feb 8;1(8476):307-10
2868172
Infect Immun. 1998 Apr;66(4):1735-9
9529104
Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9236-41
10430926
Gastroenterology. 2003 Feb;124(2):392-400
12557145
J Appl Microbiol. 2012 Nov;113(5):1014-26
22747964
Inflamm Bowel Dis. 2014 May;20(5):821-34
24704822
J Clin Microbiol. 2016 Jan;54(1):239-40
26491187
Clin Microbiol Infect. 2006 Aug;12(8):782-6
16842574
Anaerobe. 2012 Feb;18(1):14-8
22138361
Infect Immun. 2004 Oct;72(10):5832-9
15385484
Infect Immun. 2009 Apr;77(4):1708-18
19188353
J Clin Epidemiol. 2008 Oct;61(10):978-82
18468854
J Microbiol Biotechnol. 2015 Jul;25(7):1136-45
25737115
Nat Med. 2009 Sep;15(9):1016-22
19701202
Anaerobe. 2016 Aug;40:50-3
27166180
Clin Microbiol Rev. 2009 Apr;22(2):349-69, Table of Contents
19366918
PLoS One. 2014 Nov 20;9(11):e113896
25411788