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Indian Journal of Microbiology logoLink to Indian Journal of Microbiology
. 2009 Jun 5;49(3):283–289. doi: 10.1007/s12088-009-0032-y

Simultaneous detection of pathogenic B. cereus, S. aureus and L. monocytogenes by multiplex PCR

T D Kalyan Kumar 1,✉, H S Murali 1, H V Batra 1
PMCID: PMC3450020  PMID: 23100783

Abstract

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus are of major concern for food safety in terms of frequency and seriousness of the disease. The occurrence these three important pathogens and their coexistence in food matrices are predominant. Moreover, symptoms associated with B. cereus and S. aureus food poisoning not only closely resembles each other but can also be overlapping with other foodborne infections. In this context, detection of these three pathogens simultaneously in food samples by a single multiplex PCR (mPCR) would have advantages in terms of rapidity and cost saving, when compared with single organism specific PCRs. mPCR has been standardized by targeting three major diarrheal enterotoxin genes hbl A, cyt K and nhe A of B. cereus, virulence associated nuc and Ent B genes of S. aureus and virulence associated hly and iap genes of L. monocytogenes along with internal amplification control (IAC). The results showed that mPCR accurately identified all the three organisms individually or in combination without non-specificity. The mPCR was able to detect as low as 10 to 100 organisms per ml of growth following overnight enrichment of spiked food samples (vegetable biriyani and milk) and their presence in naturally contaminated samples also. The high throughput and cost effective multiplex PCR method developed in this study could provide a powerful tool for simultaneous, rapid and reliable detection of B. cereus, S. aureus and L. monocytogenes in food samples.

Keywords: Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, mPCR, IAC, Pathogens

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References

  • 1.Choma C., Guinebretiere M.H., Carlin F., Schmitt P., Velge P., Granum P.E., Nguyen-The C. Prevalence, characterization and growth of B. cereus in commercial cooked chilled foods containing vegetables. J Appl Microbiol. 2000;88:617–625. doi: 10.1046/j.1365-2672.2000.00998.x. [DOI] [PubMed] [Google Scholar]
  • 2.Kathariou S. L. monocytogenes virulence and pathogenicity, a food safety perspective. J Food Protection. 2002;65:1811–1829. doi: 10.4315/0362-028x-65.11.1811. [DOI] [PubMed] [Google Scholar]
  • 3.Balaban N., Rasooly A. Staphylococcal enterotoxins. Int J Food Microbiol. 2000;61:1–10. doi: 10.1016/S0168-1605(00)00377-9. [DOI] [PubMed] [Google Scholar]
  • 4.Holeckova B., Holoda E., Fotta M., Kalinacova V., Gondol M., Grolmus J. Occurrence of enterotoxigenic S. aureus in food. Annals Agricultural Environ Med. 2002;9:179–182. [PubMed] [Google Scholar]
  • 5.Loir Y., Baron F., Gautier M. S. aureus and food poisoning. Gen Mol Res. 2003;2:63–76. [PubMed] [Google Scholar]
  • 6.Granum P.E., Lund T. B. cereus and its food poisoning toxins. FEMS Microbiol Lett. 1997;157:223–228. doi: 10.1111/j.1574-6968.1997.tb12776.x. [DOI] [PubMed] [Google Scholar]
  • 7.McKillip J.L. Prevalence and expression of enterotoxins in B. cereus and other B. spp., a literature review. Antonie van Leeuwenhoek. 2000;77:393–399. doi: 10.1023/A:1002706906154. [DOI] [PubMed] [Google Scholar]
  • 8.Ryser E.T. Foodborne listeriosis. In: Ryser E.T., Marth E.H., editors. L. listeriosis, and food safety. 2nd ed. New York, NY: Marcel Dekker, Inc.; 1999. pp. 299–358. [Google Scholar]
  • 9.Gilmore M.S., Cruz-Rodz A.L., Leimeister-Wachter M., Kreft J., Goebel W. A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage. J Bacteriol. 1989;171:744–753. doi: 10.1128/jb.171.2.744-753.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Hardy S.P., Lund T., Granum P.E. CytK toxin of B. cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia. FEMS Microbiol Lett. 2001;197:47–51. doi: 10.1111/j.1574-6968.2001.tb10581.x. [DOI] [PubMed] [Google Scholar]
  • 11.Lindbäck T., Fagerlund A., Rodland M.S., Granum P.E. Characterization of the B. cereus Nhe enterotoxin. SGM Microbiology (UK) 2004;150:3959–3967. doi: 10.1099/mic.0.27359-0. [DOI] [PubMed] [Google Scholar]
  • 12.Brakstad O.G., Aasbakk K., Maeland J.A. Detection of S. aureus by polymerase chain reaction amplification of the nuc gene. J Clin Microbiol. 1992;30:1654–1660. doi: 10.1128/jcm.30.7.1654-1660.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Kim C.H., Khan M., Morin D.E., Hurley W.L., Tripathy D.N., Kehrli M., Oluoch A.O., Kakoma I. Optimization of the PCR for detection of S. aureus nuc gene in bovine milk. J Dairy Sci. 2001;84:74–83. doi: 10.3168/jds.S0022-0302(01)74454-2. [DOI] [PubMed] [Google Scholar]
  • 14.Johnson W.M., Tyler S.D., Ewan E.P., Ashton E., Wang G., Rozee K.R. Detection of genes coding for listeriolysin and L. monocytogenes antigen A (lmaA) in L. spp. by the polymerase chain reaction. Microb Pathog. 1992;12:79–86. doi: 10.1016/0882-4010(92)90068-Y. [DOI] [PubMed] [Google Scholar]
  • 15.Hein I., Klein D., Lehner A., Bubert A., Brandl E., Wagner M. Detection and quantification of the iap gene of L. monocytogenes and L. innocua by a new real-time quantitative PCR assay. Res Microbiol. 2001;152:37–46. doi: 10.1016/S0923-2508(00)01166-9. [DOI] [PubMed] [Google Scholar]
  • 16.Köhler S., Leimeister-Wachter M., Chakraborty T., Lottspeich F., Goebel W. The gene coding for protein p60 of L. monocytogenes and its use as a specific probe for L. monocytogenes. Infect Immun. 1990;58:1943–1950. doi: 10.1128/iai.58.6.1943-1950.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Hoorfar J., Cook N., Malorny B., Wagner M., Medici D., Abulmawjood A. Making internal amplification control mandatory for diagnostic PCR. J Clin Microbiol. 2003;41:5835. doi: 10.1128/JCM.41.12.5835.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Kumar S., Balakrishna K., Batra H.V. Detection of Salmonella enterica serovar typhi (S. typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in multiplex format. Lett Appl Microbiol. 2006;42:149–154. doi: 10.1111/j.1472-765X.2005.01813.x. [DOI] [PubMed] [Google Scholar]
  • 19.Theron J., Morar D., Preez M., Brözel V.S., Venter S.N. A sensitive seminested PCR method for the detection of Shigella in spiked environmental water samples. Water Res. 2001;35:869–874. doi: 10.1016/S0043-1354(00)00348-1. [DOI] [PubMed] [Google Scholar]
  • 20.Furrer B., Candrian U., Hoefelein C., Luethy J. Detection and identification of L. monocytogenes in cooked sausage products and in milk by in vitro amplification of hemolysin gene fragments. J Appl Bacteriol. 1991;70:372–379. doi: 10.1111/j.1365-2672.1991.tb02951.x. [DOI] [PubMed] [Google Scholar]
  • 21.Andersen Borge G.I., Skeie M., Sorhaug T., Langsrud T., Granum P.E. Growth and toxin profiles of B. cereus isolated from different food sources. Int J Food Microbiol. 2001;69:237–246. doi: 10.1016/S0168-1605(01)00500-1. [DOI] [PubMed] [Google Scholar]

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