Designing a SYBR Green Absolute Real time PCR Assay for Specific Detection and Quantification of Bacillus subtilis in Dough Used for Bread Making

Journal of Cell and Molecular Research

مقاله 4، دوره 6، شماره 2 - شماره پیاپی 12، فروردین 2014، صفحه 83-92   

نوع مقاله: Research Articles

شناسه دیجیتال (DOI): 10.22067/jcmr.v6i2.39090

نویسندگان

Seyed Ali Mortazavi¹؛ Ahmad Reza Bahrami²؛ Balal Sadeghi^{* 3}؛ Maryam Moghaddam Matin⁴

¹Gorgan Uni of Agri Sci and Natu Resources

²Ferdowsi University of Mashhad, Iran

³University of kerman

⁴Ferdowsi University of Mashhad

چکیده

In present study, a SYBR green based real time PCR assay was developed for specific detection and quantification of Bacillus subtilis in dough used for bread making. New primer pairs were designed to amplify a 212 base pair fragment of the aprE gene. Specificity of these primer pairs was confirmed with conventional and real time PCR methods. Standard curves constructed using the threshold cycle (CT) versus copy numbers of B. subtilis showed good linearity for reference standards of cloned insert (R2=0.999, slope=-3.035) and also induced contaminated dough (R2=0.988, slope=-3.142), and the melting temperature (Tm=82.2 oC) was consistently specific for the amplicon. Limits of detection were 200 and 2000 colony forming units (CFUs) per ml or g of these samples, respectively. This real time PCR offers a fast tool with high sensitivity and specificity for detection and quantification of this rope-forming pathogen in dough used for bread making.

کلیدواژه‌ها

Real time PCR؛ Bacillus subtilis؛ New primer pairs؛ Contaminated dough

مراجع

Aarts H.J., Joosten, R.G., Henkens, M.H., Stegeman, H. and van Hoek Angela, H. (2001) Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains. Journal of Microbiological Methods47: 209-217.

Blackburn C.W. (2006) Food spoilage microorganisms. CRC Press. USA, 194-209 pp.

Bustin S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W. and Shipley, G.L. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical chemistry55: 611-622.

Dhanasekaran S., Doherty, T.M. and Kenneth, J. (2010) Comparison of different standards for real-time PCR-based absolute quantification. Journal of immunological methods354: 34-39.

Fenicia L., Anniballi, F., De Medici, D., Delibato, E. and Aureli, P. (2007) SYBR green real-time PCR method to detect Clostridium botulinum type A. Applied and Environmental Microbiology73: 2891-2896.

Fernandez-No I., Guarddon, M., Böhme, K., Cepeda, A., Calo-Mata, P. and Barros-Velazquez, J. (2011) Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR. Food microbiology28: 605-610.

Fricker M., Messelhäußer, U., Busch, U., Scherer, S. and Ehling-Schulz, M. (2007) Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks. Applied and Environmental Microbiology73: 1892-1898.

Fukushima H., Tsunomori, Y. and Seki, R. (2003) Duplex real-time SYBR green PCR assays for detection of 17 species of food-or waterborne pathogens in stools. Journal of Clinical Microbiology41: 5134-5146.

Fusco V., Quero, G.M., Morea, M., Blaiotta, G. and Visconti, A. (2011) Rapid and reliable identification of Staphylococcus aureusharbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR. International Journal of Food Microbiology144: 528-537.

Gibson U., Heid, C.A. and Williams, P.M. (1996) A novel method for real time quantitative RT-PCR. Genome research6: 995-1001.

Gore H.M., Wakeman, C.A., Hull, R.M. and McKillip, J.L. (2003) Real-time molecular beacon NASBA reveals hblC expression from Bacillus spp. in milk. Biochemical and biophysical research communications311: 386-390.

Gu W. and Levin, R.E. (2008) Innovative methods for removal of PCR inhibitors for quantitative detection of Plesiomonasshigelloides in oysters by real-time PCR. Food Biotechnology22: 98-113.

Inglis G.D. and Kalischuk, L.D. (2004) Direct quantification of Campylobacter jejuni and Campylobacter lanienae in feces of cattle by real-time quantitative PCR. Applied and Environmental Microbiology70: 2296-2306.

Jay J.M., Loessner, M.J. and Golden, D.A. (2005) Modern food microbiology. Springer. USA, 204-228 pp.

Jothikumar N. and Griffiths, M.W. (2002) Rapid detection of Escherichia coli O157: H7 with multiplex real-time PCR assays. Applied and Environmental Microbiology68: 3169-3171.

Jothikumar N., Wang, X. and Griffiths, M.W. (2003) Real-time multiplex SYBR green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes. Journal of Food Protection66: 2141-2145.

Juste A., Thomma, B. and Lievens, B. (2008) Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes. Food microbiology25: 745-761.

Kennedy S. and Oswald, N. (2011) PCR troubleshooting and optimization: the essential guide. Caister academic. USA, 139-149 pp.

Klein D. (2002) Quantification using real-time PCR technology: applications and limitations. Trends in molecular medicine8: 257-260.

Levin R.E. (2010) Rapid detection and characterization of foodborne pathogens by molecular techniques. CRC Press. USA, 8-20 pp.

Liu D. (2010) Molecular detection of foodborne pathogens. CRC Press. USA, pp 129-145.

Liu Y., Cai, X., Zhang, X., Gao, Q., Yang, X., Zheng, Z., Luo, M. and Huang, X. (2006) Real time PCR using TaqMan and SYBR Green for detection ofEnterobactersakazakii in infant formula. Journal of Microbiological Methods65: 21-31.

Mackay I.M. (2004) Real-time PCR in the microbiology laboratory. Clinical Microbiology and Infection10: 190-212.

Martinez-Blanch J., Sanchez, G., Garay, E. and Aznar, R. (2009) Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples. International Journal of Food Microbiology135: 15-21.

Martinez N., Martin, M.C., Herrero, A., Fernandez, M., Alvarez, M.A. and Ladero, V. (2011) qPCR as a powerful tool for microbial food spoilage quantification: Significance for food quality. Trends in Food Science & Technology22: 367-376.

McMeekin T.A. (2000) Detecting pathogens in food. CRC press. USA, pp 347-350.

Meroth C.B., Walter, J., Hertel, C., Brandt, M.J. and Hammes, W.P. (2003) Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis. Applied and Environmental Microbiology69: 475-482.

Nam H.-M., Srinivasan, V., Gillespie, B.E., Murinda, S.E. and Oliver, S.P. (2005) Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples. International Journal of Food Microbiology102: 161-171.

Nicholson W.L., Munakata, N., Horneck, G., Melosh, H.J. and Setlow, P. (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiology and Molecular Biology Reviews64: 548-572.

O'Mahony J. and Hill, C. (2002) A real time PCR assay for the detection and quantitation of Mycobacterium avium subsp. paratuberculosis using SYBR Green and the Light Cycler. Journal of Microbiological Methods51: 283-293.

Panicker G., Myers, M.L. and Bej, A.K. (2004) Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR. Applied and Environmental Microbiology70: 498-507.

Pavic S., Brett, M., Petric, I., Lastre, D., Smoljanovic, M., Atkinson, M., Kovacic, A., centinic, E. and Ropac, D. (2005) An outbreak of food poisoning in a kindergarten caused by milk powder containing toxigenic Bacillus subtilis and Bacillus licheniformis. ArchivfürLebensmittelhygiene56: 20-22.

Ponchel F., Toomes, C., Bransfield, K., Leong, F.T., Douglas, S.H., Field, S.L., Bell, S.M., Combaret, V., Puisieux, A. and Mighell, A.J. (2003) Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC biotechnology3: 18.

Postollec F., Falentin, H., Pavan, S., Combrisson, J. and Sohier, D. (2011) Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food microbiology28: 848-861.

Rosenkvist H. and Hansen, Å. (1995) Contamination profiles and characterisation of Bacillus species in wheat bread and raw materials for bread production. International Journal of Food Microbiology26: 353-363.

Sadeghi A., Mortazavi S.A., Bahrami A.R. and Sadeghi B. (2012) Design of multiplex PCR for simultaneous detection of rope forming Bacillus strains in Iranian bread dough. Journal of the Science of Food and Agriculture92: 2652-2656.

Sorokulova I., Reva, O., Smirnov, V., Pinchuk, I., Lapa, S. and Urdaci, M. (2003) Genetic diversity and involvement in bread spoilage of Bacillus strains isolated from flour and ropy bread. Letters in applied microbiology37: 169-173.

Sun F., Wu D., Qiu Z., Jin M. and Li J. (2010) Development of real-time PCR systems based on SYBR Green for the specific detection and quantification ofKlebsiellapneumoniae in infant formula. Food Control21: 487-491.

Wehrle E., Didier, A., Moravek, M., Dietrich, R. and Märtlbauer, E. (2010) Detection of Bacillus cereus with enteropathogenic potential by multiplex real-time PCR based on SYBR green I. Molecular and cellular probes24: 124-130.