Abstract
Detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and blood has the potential to improve diagnostic tools in microbiology. A novel real-time PCR-based assay was developed and its performance and robustness were evaluated for a panel of spiked suspensions of 15 clinically relevant bacteria. As low as ten colony forming units (CFU)/100 μl were detectable. No cross-reactivity was observed, except for Staphylococcus aureus and Staphylococcus epidermidis. Nevertheless, S. aureus and S. epidermidis were reliably differentiated by melting curve analysis. The protocol was also validated with three groups containing a mixture of five spiked bacteria each, with the result of reliable differentiation. This novel assay allows an exact identification of 15 microbes relevant in intensive care medicine, including mixed infections, in a one run experiment in less than 4 h.
References
Brakstad OG, Aasbakk K, Maeland JA (1992) Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clin Microbiol 30:1654–1660
Brisson-Noel A, Gicquel B, Lecossier D, Levy-Frebault V, Nassif X, Hance AJ (1989) Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet 2:1069–1071
Cantral DE, Tape TG, Reed EC, Spurzem JR, Rennard SI, Thompson AB (1993) Quantitative culture of bronchoalveolar lavage fluid for the diagnosis of bacterial pneumonia. Am J Med 95:601–607
Carroll NM, Jaeger EE, Choudhury S, Dunlop AA, Matheson MM, Adamson P, Okhravi N, Lightman S (2000) Detection of and discrimination between gram-positive and gram-negative bacteria in intraocular samples by using nested PCR. J Clin Microbiol 38:1753–1757
Cursons RT, Jeyerajah E, Sleigh JW (1999) The use of polymerase chain reaction to detect septicemia in critically ill patients. Crit Care Med 27:937–940
da Silva F, Levi JE, Oda BC, da SRS, Rozov T (1999) PCR identification of Pseudomonas aeruginosa and direct detection in clinical samples from cystic fibrosis patients. J Med Microbiol 48:357–361
Dagan R, Shriker O, Hazan I, Leibovitz E, Greenberg D, Schlaeffer F, Levy R (1998) Prospective study to determine clinical relevance of detection of pneumococcal DNA in sera of children by PCR. J Clin Microbiol 36:669–673
Fredricks DN, Relman DA (1999) Application of polymerase chain reaction to the diagnosis of infectious diseases. Clin Infect Dis 29:475–486
Greisen K, Loeffelholz M, Purohit A, Leong D (1994) PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol 32:335–351
Heininger A, Binder M, Schmidt S, Unertl K, Botzenhart K, Doring G (1999) PCR and blood culture for detection of Escherichia coli bacteremia in rats. J Clin Microbiol 37:2479–2482
Hykin PG, Tobal K, McIntyre G, Matheson MM, Towler HM, Lightman SL (1994) The diagnosis of delayed post-operative endophthalmitis by polymerase chain reaction of bacterial DNA in vitreous samples. J Med Microbiol 40:408–415
Ibrahim EH, Sherman G, Ward S, Fraser VJ, Kollef MH (2000) The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting. Chest 118:146–155
Isaacman DJ, Zhang Y, Rydquist-White J, Wadowsky RM, Post JC, Ehrlich GD (1995) Identification of a patient with Streptococcus pneumoniae bacteremia and meningitis by the polymerase chain reaction (PCR). Mol Cell Probes 9:157–160
Kane TD, Alexander JW, Johannigman JA (1998) The detection of microbial DNA in the blood: a sensitive method for diagnosing bacteremia and/or bacterial translocation in surgical patients. Ann Surg 227:1–9
Klaschik S, Lehmann LE, Raadts A, Book M, Hoeft A, Stuber F (2002) Real-time PCR for detection and differentiation of gram-positive and gram-negative bacteria. J Clin Microbiol 40:4304–4307
Klaschik S, Lehmann LE, Raadts A, Book M, Gebel J, Hoeft A, Stuber F (2004) Detection and differentiation of in vitro-spiked bacteria by real-time PCR and melting-curve analysis. J Clin Microbiol 42:512–517
Kumar Y, Qunibi M, Neal TJ, Yoxall CW (2001) Time to positivity of neonatal blood cultures. Arch Dis Child Fetal Neonatal Ed 85:F182–F186
Lu JJ, Perng CL, Lee SY, Wan CC (2000) Use of PCR with universal primers and restriction endonuclease digestions for detection and identification of common bacterial pathogens in cerebrospinal fluid. J Clin Microbiol 38:2076–2080
Mariani BD, Martin DS, Levine MJ, Booth REJ, Tuan RS (1996) The coventry award. Polymerase chain reaction detection of bacterial infection in total knee arthroplasty. Clin Orthop 331:11–22
Pahl A, Kuhlbrandt U, Brune K, Rollinghoff M, Gessner A (1999) Quantitative detection of Borrelia burgdorferi by real-time PCR. J Clin Microbiol 37:1958–1963
Pingle MR, Granger K, Feinberg P, Shatsky R, Sterling B, Rundell M, Spitzer E, Larone D, Golightly L, Barany F (2007) Multiplexed identification of blood-borne bacterial pathogens by use of a novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis assay. J Clin Microbiol 45:1927–1935
Pizzo PA, Rubin M, Freifeld A, Walsh TJ (1991) The child with cancer and infection. I. Empiric therapy for fever and neutropenia, and preventive strategies. J Pediatr 119:679–694
Rantakokko-Jalava K, Nikkari S, Jalava J, Eerola E, Skurnik M, Meurman O, Ruuskanen O, Alanen A, Kotilainen E, Toivanen P, Kotilainen P (2000) Direct amplification of rRNA genes in diagnosis of bacterial infections. J Clin Microbiol 38:32–39
Rogers NK, Fox PD, Noble BA, Kerr K, Inglis T (1994) Aggressive management of an epidemic of chronic pseudophakic endophthalmitis: results and literature survey. Br J Ophthalmol 78:115–119
Speich R, Hauser M, Hess T, Wust J, Grebski E, Kayser FH, Russi EW (1998) Low specificity of the bacterial index for the diagnosis of bacterial pneumonia by bronchoalveolar lavage. Eur J Clin Microbiol Infect Dis 17:78–84
van Kuppeveld FJ, Johansson KE, Galama JM, Kissing J, Bolske G, Hjelm E, van der Logt JT, Melchers WJ (1994) 16S rRNA based polymerase chain reaction compared with culture and serological methods for diagnosis of Mycoplasma pneumoniae infection. Eur J Clin Microbiol Infect Dis 13:401–405
Zucol F, Ammann RA, Berger C, Aebi C, Altwegg M, Niggli FK, Nadal D (2006) Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification. J Clin Microbiol 44:2750–2759
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Klaschik, S., Lehmann, L.E., Gebel, J. et al. Species-specific probes and real-time PCR as a tool for fast detection and differentiation of 15 bacteria relevant in intensive care medicine. World J Microbiol Biotechnol 27, 2217–2222 (2011). https://doi.org/10.1007/s11274-011-0661-2
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DOI: https://doi.org/10.1007/s11274-011-0661-2