ARS | Publication request: Development of a Rapid Multiplex PCR Technique for Determination of Salmonella enterica Serotypes Isolated from Pork and Poultry

Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: March 5, 2008
Publication Date: June 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.

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.