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Title: Development of a Rapid Multiplex PCR Technique for Determination of Salmonella enterica Serotypes Isolated from Pork and Poultry

Author
item Frye, Jonathan
item LEADER, BRANDON
item Cray, Paula
item HU, JINXIN
item BOYLE, DAVID

Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 3/5/2008
Publication Date: 6/4/2008
Citation: Frye, J.G., Leader, B.T., Cray, P.J., Hu, J., Boyle, D.S. 2008. Development of a Rapid Multiplex PCR Technique for Determination of Salmonella enterica Serotypes Isolated from Pork and Poultry. American Society for Microbiology Annual Meeting. CD-ROM. C-294.

Interpretive Summary:

Technical Abstract: Background: A multiplex PCR technique to discriminate Salmonella enterica serotypes was adapted to a high-throughput, automated assay. Methods: Fifteen target genes were chosen that varied in distribution among common Salmonella enterica serotypes isolated from various hosts. These targets were detected in a single multiplex PCR reaction with specific primer sets for each target. Each forward primer included a universal sequence complementary to a carboxyfluorescein (FAM) linked primer which was used to label all products for detection. Primer pairs, universal primer and master mix containing a Hot Start Taq polymerase (Bioline, USA Inc, Randolph, MA, USA) were combined with template DNA prepared from isolates by the boiled colony method. Thermocycling parameters were 94°C 15 min, (94°C 30s, 57°C 90s, 72°C 30s) x 25, 72°C 5min, (94°C 30s, 68°C 90s, 72°C 30s) x 15, 72°C 5min. Samples were diluted 1:50 (v/v) in formamide containing carboxy-X-rhodamine (ROX) labeled GENEFLO 625 DNA Ladder (CHIMERx, Milwaukee, WI, USA) and separated in an ABI 3100 Avant gene analyzer on a 50 cm capillary. Results: Salmonella isolates from raw pork (54) and poultry (68) were analyzed by the multiplex PCR analysis technique (PCR) and compared to traditional serotyping (TS). PCR resulted in serotypes for 49/54 pork isolates, all in agreement with TS (92% accuracy). PCR analysis of poultry isolates resulted in correct serotypes for 59/68 isolates (87% accuracy) as compared to TS. Overall, PCR resulted in 89% (108/122) accuracy, lower cost (~$5 for PCR versus ~$39 for TS), and quicker turn-around time of about 6 h for PCR versus weeks for TS. Conclusions: Automated multiplex PCR determined the serotype of Salmonella enterica isolated from pork 92% of the time and poultry 87% of the time as compared to traditional serotyping and is a rapid and more economical alternative to TS.