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Real-Time PCR to Identify Staphylococci and Assay for Virulence from Blood

  • Charles E. Okolie
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1616)

Abstract

The genus Staphylococcus includes pathogenic and non-pathogenic facultative anaerobes. Due to the plethora of virulence factors encoded in its genome, the species Staphylococcus aureus is known to be the most pathogenic. S. aureus strains harboring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, however, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbor mecA, the genetic driver for staphylococcal methicillin-resistance. In this chapter, the detailed practical procedure for operating a real-time pentaplex PCR assay in blood cultures is described. The pentaplex real-time PCR assay simultaneously detects markers for the presence of bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl), and methicillin resistance (mecA).

Key words

Staphylococcus Virulence Staphylococcal proteinA Panton-valentine leukocidin Blood culture Differential diagnosis 

References

  1. 1.
    Vu BN, Jafari AJ, Aardema M, Tran HK, Nguyen DN, Dao TT, Nguyen TV, Tran TK, Nguyen CK, Fox A, Bañuls AL, Thwaites G, Nguyen KV, Wertheim HF (2016) Population structure of colonizing and invasive Staphylococcus aureus strains in northern Vietnam. J Med Microbiol 65(pt 4):298–305. doi: 10.1099/jmm.0.000220
  2. 2.
    Kolawole DO, Adeyanju Al, Schaumburg F, Akinyoola AL, Lawal OO, Amusa YB, Köck R, Becker K (2013) Characterization of colonizing Staphylococcus aureus isolated from surgical wards’ patients in a Nigerian university hospital. PLoS One 8(7):e68721. doi: 10.1371/journal.pone.0068721 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Ubukata K, Nakagami S, Nitta A, Yamane A, Kawakami S, Sugiura M, Konno M (1992) Rapid detection of the mecA gene in methicillin-resistant staphylococci by enzymatic detection of polymerase chain reaction products. J Clin Microbiol 30:1728–1733PubMedPubMedCentralGoogle Scholar
  4. 4.
    Murakami K, Nomura K, Doi M, Yoshida T (1989) Production of low-affinity penicillin-binding protein by low- and high-resistance groups of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 32:1307–1311Google Scholar
  5. 5.
    Foster TJ, Geoghegan JA, Ganesh VK, Hook M (2013) Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus. Nat Rev Microbiol 12(1):49–62CrossRefGoogle Scholar
  6. 6.
    Chambers HF (2005) Community-associated MRSA—resistance and virulence converge. N Engl J Med 352:1485–1487CrossRefPubMedGoogle Scholar
  7. 7.
    David, M.Z., and Daum, R.S (2010) Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev 23(3), 616–687.Google Scholar
  8. 8.
    Geha DJ, Uhl JR, Gustaferro CA, Persing DH (1994) Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory. J Clin Microbiol 32(7):1768–1772PubMedPubMedCentralGoogle Scholar
  9. 9.
    Okolie CE, Wooldridge KG, Turner DP, Cockayne A, James R (2015a) Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes. BMC Microbiol 15:157CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Roche Applied Science. The LightCycler® 480 real-time PCR system catalogue. https://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/05237645001_10.12.pdf. Accessed 4 Apr 2016
  11. 11.
    Okolie CE (2009) Development of diagnostic and therapeutic tools for Staphylococcus aureus infections. Ph.D. Thesis, University of Nottingham. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.517830
  12. 12.
    Okolie CE, Wooldridge KG, Turner DP, Cockayne A, James R (2015b) Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers. Mol Cell Probes 29(3):144–150CrossRefPubMedGoogle Scholar
  13. 13.
    Johnsson DP, Stralin MK, Soderquist B (2004) Detection of Panton-valentine leukocidin gene in Staphylococcus aureus by LightCycler PCR: clinical and epidemiological aspects. Clin Microbiol Infect 10:884–889CrossRefPubMedGoogle Scholar
  14. 14.
    Louie L, Goodfellow J, Mathieu P, Glatt A, Louie M, Simor AE (2002) Rapid detection of methicillin-resistant staphylococci from blood culture bottles by using a multiplex PCR assay. J Clin Microbiol 40(8):2786–2790CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Biological SciencesLandmark UniversityOmu-AranNigeria

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