#08-007

Complete

Date Full Report Received

08/31/2009

Date Abstract Report Received

08/31/2009

Investigation

Institution:
Primary Investigator:

Novel rapid, sensitive, and simple detection assays that can be routinely used to prevent the transmission of Salmonella in the pork industry are in high-demand. Reverse-transcriptase Loop-mediated isothermal Amplification (RT-LAMP) assay is a novel molecular method that has advantages over real-time reverse-transcriptase Polymerase Chain Reaction (RT-PCR) in that it does not require expensive equipment such as a real-time PCR machine as the reaction occurs in a waterbath at one temperature and detection is by turbidity or fluorescence after 2 h. The objectives of this research were to develop and apply novel assays such as RT-LAMP and RT-PCR to pork products and the pork environment for the rapid and sensitive detection of Salmonella Typhimurium and for comparison to traditional cultural methods. Another objective was to apply the RT-LAMP assay to detect Salmonella from pork products obtained from grocery stores and from pork processing facilities to test the suitability of the novel assays for routine testing by the pork industry in real-world scenarios. Twenty-five gram pork chop, pork sausage, and ground pork samples were spiked with S. Typhimurium at high (108 to 106 CFU) and low (105 to 100 CFU) inocula levels. Carcass rinse samples (500 ml; that were concentrated through sequential filtration) and carcass swabs (100 cm2) were spiked with only high inocula levels. Samples were stomached in 225 ml of Tetrathionate (TT) broth, portions were serially diluted and plated on Xylose Lysine Tergitol (XLT4) agar for traditional cultural detection and RNA was extracted and assayed by RT-PCR and RT-LAMP using previously described primers. Each experiment in duplicate was replicated at least twice.

The Trizol® RNA extraction method provided improved RNA quality over the Qiagen RNA extraction method and was used in these studies. The RT-PCR assay on pork products spiked with high inocula S. Typhimurium showed detection of 106 CFU/25g within one 8 h shift. Selective enrichment in TT broth for 10 h was necessary to obtain detection of 101 CFU/25g for pork chop and pork sausage, which required at least two 8 h work shifts. The previously described LAMP assay was developed into a RT-LAMP assay that gave detection sensitivities of 101 CFU/ml after gel electrophoresis for S. Typhimurium in pure culture and was better than RT-PCR by 1 log CFU. Spiked pork chop and pork sausage without enrichment gave detection sensitivities of 106 CFU/25g similar to traditional plating and RT-PCR. The lower inocula levels required selective enrichment in TT broth, to obtain detection limits of 102 CFU/25g by RT-LAMP which was one-log less sensitive than RT-PCR and traditional plating. However, this RT-LAMP method is faster than RT-PCR by ~2 h. For both assays, negative controls including sterile water, sterile TT broth, and un-inoculated samples did not show any positive results, eliminating cross-reactivity or false positives. Background flora and autoclaved Salmonella cultures inoculated on to pork samples did not show any positive results as well, again eliminating false positives, indicating the robustness of these assays. Pork products spiked with cold stressed cells to simulate conditions associated with storage and transport gave detection limits of ~102 CFU/25g, after pre-enrichment for 3 h in buffered peptone water and selective enrichment in TT broth for 10-12 h. Screening of 57 natural samples from pork processing facilities and grocery stores resulted in 9 positives by traditional cultural plating, 5 positives by RT-PCR and 10 positives RT-LAMP assays. These results indicate that RT-LAMP shows potential for application in routine testing of Salmonella from pork products that can obtain results within two 8 h work shifts, being faster and simpler than RT-PCR and traditional cultural assays, but less sensitive by 1-log CFU. Further work using fluorescence dyes is necessary to improve detection sensitivity that will also allow conversion of the RT-LAMP to a quantitative assay by using automated portable fluorometers. This will further simplify the assay for routine use by the pork industry.

A cost-effective, rapid, sensitive, Salmonella detection assay was developed for routine testing of pork commodities that can obtain results within two 8 h work shifts. Using this sensitive portable assay that will allow rapid detection will help to prevent contaminated products from being released in the market and contaminated products can also be isolated to prevent cross contamination of other pork commodities and food contact surfaces. The pork industry will be economically and socially benefited as early detection will prevent expensive recalls and associated litigation costs and will protect brand name. Also, any processing areas or equipment found contaminated with Salmonella can be attended to immediately, properly cleaned and improved mitigation strategies and HACCP plans can be implemented. Use of this novel rapid assay in routine testing and surveillance will therefore aid in the prevention of Salmonella transmission by the pork industry as well as pork-related Salmonella outbreaks in order to protect public health.

Contact Information: Dr. Doris H. D’Souza, University of Tennessee-Knoxville, 2605 River Drive, Room 102 FSPB, Department of Food Science and Technology, Knoxville, TN 37996-4591. Tel.: 865-974-2753. Fax: 865-974-7332. E-mail: ddsouza@utk.edu