Use of Real-Time PCR and High-Resolution Melting Analysis for Detection and Discrimination of Salmonella typhimurium and Salmonella enteritidis in Contaminated Raw-Egg Samples

الموضوعات :

H. Ahari ¹ (Associate Professor of the Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran.)
B. Fahimi ² (MSc Student of the Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran.)
N. Sheikhi ³ (Assistant Professor of the Department of Hygiene, Science and Research Branch, Islamic Azad University, Tehran, Iran.)
A.A. Anvar ⁴ (Assistant Professor of the Department of Hygiene, Science and Research Branch, Islamic Azad University, Tehran, Iran.)
S. Paidari ⁵ (MSc Student of the Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran.)

تاريخ الإرسال : 15 الأربعاء , جمادى الأولى, 1442 تاريخ التأكيد : 15 الأربعاء , جمادى الأولى, 1442 تاريخ الإصدار : 17 الجمعة , جمادى الأولى, 1442

الکلمات المفتاحية: HRM, Melting Curve, Real Time-PCR, Salmonella enteritidis, Salmonella typhimurium,

ملخص المقالة :

In order to evaluate the efficacy of high-resolution melting (HRM) analysis in the detection and discrimination of S. typhimurium and S. enteritidis, this study was conducted on 30 samples of raw eggs, where 14 treatments were employed: 4 groups of samples contaminated with Salmonella typhimurium, 4 with S. enteritidis, 4 with a mixture of both, as well as 2 control groups. DNA was extracted from the contaminated samples and used for preparation of a target DNA sequence using an invA gene-specific primer pair. RT-PCR identified and detected Salmonella in the contaminated egg samples within less than 24 hours, and HRM analysis proved that this method is suitable for the discrimination of the S. enteritidis and S. typhimurium species. The sensitivity of this method was equal to 200 Salmonella cells per microliter, and HRM analysis could distinguish S. enteritidis and S. typhimurium and their combination with high consistency (R2 > 0.993).

المصادر:

Ahari, H., Karim, G., Anvar, S. A., Paidari, S., Mostaghim, S. A. & Mazinani, A. S. (2020) Method for producing antimicrobial nanofilms packaging cover based on titanium nano-dioxide through extrusion for extension of food shelf-life. Google Patents

Anvar, A., Haghighat Kajavi, S., Ahari, H., Sharifan, A., Motallebi, A., Kakoolaki, S. & Paidari, S. (2019). Evaluation of the antibacterial effects of Ag-TiO2 nanoparticles and optimization of its migration to sturgeon caviar (Beluga). Iranian Journal of Fisheries Sciences, 18 (4), 954-967.

Bratchikov, M. & Mauricas, M. (2011). Development of a multiple-run high-resolution melting assay for Salmonella spp. genotyping: HRM application for Salmonella spp. subtyping. Diagnostic Microbiology and Infectious Disease, 71, 192-200.

Chen, W., Martinez, G. & Mulchandani, A. (2000.) Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella. Analytical Biochemistry, 280, 166-172.

Day, J., Basavanna, U. & Sharma, S. ( 2009). Development of a cell culture method to isolate and enrich Salmonella enterica serotype enteritidis from shell eggs for subsequent detection by real-time PCR. Applied and Environmental Microbiology, 75, 5321-5327.

De Medici, D., Croci, L., Delibato, E., Di Pasquale, S., Filetici, E. & Toti, L. ( 2003). Evaluation of DNA extraction methods for use in combination with SYBR green I real-time PCR to detect Salmonella enterica serotype enteritidis in poultry. Applied Environtal Microbiology, 69, 3456-3461.

De Smedt, J. M., Bolderdijk, R. F., Rappold, H. & Lautenschlaeger, D. (1986). Rapid Salmonella detection in foods by motility enrichment on a modified semi-solid Rappaport-Vassiliadis medium. Journal of Food Protection, 49, 510-514.

Gbassi, G. K. & Vandamme, T. (2012). Probiotic encapsulation technology: from microencapsulation to release into the gut. Pharmaceutics, 4(1), 149-163.

Hein, I., Flekna, G., Krassnig, M. & Wagner, M. (2006). Real-time PCR for the detection of Salmonella spp. in food: an alternative approach to a conventional PCR system suggested by the Food-PCR project. Journal of Microbiology Methods, 66, 538-547.

Hosseini, R., Ahari, H., Mahasti, P. & Paidari, S. (2017). Measuring the migration of silver from silver nanocomposite polyethylene packaging based on (TiO 2) into penaeus semisulcatus using titration comparison with migration methods. Fisheries Science, 83(4), 649-659.

Janda, J. M. & Abbott, S. L. (2006). The genus Hafnia: from soup to nuts. Clin. Microbiology Reviewes, 19, 12-28.

Malorny, B., Paccassoni, E., Fach, P., Bunge, C., Martin, A. & Helmuth, R. (2004). Diagnostic real-time PCR for detection of Salmonella in food. Applied and Environmental Microbiology, 70, 7046-7052.

Seo, K. & Brackett, R. (2005). Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay. Journal of Food Protection, 68, 59-63.

 Singh, P. & Mustapha, A. (2014). Development of a real-time PCR melt curve assay for simultaneous detection of virulent and antibiotic resistant Salmonella. Food Microbiology, 44, 6-14.

Souza, R. A., Frazão, M. R., Almeida, A. M. & Falcão, J. P. (2015). Rapid and efficient differentiation of Yersinia species using high-resolution melting analysis. Journal of  Microbiology Methods, 115, 6-12.

van Blerk, G., Leibach L., Mabunda, A., Chapman, A. & Louw, D. (2011). Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis. Water Science and Technology, 64, 2453-2459.

Vellai, T. & Vida, G. (1999). The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells. Proc. R. Soc. London, Ser. B. 266: 1571-1577.

Vossen, R. H., Aten, E., Roos, A. & den Dunnen, J. T. (2009). High‐Resolution Melting Analysis (HRMA)—More than just sequence variant screening. Human Mutation, 30, 860-866.

Wolffs, P. F., Glencross, K., Thibaudeau, R. & Griffiths, M. W. (2006). Direct quantitation and detection of salmonellae in biological samples without enrichment, using two-step filtration and real-time PCR. Applied and Environmental Microbiology, 72, 3896-3900.

Xiao, X., Zhang, L., Wu, H., Yu, Y. G., Tang, Y. Q. & Liu, D. M. (2014). Simultaneous detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus by multiplex real-time PCR assays using high-resolution melting. Food and Methods, 7, 1960-1972.

Zhang, G., Brown, E. W. & González-Escalona, N. (2011). Comparison of real-time PCR, reverse transcriptase real-time PCR, loop-mediated isothermal amplification, and the FDA conventional microbiological method for the detection of Salmonella spp. in produce. Applied and Environmental Microbiology, 77, 6495-6501.