Multiplex PCR for detection of botulinum neurotoxin-producing clostridia in clinical, food, and environmental samples.
Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.
De Medici
Dario
D
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. dario.demedici@iss.it
Anniballi
Fabrizio
F
Wyatt
Gary M
GM
Lindström
Miia
M
Messelhäusser
Ute
U
Aldus
Clare F
CF
Delibato
Elisabetta
E
Korkeala
Hannu
H
Peck
Michael W
MW
Fenicia
Lucia
L
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay / Q000706:statistics & numerical data; D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000235:genetics*; D003013:Clostridium / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003014:Clostridium botulinum / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004783:Environmental Microbiology; D005516:Food Microbiology; D005798:Genes, Bacterial; D006801:Humans; D051379:Mice; D009498:Neurotoxins / Q000096:biosynthesis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D009498:Neurotoxins; D001905:Botulinum Toxins
Biotechniques. 1997 Sep;23(3):504-11
9298224
J Bacteriol. 2007 Feb;189(3):818-32
17114256
Foodborne Pathog Dis. 2004 Winter;1(4):247-57
15992287
J Infect Dis. 1986 Aug;154(2):207-11
3722863
Mol Biotechnol. 2004 Feb;26(2):133-46
14764939
J Clin Microbiol. 1997 Aug;35(8):2160-2
9230405
Appl Environ Microbiol. 2007 May;73(9):2891-6
17369349
Orv Hetil. 1995 Jul 9;136(28):1497-9
7637966
Mol Cells. 2007 Dec 31;24(3):329-37
18182847
Meat Sci. 2005 Jul;70(3):461-75
22063745
Scand J Clin Lab Invest. 2008;68(1):77-80
18224558
Int J Food Microbiol. 2003 Dec 31;89(2-3):241-9
14623390
J AOAC Int. 2002 Sep-Oct;85(5):1025-8
12374399
Int J Food Microbiol. 2004 Mar 1;91(2):141-5
14996457
Adv Microb Physiol. 2009;55:183-265, 320
19573697
Clin Microbiol Rev. 2006 Apr;19(2):298-314
16614251
BMC Genomics. 2009 Mar 19;10:115
19298644
Pediatr Neurol. 2008 Jan;38(1):55-7
18054696
Clin Infect Dis. 1999 Dec;29(6):1381-7
10585782
J Food Prot. 2001 Feb;64(2):201-7
11271768
APMIS. 2004 Nov-Dec;112(11-12):808-14
15688523
Mol Cell Probes. 2002 Oct;16(5):335-9
12477437
J Protein Chem. 1993 Jun;12(3):351-63
8397793
Infect Immun. 2005 Sep;73(9):5450-7
16113261
J AOAC Int. 1996 Jul-Aug;79(4):861-5
8757444
Br J Biomed Sci. 1997 Sep;54(3):166-73
9499593
Emerg Infect Dis. 1998 Jul-Sep;4(3):506-7
9716988
Curr Microbiol. 1998 Nov;37(5):312-8
9767710
J Appl Microbiol. 2006 Sep;101(3):556-70
16907806
J Clin Microbiol. 1994 Aug;32(8):1911-7
7989542
J Food Prot. 2002 Aug;65(8):1267-70
12182478
J Mol Biol. 2006 Sep 29;362(4):733-42
16938310
J Clin Microbiol. 2005 Aug;43(8):4280-2
16082001
FEMS Microbiol Lett. 2006 Aug;261(1):88-94
16842364
J Appl Bacteriol. 1993 Sep;75(3):234-9
8244901
J Bacteriol. 2007 Dec;189(23):8643-50
17905976
Appl Environ Microbiol. 2001 Dec;67(12):5694-9
11722924
J Clin Microbiol. 2004 May;42(5):1863-8
15131141
Appl Environ Microbiol. 2004 Dec;70(12):7192-9
15574917
J Clin Microbiol. 2002 Jun;40(6):2260-2
12037104
Appl Environ Microbiol. 2002 Dec;68(12):5870-6
12450805
Appl Environ Microbiol. 1995 Jan;61(1):389-92
7887623