A simple and rapid molecular detection of Staphylococcus aureus strain B using multiplex PCR

Fatemeh  Sheikhi; Mehdi  Zeinoddini; Afshin  Samimi Nemati; Saeed  Veysi

Authors

Fatemeh Sheikhi Malek Ashtar University of Technology, Tehran, Iran
Mehdi Zeinoddini Malek Ashtar University of Technology, Tehran, Iran
Afshin Samimi Nemati Malek Ashtar University of Technology, Tehran, Iran
Saeed Veysi Young Researchers and Elite Club, Rasht Branch, Islamic Azad University, Rasht, Iran

Keywords:

Staphylococcucs, detection, multiplex PCR, sensitivity

Abstract

Background: Staphylococcucs aureus (S. aureus) is one of the most important and pathogenic species from the Staphylococcaceae family. S. aureus is capable of producing several enterotoxins (SEs) that are classified in different groups from A-R.

Objective: The present study aimed to set up simple and rapid detection of S. aureus strain B to develop a multiplex polymerase chain reaction (PCR) assay for the identification of genus and strain B of this bacterium.

Methods: In this work, after designing specific primers for 23S rRNA and SEB, one step multiplex PCR was carried out and the PCR product analyzed by agarose gel electrophoresis. The specificity of this method was determined using different strains of S. aureus A (SEA), S.aureus C (SEC), Bacillus subtilis, Bacillus cereus, and Listeria monocytogenes. Also, the sensitivity was distinguished by serial dilution of the DNA genome from S. aureus.

Results: The PCR product was indicated at 226 and 1,250 bp specific amplified fragments according to amplification of SEB and 23SrRNA, respectively. The sensitivity of this method determined about 2 $gif.latex?\times$ 10³ CFU/ml and also the results showed that according to specific primers, the PCR is very specific.

Conclusion: This multiplex PCR assay can be used as a simple diagnostic test in clinical laboratories for identification of S. aureus.

Downloads

Download data is not yet available.

References

Vannuffel P, Gigi J, Ezzedine H, Vandercam B, DelmeeM, Wauters G, et al. Specific detection of methicillinresistant Staphylococcus species by multiplex PCR. J Clin Microbiol 1995;33:2864-7.

https://doi.org/10.1128/jcm.33.11.2864-2867.1995

Abdel-moein KA, El-Hariri M, Samir A. Methicillinresistant Staphylococcus aureus: an emerging pathogen of pets in Egypt with a public health burden. Transbound Emerg Dis 2012;59:331-5.

https://doi.org/10.1111/j.1865-1682.2011.01273.x

Ahari H, Shahbazzadeh D, Misaghi A. Selective amplification of SEA, SEB and SEC genes by multiplex PCR for rapid detection of Staphylococcus aureus. Pak J Nutr 2009;8:1224-8

https://doi.org/10.3923/pjn.2009.1224.1228

Alizadeh S, Amini K. Determining the presence of virulence genes Panton Valentine Leukocidin PVL and methicillin resistance gene MECA in Staphylococcus aureus strains isolated from food samples by multiplex PCR and antibiotic resistance. J Food Microbiol 2015;2:49-58.

Balaban N, Rasooly A. Staphylococcal enterotoxins. Int J Food Microbiol 2000;61:1-10.

https://doi.org/10.1016/S0168-1605(00)00377-9

Salari Sharif A, Sattari M, Moradi M, Shahrokhabad R. Detection of Staphylococcus aureus Entrotoxin Genes A & B in Clinical Samples of the Patients Referring to the Medical Centers of Kerman and Rafsanjan Cities by PCR Technique. JRUMS 2012; 11:128-36.

Ertas N, Gonulalan Z, Yildirim Y, Kum E. Detection of Staphylococcus aureus enterotoxins in sheep cheese and dairy desserts by multiplex PCR technique. Int J Food Microbiol 2010;142:74-7.

https://doi.org/10.1016/j.ijfoodmicro.2010.06.002

Bianchi DM, Gallina S, Bellio A, Chiesa F, Civera T, Decastelli L. Enterotoxin gene profiles of Staphylococcus aureus isolated from milk and dairy productsin Italy. Lett Appl Microbiol 2014;58:190-6.

https://doi.org/10.1111/lam.12182

Lues AJ, Human I. The occurrence of indicator bacteria on hands and aprons of food handlers in the delicatessen sections of a retail group. Food Control 2007;18:326-32.

https://doi.org/10.1016/j.foodcont.2005.10.010

Eshraghi S, Salehipour Z, Pourmand M, Rahimi FA, Zahraei SMT, Agha AS, et al. Prevalence of tst, entC, entA and entA/C genes in Staphylococcus aureus strains isolated from different foods. Tehran Univ Med J 2009;67:470-6.

Akineden O, Hassan AA, Schneider E, Usleber E. Enterotoxigenic properties of Staphylococcus aureus isolated from goats' milk cheese. Int J Food Microbiol 2008;124:211-6.

https://doi.org/10.1016/j.ijfoodmicro.2008.03.027

Sospedra I, Soriano JM, Manes J. Enterotoxinomics: The omic sciences in the study of staphylococcal toxins analyzed in food matrices. Food Res Int 2013; 54:1052-60.

https://doi.org/10.1016/j.foodres.2013.03.002

Saadati M, Barati B, Doroudian M, Shirzad H, Hashemi M, Mostafa Hosseini S, et al. Detection of Sea, Seb, Sec, Seq genes in Staphylococcus aureus isolated from nasal carriers in Tehran province, Iran; by multiplex PCR. J Paramed Sci 2011;2:34-40.

Ranjbar R. A review of different methods used for detection of toxins. J Mil Med 2008;10:1-10.

Mousa WS. Prevalence and multiplex PCR for enterotoxin genes of Staphylococcus aureus isolates from subclinical mastitis and kareish cheese. IJVSM 2017;2:29-33.

https://doi.org/10.4172/2329-8731.1000174

Mousa WS, Abdeen E, Hussein H, Hadad G. Prevalence and multiplex PCR for enterotoxin genes of Staphylococcus aureus isolates from Subclinical Mastitis and Kareish Cheese. J Infect Dis Preve Med 2017;5:174. https://doi.org/10.4172/2329-8731.1000174

Omoe K, Ishikawa M, Shimoda Y, Hu DL, Ueda S, Shinagawa K. Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates Harboring seg, seh, or sei genes. J Clin Microbiol 2002;40:857-62.

https://doi.org/10.1128/JCM.40.3.857-862.2002

Yang H, Ma X, Zhang X, Wang Y, Zhang W. Development and evaluation of a loop-mediated isothermal amplification assay for the rapid detection of Staphylococcus aureus in food. Eur Food Res Technol 2011;232:769-76. https://doi.org/10.1007/s00217-011-1442-8

Imani Fooladi A, Tavakoli H, Naderi A. Detection of enterotoxigenic Staphylococcus aureus isolates in domestic dairy products. Iran J Microbiol 2010;2:137-42.

Bokaeian M, Saeidi S, Hassanshahian M. Molecular detection of Staphylococcus aureus enterotoxin A and B genes in clinical samples from patients referred to health centers in Zahedan City. Res Mol Med 2016;4:44-6. https://doi.org/10.18869/acadpub.rmm.4.2.44

Sheikhi F, Zeinoddini M, Saeedinia AR, Rouhani NH, Monazah A. Molecular detection of enterotoxin B from Staphylococcus aureus. J Infect Dis Diagn 2017;2:100117.

Sowmya N, Thakur MS, Manonmani HK. Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods. J Appl Microbiol 2012;113:106-13. https://doi.org/10.1111/j.1365-2672.2012.05315.x

Ahani N, Alipour-Eskandani M. Detection ofenterotoxigenic Staphylococcus aureus in Schizothorax zarudnyi using PCR method. Zahedan J Res Med Sci 2014;16:29-31.

https://doi.org/10.1007/s12032-014-0252-9

Najera-Sanchez G, Maldonado-Rodriguez R, Ruiz Olvera P, de la Garza LM. Development of two multiplex polymerase chain reactions for the detection of enterotoxigenic strains of Staphylococcus aureus isolated from foods. J Food Prot 2003;66:1055-62. https://doi.org/10.4315/0362-028X-66.6.1055

Nagaraj S, Ramlal S, Sripathy MH, Batra HV. Development and evaluation of a novel combinatorial selective enrichment and multiplex PCR technique for molecular detection of major virulence-associated genes of enterotoxigenic Staphylococcus aureus in food samples. J Appl Microbiol 2014;116:435-46.

https://doi.org/10.1111/jam.12364