24. DETECTION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS USING LOOP MEDIATED ISOTHERMAL AMPLIFICATION
Main Article Content
Abstract
Objective: To develop a loop-mediated isothermal amplification (LAMP) method for detecting Methicillin - resistant Staphylococcus aureus (MRSA) using the characteristic mecA gene, aimed at rapid MRSA detection in nasal swab samples.
Methods: An experimental study was conducted on 60 nasal swab samples from healthy individuals aged 18-25. Specific primers for the LAMP reaction were designed using primer design software. The LAMP reaction conditions were optimized in the laboratory to determine the temperature, specificity, and minimum DNA detection limit (LOD). The optimized LAMP reaction was then tested on 60 DNA extraction samples from nasal swabs, including 30 MRSA-positive and 30 MRSA-negative samples.
Results: Specific primers for mecA gene were successfully designed, and the amplification reaction was optimized at 65°C for 30 minutes. The LAMP method demonstrated high sensitivity and specificity, with the lowest detection limit (LOD) of 1 pg of DNA extracted from MRSA per reaction, and no cross-reactivity with other species was observed. When applied to 60 DNA samples extracted from nasal swabs (30 MRSA-positive and 30 MRSA-negative), the LAMP reaction achieved 100% sensitivity, 100% specificity, and a Kappa coefficient of 1, indicating a high degree of agreement compared to traditional microbiological culture and PCR methods.
Conclusions: The study successfully applied the LAMP method to detect the presence of MRSA in clinical samples with high sensitivity and specificity using the mecA gene. Furthermore, the study demonstrated a high level of agreement between the LAMP method, microbiological culture, and PCR diagnostics. LAMP is a promising method for MRSA detection, serving as a rapid diagnostic tool with high applicability to support effective treatment.
Article Details
Keywords
LAMP, MRSA, nasal swab, isothermal amplification, mecA gene
References
[2] Adams C.E, Dancer S.J, Dynamic transmission of Staphylococcus aureus in the intensive care unit, International Journal of Environmental Research and Public Health, 17, Epub ahead of print 2020, doi: 10.3390/ijerph17062109.
[3] Howden B.P, Giulieri S.G, Wong Fok Lung T et al, Staphylococcus aureus host interactions and adaptation, Nature Reviews Microbiology, 21, Epub ahead of print 2023, doi: 10.1038/s41579-023-00852-y.
[4] David M.Z.S, Treatment of Staphylococcus aureus, Assess Eval High Educ, 2012, 37: 435.
[5] David M.Z, Daum R.S, Treatment of Staphylococcus aureus Infections, In: Bagnoli F, Rappuoli R, Grandi G (eds) Staphylococcus aureus: Microbiology, Pathology, Immunology, Therapy and Prophylaxis. Cham: Springer International Publishing, 2017, pp. 325-383.
[6] Kadariya J, Smith T.C, Thapaliya D, Staphylococcus aureus and Staphylococcal Food-Borne Disease: An Ongoing Challenge in Public Health, Biomed Res Int 2014, 2014: 827965.
[7] Lam J.C, Stokes W, The Golden Grapes of Wrath - Staphylococcus aureus Bacteremia: A Clinical Review, American Journal of Medicine, 136, Epub ahead of print 2023, doi: 10.1016/j.amjmed.2022.09.017.
[8] Misra N, Pu X, Holt D.N et al, Immunoproteomics to identify Staphylococcus aureus antigens expressed in bovine milk during mastitis, J Dairy Sci 2018, 101: 6296-6309.
[9] Lee A.S, De Lencastre H, Garau J et al, Methicillin-resistant Staphylococcus aureus, Nat Rev Dis Primers 2018, 4: 1-23.
[10] Cheung G.Y.C, Bae J.S, Otto M, Pathogenicity and virulence of Staphylococcus aureus, Virulence, 12, Epub ahead of print 2021, doi: 10.1080/21505594.2021.1878688.
[11] Huletsky A, Giroux R, Rossbach V et al, New Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococcus aureus Directly from Specimens Containing a Mixture of Staphylococci, J Clin Microbiol, 2004, 42: 1875-1884.
[12] Luteijn J.M, Hubben G.A.A, Pechlivanoglou P et al, Diagnostic accuracy of culture-based and PCR-based detection tests for methicillin-resistant Staphylococcus aureus: A meta-analysis, Clinical Microbiology and Infection, 2011, 17: 146-154.
[13] Okolie C.E, Wooldridge K.G, Turner D.P.J.J et al, Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes, BMC Microbiol, 2015, 15: 157.
[14] Milheiriço C, Oliveira D.C, De Lencastre H, Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus, Antimicrob Agents Chemother, Epub ahead of print, 2007, doi: 10.1128/AAC.00275-07.
[15] DeLeo F.R, Otto M, Kreiswirth B.N et al, Community-associated meticillin-resistant Staphylococcus aureus, The Lancet, 2010, 375: 1557-1568.
[16] Sheet O.H, Grabowski N.T, Klein G et al, Development and validation of a loop mediated isothermal amplification (LAMP) assay for the detection of Staphylococcus aureus in bovine mastitis milk samples, Mol Cell Probes, 2016, 30: 320-325.
[17] Ou A, Wang K, Mao Y et al, First Report on the Rapid Detection and Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) in Viable but Non-culturable (VBNC) Under Food Storage Conditions, Front Microbiol, 2021, 11: 1-7.
[18] Liu W, Dong D, Yang Z et al, Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method, Sci Rep, 2015, 5: 12723.
[19] Geng Y, Liu S, Wang J et al, Rapid Detection of Staphylococcus aureus in Food Using a Recombinase Polymerase Amplification-Based Assay, Food Anal Methods, 2018, 11: 2847-2856.
[20] Tran D.H, Tran H.T, Le U.P et al, Direct colorimetric LAMP assay for rapid detection of African swine fever virus: A validation study during an outbreak in Vietnam, Transbound Emerg Dis, 2021, 68: 2595-2602.
[21] Abdullah J, Saffie N, Sjasri F.A.R et al, Rapid detection of Salmonella typhi by loop-mediated isothermal amplification (LAMP) method, Brazilian Journal of Microbiology, 2014, 45: 1385-1391.
[22] Torigoe H, Seki M, Yamashita Y et al, Detection of Haemophilus influenzae by loop-mediated isothermal amplification (LAMP) of the outer membrane protein P6 gene, Jpn J Infect Dis, 2007, 60: 55-58.
[23] Kim H.J, Kim YJ, Yong D.E et al, Loop-mediated isothermal amplification of vanA gene enables a rapid and naked-eye detection of vancomycin-resistant enterococci infection, J Microbiol Methods, 2014, 104: 61-66.
[24] Tang C.T, Nguyen D.T, Hoa N.T et al, An outbreak of severe infections with community-acquired MRSA carrying the panton-valentine leukocidin following vaccination, PLoS One, 2, Epub ahead of print, 2007, doi: 10.1371/journal.pone.0000822.
[25] Otto M, Community-associated MRSA: What makes them special? International Journal of Medical Microbiology, Epub ahead of print, 2013, doi: 10.1016/j.ijmm.2013.02.007.
[26] Notomi T, Okayama H, Masubuchi H et al, Loop-mediated isothermal amplification of DNA, Nucleic Acids Res, 2000, 28: e63-e63.
[27] Abusheraida N.S.A, AlBaker A.A.H, Aljabri A.S.A et al, Rapid Visual Detection of Methicillin-Resistant Staphylococcus aureus in Human Clinical Samples via Closed LAMP Assay Targeting mecA and spa Genes, Microorganisms, 12, Epub ahead of print 2024, doi: 10.3390/microorganisms12010157.
[28] Emara M.M.M, Ghoname N.F, Ramadan M.O et al, Evaluation of loop mediated isothermal amplification (LAMP) assay for rapid detection of methicillin resistant Staphylococcus aureus in Tanta University Hospitals in Egypt, Microbes and Infectious Diseases, 2023, 4: 1219-1231.