Submitted to: Journal of Microbiological Methods
Publication Type: Peer reviewed journal
Publication Acceptance Date: 9/4/2008
Publication Date: 1/1/2009
Citation: Wise, M., Siragusa, G.R., Plumblee, J., Healy, M., Cray, P.J., Seal, B.S. 2009. Predicting Salmonella enterica serotypes by repetitive sequence-based PCR. Journal of Microbiological Methods. 76(1):18-24. Interpretive Summary: Salmonella bacteria remain one of the leading causes of bacterial food-borne disease worldwide. Epidemiological tracking of these bacteria is still conducted by first determining the serological type by a standard methodology that can take from one to several weeks to obtain results. Consequently, a new molecular technique that has been semi-automated was evaluated as a quicker, more reliable method to predict serotype of Salmonella isolates. The technique of rep-PCR or repetitive extragenic palindromic sequence-based polymerase chain reaction takes advantage of the fact that the Salmonella genome as well as other bacterial genomes contains DNA sequences that are present in multiple copies and can be utilized to generate a reproducible gel pattern of the resultant DNA amplification product. The technique has now been semi-automated so that rep-PCR DNA amplification products are placed into micro-capillaries wherein a computer is used to generate a pattern that was previously completed in more cumbersome agarose gels. Patterns are stored in a computer data-base that can be made available to other investigators via the internet. Following rep-PCR of the bacterial DNA, resultant gel patterns can be utilized to characterize isolates into groups that can reliably predict serotypes of Salmonella from different environmental sources. Two hundred sixteen different Salmonella isolates along with a blinded-set of 44 isolates were examined utilizing the rep-PCR technique and serotype of the isolates could be reliably predicted utilizing the molecular technique within a two-day timeframe. Consequently, the rep-PCR System holds promise as a more rapid classification scheme to predict serotype for members of this group of disease-causing bacteria.
Technical Abstract: Repetitive extragenic palindromic sequence-based PCR (rep-PCR) utilizing a semi-automated system, was evaluated as a method to determine Salmonella serotypes. A group of 216 Salmonella isolates belonging to 13 frequently isolated serotypes and one rarer serotype from poultry were used to create a DNA fingerprint library with the rep-PCR system software. Subsequently, a blinded set of 44 poultry isolates were fingerprinted by rep-PCR and queried against the library to predict a serotype designation for each Salmonella isolate. The query isolates were previously typed employing standard Kaufmann-White serological techniques. Utilizing pair-wise similarity percentages as calculated by the Pearson correlation coefficient, the rep-PCR predicted serotype of 28 isolates matched the serological typing result. For eight isolates, rep-PCR results were interpreted as one of two very closely related serotypes, Hadar and the rarer Istanbul. Traditional serological assays have difficulty distinguishing between these groups, and sequencing inter-spacer regions of the rrfH gene was unable to differentiate among isolates of these two serovars. Six of the remaining isolates resulted in no match to the database (similarity values < 95%) and these indeed proved to be serotypes not included in the original library. The two remaining samples proved discrepant at the 95% similarity threshold, however examination of electropherograms clearly indicated fingerprint variability between query and library samples, suggesting an expanded rep-PCR library will be necessary for increased utility. Since serological assays take several days or weeks to provide information, the rep-PCR System holds promise as a more rapid classification scheme to predict serotype for members of this group of bacteria.