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From Primer Generation to Chromosome Identification: A Primer Generation Genotyping Method for Bacterial Identification and Typing

Authors: Wisam H. Benamer, Ehab A. Elfallah, Mohamed A. Elshaari, Farag A. Elshaari


A challenge for laboratories is to provide bacterial identification and antibiotic sensitivity results within a short time. Hence, advancement in the required technology is desirable to improve timing, accuracy and quality. Even with the current advances in methods used for both phenotypic and genotypic identification of bacteria the need is there to develop method(s) that enhance the outcome of bacteriology laboratories in accuracy and time. The hypothesis introduced here is based on the assumption that the chromosome of any bacteria contains unique sequences that can be used for its identification and typing. The outcome of a pilot study designed to test this hypothesis is reported in this manuscript. Methods: The complete chromosome sequences of several bacterial species were downloaded to use as search targets for unique sequences. Visual basic and SQL server (2014) were used to generate a complete set of 18-base long primers, a process started with reverse translation of randomly chosen 6 amino acids to limit the number of the generated primers. In addition, the software used to scan the downloaded chromosomes using the generated primers for similarities was designed, and the resulting hits were classified according to the number of similar chromosomal sequences, i.e., unique or otherwise. Results: All primers that had identical/similar sequences in the selected genome sequence(s) were classified according to the number of hits in the chromosomes search. Those that were identical to a single site on a single bacterial chromosome were referred to as unique. On the other hand, most generated primers sequences were identical to multiple sites on a single or multiple chromosomes. Following scanning, the generated primers were classified based on ability to differentiate between medically important bacterial and the initial results looks promising. Conclusion: A simple strategy that started by generating primers was introduced; the primers were used to screen bacterial genomes for match. Primer(s) that were uniquely identical to specific DNA sequence on a specific bacterial chromosome were selected. The identified unique sequence can be used in different molecular diagnostic techniques, possibly to identify bacteria. In addition, a single primer that can identify multiple sites in a single chromosome can be exploited for region or genome identification. Although genomes sequences draft of isolates of organism DNA enable high throughput primer design using alignment strategy, and this enhances diagnostic performance in comparison to traditional molecular assays. In this method the generated primers can be used to identify an organism before the draft sequence is completed. In addition, the generated primers can be used to build a bank for easy access of the primers that can be used to identify bacteria.

Keywords: Bacteria chromosome, bacterial identification, sequence, primer generation.

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[1] Wolk DM, Kaleta EJ, and Wysocki VH. PCR-Electrospray Ionization Mass Spectrometry, The Potential to Change Infectious Disease Diagnostics in Clinical and Public Health Laboratories. Journal of Molecular Diagnostics 2012;14(4), 295-304
[2] Li W, Raoult D, Fournier P. Bacterial strain typing in the genomic era. FEMS Microbiol Rev 2009;33:892-916.
[3] Tenover FC, Arbeit RD, Goering RV. How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: a review for healthcare epidemiologists. Infect Control Hosp Epidemiol 1997;18:426-439.
[4] Jackson GW, McNichols RJ, Fox GE, Wilson RC. Bacterial genotyping by 16S rRNA mass cataloging. BMC Bioinformatics 2006;7:321 doi:10.1 186/147-2105/7/321.
[5] Taha MK, Olcen P. Molecular genetic methods in diagnosis and direct characterization of acute bacterial central nervous system infections. APMIS 2004;112:753-770.
[6] Versalovic J, Lupski JR, Molecular detection and genotyping of pathogens: more accurate and rapid answers. Trends Microbiol 2002;10:S15-S21.
[7] Forrest GN, Mehta S, Weekes E, Lincalis DP, et al. Impact of rapid in situ hybridization testing on coagulase­negative staphylococci positive blood cultures. J Antimicrob Chemother 2006;58:154–8
[8] Kaleta EJ, Clark AE, Cherkaoui A, Wysocki VH, et al. Comparative analysis of pcr­electrospray ionization/mass spectrometry (ms) and maldi­tof/ms for the identification of bacteria and yeast from positive blood culture bottles. Clin Chem 2011a;57:1057–67.
[9] Kaleta EJ, Clark AE, Johnson DR, Gamage DC, et al. Use of pcr coupled with electrospray ionization mass spectrometry for rapid identification of bacterial and yeast bloodstream pathogens from blood culture bottles. J Clin Microbiol 2011b;49:345–53.
[10] Elshaari, FA, Elshaari, FA and Elshaari, MA. New Algorithm for Selecting Degenerate Primers to Clone DNA Using a Short Peptide Sequence. ISAC Proceeding for Computational Biology, USA, 2012
[11] Accessed on May the 11th 2016
[12] Barghouthi S. A. A Universal Method for the Identification of Bacteria Based on General PCR Primers. Indian J Microbiol 2011; 51(4):430–444
[13] Mitterer G, Huber M, Leidinger E, Kirisits C, Lubitz W, Mueller MW, Schmidt WM Microarray-based identification of bacteria in clinical samples by solid-phase PCR amplification of 23S ribosomal DNA sequences. J Clin Microbiol 2004;42:1048–1057.
[14] Yasuoka MO A multiplex polymerase chain reaction– based diagnostic method for bacterial vaginosis. Obst Gynecol 2002; 100:759–764
[15] Muldrew K. L. Molecular diagnostics of infectious disease. Curr Opin Pediatr 2009; 21:102–111