A new multiplex polymerase chain reaction assay for the identification a panel of bacteria involved in bacteremia

Authors
Hossein Fazzeli 1
Mohammad R Arabestani 1
Bahram N Esfahani 1
Farzin Khorvash 2
Mohammad R Pourshafie 3
Sharareh Moghim 1
Hajieh G Safaei 1
Jamshid Faghri 1
Amir Azimian 1
1 Department of Microbiology, Isfahan University of Medical Sciences, Isfahan, Iran
2 Nosocomial Infection Research Centre, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran
Abstract
Background: Throughout the world, bloodstream infections (BSI s ) are associated with high rates of morbidity and mortality. Rapid pathogens identification is central significance for the outcome of the patient than culture techniques for microbial identification. To develop an end point multiplex PCR to identify a group of bacteria including Enterococcus spp., Pseudomons aeruginosa, Staphylococcus spp., Acinetobacter baumannii, 16S rDNA, and Drosophila Melanogaster were used as internal control (IC).
Materials and Methods: Design of primers was done using Mega4, Allel ID6, Oligo6 and Oligo analyzer softwares. Genetic targets for primer designing and identification of genus Enterococcus spp., Staphylococcus spp., and species of Acinetobacter baumannii, Pseudomons aeruginosa, included the rpoB, rpoB and gyrA, sss respectively. Then PCR and multiplex PCR were performed
Results: The intended specificity was obtained for the bacteria, which used in this study and there wasn't seen any unspecific amplification by the multiplex PCR. The test showed a sensitivity ranging from 1 to 100 target copies per reaction depending on the bacterial species.
Conclusions: The presented multiplex PCR offers a rapid and accurate molecular diagnostic tool for simultaneous detection of some pathogenic microorganisms. The IC exists in the multiplex PCR accompanied by other primers in the system, can serve as a simple, cost- effective internal control for the multiplex PCR assay.

Keywords

1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001;29:1303-10.  Back to cited text no. 1
    
2. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003;348:1546-54.  Back to cited text no. 2
    
3. Magadia RR, Weinstein MP. Laboratory diagnosis of bacteremia and fungemia. Infect Dis Clin North Am 2001; 15:1009-24.  Back to cited text no. 3
    
4. Søgaard M, Stender H, Schønheyder HC. Direct identification of major blood culture pathogens, including Pseudomonas aeruginosa and Escherichia coli, by a panel of fluorescence in situ hybridization assays using peptide nucleic acid probes. J Clin Microbiol 2005;43:1947  Back to cited text no. 4
    
5. Jansen GJ, Mooibroek M, Idema J, Harmsen HJ, Welling GW, Degener JE. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes. J Clin Microbiol 2000;38:814-7  Back to cited text no. 5
    
6. Christensen JE, Stencil JA, Reed KD. Rapid identification of bacteria from positive blood cultures by terminal restriction fragment length polymorphism profile analysis of the 16S rRNA gene. J Clin Microbiol 2003;41:3790-800.  Back to cited text no. 6
    
7. Turenne CY, Witwicki E, Hoban DJ, Karlowsky JA, Kabani AM. Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene. J Clin Microbiol 2000; 38:513-20.  Back to cited text no. 7
    
8. de Cueto M, Ceballos E, Martinez-Martinez L, Perea EJ, Pascual A. Use of positive blood cultures for direct identification and susceptibility testing with the vitek 2 system. J Clin Microbiol 2004; 42:3734-8.  Back to cited text no. 8
    
9. Kurupati P, Chow C, Kumarasinghe G, Poh CL. Rapid detection of Klebsiella pneumoniae from blood culture bottles by real-time PCR. J Clin Microbiol 2004; 42:1337-40.  Back to cited text no. 9
    
10. Laforgia N, Coppola B, Carbone R, Grassi A, Mautone A, Iolascon A. Rapid detection of neonatal sepsis using polymerase chain reaction. Acta Paediatr 1997; 86:1097-9.  Back to cited text no. 10
    
11. Louie L, Goodfellow J, Mathieu P, Glatt A, Louie M, Simor AE. Rapid detection of methicillin-resistant staphylococci from blood culture bottles by using a multiplex PCR assay. J Clin Microbiol 2002; 40:2786-90.  Back to cited text no. 11
    
12. Rothman RE, Majmudar MD, Kelen GD, Madico G, Gaydos CA, Walker T, et al. Detection of bacteremia in emergency department patients at risk for infective endocarditis using universal 16S rRNA primers in a decontaminated polymerase chain reaction assay. J Infect Dis 2002;186:1677-81  Back to cited text no. 12
    
13. Fujita S, Senda Y, Iwagami T, Hashimoto T. Rapid identification of staphylococcal strains from positive-testing blood culture bottles by internal transcribed spacer PCR followed by microchip gel electrophoresis. J Clin Microbiol 2005;43:1149-57  Back to cited text no. 13
    
14. Kang CI, Kim SH, Kim HB, Park SW, Choe YJ, Oh MD, et al. Pseudomonas aeruginosa bacteremia: Risk factors for mortality and influence of delayed receipt of effective antimicrobial therapy on clinical outcome. Clin Infect Dis 2003;37:745-51.  Back to cited text no. 14
    
15. Bergogne-Bérézin E, Towner KJ. Acinetobacter spp. as nosocomial pathogens: Microbiological, clinical, and epidemiological features. Clin Microbiol Rev 1996;9:148-65.  Back to cited text no. 15
    
16. Caðatay AA, Ozcan PE, Gulec L, Ince N, Tugrul S, Ozsut H, et al. Risk factors for mortality of nosocomial bacteraemia in intensive care units. Med Princ Pract 2007;16:187-92.  Back to cited text no. 16
    
17. Cheng AC, West TE, Peacock SJ. Surviving sepsis in developing countries. Crit Care Med 2008;36:2487-8.  Back to cited text no. 17
    
18. Bej AK, DiCesare JL, Haff L, Atlas RM. Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid. Appl Environ Microbiol 1991;57:1013-7.  Back to cited text no. 18
    
19. Sabet NS, Subramaniam G, Navaratnam P, Sekaran SD. Simultaneous species identification and detection of methicillin resistance in staphylococci using triplex real-time PCR assay. Diagn Microbiol Infect Dis 2006;56:13-8.  Back to cited text no. 19
    
20. Xu J, Millar BC, Moore JE, Murphy K, Webb H, Fox AJ, et al. Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis-rapid separation of 16S rRNA PCR amplicons without the need for cloning. J Appl Microbiol 2003;94:197-206.  Back to cited text no. 20
    
21. Mancini N, Carletti S, Ghidoli N, Cichero P, Burioni R, Clementi M. The era of molecular and other non-culture-based methods in diagnosis of sepsis. Clin Microbiol Rev 2010;23:235-51  Back to cited text no. 21
    
22. Reier-Nilsen T, Farstad T, Nakstad B, Lauvrak V, Steinbakk M. Comparison of broad range 16S rDNA PCR and conventional blood culture for diagnosis of sepsis in the newborn: A case control study. BMC Pediatr 2009; 9:5.  Back to cited text no. 22
    
Advanced Biomedical Research
Volume 2013, March
March 2013
Pages 1-5