Evaluation of real time PCR for detection of clinical isolates of Staphylococcus aureus and methicillin-resistance strains based on melting curve analysis method

authors:

avatar Narges Heydari , avatar Mohammad Yousef Alikhani , avatar Farid Azizi Jalilian , avatar Hamed Tahmasebi , avatar mohammad reza arabestani , *

Koomesh: Vol.19, issue 4; 877-886
published online: December 02, 2017
article type: Research Article
received: February 05, 2017
accepted: July 02, 2017

how to cite: Heydari N, Alikhani M Y, Azizi Jalilian F, Tahmasebi H, arabestani M R. Evaluation of real time PCR for detection of clinical isolates of Staphylococcus aureus and methicillin-resistance strains based on melting curve analysis method. koomesh. 2017;19(4):e152945. 

Abstract

Introduction: Rapid and timely detection of Staphylococcus aureus can play a significant role in the treatment of staphylococcal infections.Impresingly, by precise designing methods which have acceptable sensitivity and specificity, can identify species and even antibiotic-resistant strains. The purpose of this study was to evaluate the real time PCR diagnostic method based on the melting curve analysis for the detection of clinical isolates of S. aureus and the methicilin resistance gene. Materials and Methods: In this experimental study, clinical isolated of S. aureus was used from the Microbiology Bank of Hamadan University of Medical Sciences. The primer design was done by selecting (ITS) target for S. aureus and the mecA gene for methicillin-resistant strains. Real-time PCR and DNA melting curves analysis were used to determine the analytical specificity and sensitivity of the designed primers. Results: The analytical specificity of the primers was 83.79 ° C for S. aureus and 76.6 ° C for methicillin resistant Staphylococcus aureus respectively. The analytical sensitivity of the primers was 15 CFU/ml bacteria for ITS gene and 25 CFU/ml bacteria for mecA gene. Conclusion: By selecting appropriate primers and using sensitive molecular techniques, which could be the main factors for designing of both quick and accurate method, it is possible to identify invasive bacteria such as S. aureus.

References

  • 1.

    Seyed javadi SS, Alebouyeh M, Nazem Alhosseini Mojarad E, Zali MR. Frequency of class 1 integron and multidrug resistance pattern among isolates of Staphylococcus aureus from hospitalized patients and environmental samples in an intensive care unit in Tahran, Iran. Koomesh 2014; 15: 341-348.

  • 2.

    najar-peerayeh s, jazayeri moghadas a, bakhshi b. Staphylococcus epidermidis virulence factor and ability of macroscopic biofilm production. Koomesh 2016; 17: 918-923.

  • 3.

    Abdal N, Ghaznavirad E, Hamidi A, Hosseini D. Prevalence of genes encoding aminoglycoside resistant in methicillin-sensitive Staphylococcus aurous and coagulase-negative staphylococci isolated from hospital infectious. Koomesh 2014; 16: 82-89.

  • 4.

    Mehta S, Singh C, Plata KB, Chanda PK, Paul A, Riosa S, Rosato RR, Rosato AE. -Lactams Increase the antibacterial activity of daptomycin against clinical methicillin-resistant staphylococcus aureus strains and prevent selection of daptomycin-resistant derivatives. Antimicrob Agents Chemother 2012; 56: 6192-6200.

  • 5.

    Camussone CM, Calvinho LF. [Virulence factors of Staphylococcus aureus associated with intramammary infections in cows: relevance and role as immunogens]. Rev Argent Microbiol 2013; 45: 119-130.

  • 6.

    Poulsen MO, Jacobsen K, Thorsing M, Kristensen NR, Clasen J, Lillebaek EM, et al. Thioridazine potentiates the effect of a beta-lactam antibiotic against Staphylococcus aureus independently of mecA expression. Res Microbiol 2013; 164: 181-188.

  • 7.

    Zarei Koosha R, Mahmoodzadeh Hosseini H, Mehdizadeh Aghdam E, Ghorbani Tajandareh S, Imani Fooladi AA. Distribution of tsst-1 and mecA Genes in Staphylococcus aureus Isolated From Clinical Specimens. Jundishapur J Microbiol 2016; 9: 290-257.

  • 8.

    Bokaeian M, Tahmasebi H. Molecular Identification of genes responsible for resistance to aminoglycosides and methicillin in clinical samples of staphylococcus aureus. J Babol Univ Med Sci 2017; 19: 38-46. (Persian).

  • 9.

    Zeyni B, Arabestani M, YuosefiMashof R, Tahmasebi H. Evaluation of Real-time PCR-based DNA melting method for detection of Enterococcus faecalis and Enterococcus faecium in clinical isolates. J Babol Univ Med Sci 2017; 19: 26-33. (Persian).

  • 10.

    Arabestani MR, Tahmasebi H, Zeyni B. Diagnostic value of melting curve analysis based on multiplex-real time PCR in identification of enterococci species. J Mazandaran Univ Med Sci 2017; 26: 234-247. (Persian).

  • 11.

    Tong SY, Xie S, Richardson LJ, Ballard SA, Dakh F, Grabsch EA, et al. High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms. PLoS One 2011; 6: 291-89.

  • 12.

    Tong SY, Giffard PM. Microbiological applications of high-resolution melting analysis. J Clin Microbiol 2012; 50: 3418-3421.

  • 13.

    Woksepp H, Jernberg C, Trnberg M, Ryberg A, Brolund A, Nordvall M, et al. High-resolution melting-curve aAnalysis of ligation-mediated real-time PCR for rapid evaluation of an epidemiological outbreak of extended-spectrum-beta-lactamase-producing escherichia coli. J Clin Microbiol 2011; 49: 4032-4039.

  • 14.

    Bokaeian M, Adabi J, Zeyni B, Tahmasebi H. The Presence of aac (6 ') Ie / aph (2 "), aph (3') - IIIa1, ant (4 ') - Ia1 genes and determining methicillin resistance in staphylococcus epidermidis and staphylococcus saprophyticus strains isolated from clinical specimens. Arak Med Univ J 2017; 19: 11-25. (Persian).

  • 15.

    Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; eighteenth informational supplement. CLSI document M100-18. Wayne, PA: Clinical and Laboratory Standards Institute. 2016.

  • 16.

    Tahmasebi H, Bokaeian M, Adabi J. Coagulase-negative, Beta-lactam, antibiotic resistance, methicillin resistance. Pars Jahrom Univ Med Sci 2016; 14: 55-63. (Persian).

  • 17.

    Wong YP, Chua KH, Thong KL. One-step species-specific high resolution melting analysis for nosocomial bacteria detection. J Microbiol Methods 2014; 107: 133-137.

  • 18.

    Liu Z, Zhang J, Rao S, Sun L, Zhang J, Liu R, et al. Heptaplex PCR melting curve analysis for rapid detection of plasmid-mediated AmpC beta-lactamase genes. J Microbiol Methods 2015; 110: 1-6.

  • 19.

    Xiao XL, Zhang L, & Wu H, Yu YG, Tang YQ, Liu DM, LI XF. Simultaneous Detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus by Multiplex Real-Time PCR Assays Using High-Resolution Melting. Food Analytical Methods 2014; 7: 1960-1972.

  • 20.

    Forghani F, Wei S, Oh DH. "A rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of bacillus cereus, Listeria monocytogenes, and staphylococcus aureus in Food." J Food Prot 2016; 79: 810-815.

  • 21.

    Clifford RJ, Milillo M, Prestwood J, Quintero R, Zurawski DV, Kwak YI, et al. " Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR." PLoS One 2012; 7: 48558.

  • 22.

    Yam WC, Siu GK, Ho PL, Ng TK, Que TL, Yip KT, et al. Evaluation of the LightCycler methicillin-resistant Staphylococcus aureus (MRSA) advanced test for detection of MRSA nasal colonization. J Clin Microbiol 2013; 51: 2869-2874.

  • 23.

    Bratchikov M, Mauricas M. Development of a multiple-run highresolution melting assay for Salmonella spp. genotyping: HRM application for Salmonella spp. subtyping. Diagn Microbiol Infect Dis 2011; 71: 192-200.

  • 24.

    Wongboot W, Chomvarin C, Engchanil C, Namwat W, Chaimanee P, Tirapattanun A, et al. Application of sybr green real-time quantitative pcr and conventional pcr for the detection of methicillin-resistant staphylococcus aureus in blood samples. Month 2016; 43: 129-134.

  • 25.

    Skow A, Mangold KA, Tajuddin M, Huntington A, Fritz B, Thomson RB, et al. Species-level identification of staphylococcal isolates by real-time PCR and melt curve analysis. J Clin Microbiol 2005; 43: 2876-2880.

  • 26.

    Krawczyk B, Leibner J, Baraska-Rybak W, Samet A, Nowicki R, Kur J. ADSRRS-fingerprinting and PCR MP techniques for studies of intraspecies genetic relatedness in Staphylococcus aureus. J Microbiol Methods 2007; 71: 114-122.

  • 27.

    Robinson D, Enright M. Multilocus sequence typing and the evolution of methicillinresistant Staphylococcus aureus. Clin Microbiol Infect 2004; 10: 92-97.