A Review of the development in the multiplex PCR technique for the detection of Bacillus cereus (original) (raw)
Related papers
2011
Bacillus cereus food poisoning has been implicated in outbreak episodes in the world. Bacillus cereus is a potential food safety hazard causesing two different types of food poisoning, the diarrhea type, caused by hemolysin BL and non-hemolytic enterotoxin, and the emetic type, caused by the emetic toxin cereulide. In Kenya the organism has been isolated from pasteurized milk at 41.2%, 26% of which were enterotoxigenic. The aim of this study was to, develop a multiplex polymerase chain reation (PCR) technique that can be used to detect enterotoxigenic B. cereus in food, determine the level of contamination in various foods that will enable gene detection, and to develop suitable food processing procedures that can reduce PCR inhibitory substances to enable use of direct multiplex PCR for detection of toxins genes in various foods. In this study, seven pairs of primers for multiplex PCR were used for simulteneous detection of 6 genes and a specific sequence encording hemolysin BL, no...
Antonie van Leeuwenhoek, 2010
Identification of Bacillus cereus sensu stricto is a challenge for the food industry since it is being increasingly reported as implicated in many foodborne outbreaks. So far no conclusive microbiological or biochemical traits have been described for their specific differentiation. Here a polyphasic approach aiming at identification of new isolates is presented. It was conducted on a total of 75 strains, 59 Bacillus cereus group (29 reference strains and 30 food and environmental isolates) and 16 other Bacillus species. It includes biochemical traits (API 50CH and API 20E) and genetic profiles: PCR amplification of the internal spacer region (ISR) between 23S and 16S rRNA genes (ISR-PCR), randomly amplified polymorphic DNA (RAPD-PCR) with three universal primers (M13, T3, and T7), and PCR amplification using specific primers directed to genes encoding hemolysin BL (hbl), cytotoxin K (cytK) and cereulide (ces). As expected, PCR-enterotoxin profiles revealed the toxigenic potential of strains within the B. cereus group irrespective of the species. Cluster analysis combining the three RAPD fingerprints (RAPD-M13, RAPD-T3 and RAPD-T7) allowed almost a complete separation of strains within the B. cereus group. As a result, the ISR-PCR profile is proposed for the rapid assignation of isolates to B. cereus group with the advantage over the API profile of being a specific and culture-independent technique. Following, differentiation at species level can be obtained by RAPD profiles analysis.
Current Journal of Applied Science and Technology
Aims: Experiments were conducted to evaluate the specificity and rapidity of the application of conventional, immunoassay and direct Polymerase Chain Reaction (dPCR) techniques in the detection of Bacillus cereus and its toxins in contaminated rice. Place and Duration of Study: Department of Life Sciences, Glasgow Caledonian University, UK, between May 2015 and December 2015. Methodology: Conventional testing for the presence of B. cereus and associated toxins was achieved using Polymixin Egg-yolk Mannitol Agar (PEMBA) culture plates while immunoassays were conducted using a commercially available Reverse Passive Latex Agglutination (TD0950 BCET-RPLA, Oxoid, UK) kit. Direct PCR was used to detect the HBL-E gene in the food samples and the PCR amplicons were visualised after separation by gel electrophoresis. Results: Total Mean B. cereus count was recorded as 5.3 ×107 cfu / g of the rice sample from the PEMBA plate culture. The PEMBA method was the least in terms of rapidity of assa...
International Journal of Food Microbiology, 2002
Isolates of Bacillus cereus from traditional Indian foods were detected by colony hybridization using the PCR-generated phospholipase (PL-1) probe. In all, 29 isolates picked up by the probe were confirmed as B. cereus by conventional cultural and biochemical characteristics. All the isolates reacted positively in PCR with phospholipase (PL-1) primers. Among the native isolates, 11 of them showed the discontinuous pattern of haemolysin BL activity in gel diffusion assay. Though 14 isolates reacted positively in PCR with primers (Ha-1) specific to the B gene of haemolysin BL, only four of them showed both the presence of gene and haemolysin BL activity. More than 50% of the isolates indicated their potential enterotoxigenicity by reacting positively with primers specific for the BceT gene encoding for diarrhoeal enterotoxin. PCR with primers for different inverse (IS) repeat elements revealed that isolates carrying transposon IS 231 -P 231-1 did not carry IS 240 -P 240. Some of the isolates were devoid of these IS elements. The study demonstrated the potential of using of a PCR-generated labelled PL-1 probe for the direct detection of B. cereus in food samples and PCR for characterizing the toxigenic isolates. D
International Journal of Food Microbiology, 2009
A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.
Detection and quantification of viable Bacillus cereus in food by RT–qPCR
European Food Research and Technology, 2011
A reverse-transcription real-time PCR (RT-qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C (pc-plc) mRNA, was developed for the specific detection and quantification of viable Bacillus cereus. Initial experiments focused on evaluating the performance of various RNA extraction kits and optimizing the DNase I digestion. After optimization, RNA from B. cereus was isolated, and following DNase treatment, the RNA was amplified by RT-qPCR. The assay was used to construct a calibration curve from purified RNA of B. cereus CECT 148 T , and it had a wide quantification range of 5 log units. The detection limit was 30 CFU per reaction and the efficiency 0.88. When the developed methodology was applied in artificially contaminated liquid egg, the detection limit was found to be 850 CFU per reaction or 1.1 9 10 4 CFU per mL of food sample without an enrichment step. To the best of our knowledge, this is the first time that an assay for the detection and quantification of viable B. cereus in food has been described.
Applied and Environmental Microbiology, 2000
A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5-to-3 exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probablenumber technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.
Novel Multiplex PCR Assay for Rapid Detection of Five Bacterial Foodborne Pathogens
2017
Milk and dairy products can harbor varieties of foodborne pathogens especially Bacillus cereus, Escherichia coli, Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus. In this work, a rapid multiplex polymerase chain reaction (m-PCR) method for simultaneous detection of 5 major foodborne pathogens in milk was developed. Specific primers targetting the enterotoxin FM, uspA, prfA, fimY, and eap genes were selected for specific detection of B. cereus, E. coli, L. monocytogenes, Salmonella spp., and S. aureus, respectively. The optimum concentrations of the primers in the m-PCR reaction were 0.04 μM enterotoxin FM, 0.12 μM uspA, 0.16 μM prfA, 0.04 μM fimY, and 0.2 μM eap. The expected polymerase chain reaction (PCR) products of 513, 884, 398, 315, and 230 bp were detected from the specific amplification of B. cereus, E. coli, L. monocytogenes, Salmonella spp., and S. aureus, respectively. Cross-amplifications from non-target bacteria isolated from raw milk samples were not...
Direct detection of Bacillus cereus enterotoxin genes in food by multiplex Polymerase Chain Reaction
2000
This study evaluated a direct multiplex PCR to detect food contamination with enterotoxigenic Bacillus cereus (B. cereus) in comparison with culture and multiplex gene detection using colonies. Detection of B. cereus enterotoxin genes was done on artificially contaminated and ready-to- eat market foods including cooked rice, pasteurized milk and cheese. Of the 108 food samples analysed, 51(47.2 % were found
Systematic and Applied Microbiology, 1999
Toxin production, biochemical properties and ribotypes of Bacillus cereus group (B. cereus, B. thuringiensis, B. mycoides) strains originating from industrial and environmental sources (n = 64), from food poisoning incidents (n = 22) and from reference sources (n = 7) were analysed. Forty ribotypes were found among the 93 strains. Eleven strains from food poisoning incidents produced emetic (mitochondrio) toxin, as determined by the boar spermatozoa toxicity test. These strains possessed closely similar riborypes which were rare among strains of other origins. Sperm toxin producing (cereulide positive) strains did not hydrolyse starch and did not produce haemolysin BL, as determined by the reverse passive latex agglutination test. Sixteen different ribotypes were found among B. cereus strains from board machines (n = 16) and from packaging board (n = 16), indicating many different sources of B. cereus contamination in board mills. Strains originating from packaging board had predominantly different ribotypes from those of dairy and dairy product originating strains. Nine (53%) out of 17 strains from a single dairy process shared the same ribotype whereas strains from milk and milk products from different dairies had different ribotypes indicating that B. cereus group populations were dairy specific. Twenty-two percent of strains isolated from the paperboard industry on non-selective medium were lecithinase negative, including enterotoxin producing strains. This stresses the importance of other detection methods not based on a positive lecithinase reaction.