Source: UNIV OF MASSACHUSETTS submitted to
DEVELOPMENT OF REAL-TIME PCR METHODOLOGY FOR THE ENUMERATION OF LOW-NUMBERS OF SALMONELLA AND E. COLI IN GROUND BEEF AND RAW VEGETABLES WITHOUT ENRICHMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0230925
Grant No.
(N/A)
Project No.
MAS00434
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Project Director
Levin, RO.
Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
Food Science
Non Technical Summary
There is a critical need in the meat processing and raw vegetable processing industries for the development of a rapid method for detection of infectious bacteria such as Salmonella and E. coli O157:H7 in such products well before shipping, so as to prevent infectious outbreaks and costly recalls.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240991100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Project terminated due to P.I. Retirement
Project Methods
Methodology will involve: (1) Homogenizing or stomaching 25 grams of ground beef (seeded with varying numbers of target bacteria) with at last 20 grams of beta-cyclodextrin in a total volume of 200 ml in saline to bind fat and exclude all bacteria. (2) Using differential centrifugation at 1,000g to sediments solids and 16,000g to pellet suspended bacterial cells. (3) Final pellet is then suspended in 30 ml of saline that is then mixed for 15 min with 11 grams of activated charcoal coated with bentonite in a 400 ml beaker to remove PCR inhibitors. (4) Bacterial cell suspension with charcoal is then passed through glass wool and resulting water clear filtrate is centrifuged at 16,000g to sediment bacterial cells. (5) Pellet is then made up to 1.0 ml with lysing solution and heated to boiling for 10 min. (6) Lysed cells are then centrifuged at 10,000g for 5 min to pellet debri and 10 microliters of DNA solution incorporated into PCR reactions using primers specific for the target organism. (7) Agarose gel electrophoresis will then be used to resolve the amplified band visualized under UV with "red safe" fluorescent dye, photographed and the fluorescent band quantified using the NIH 1.62 scanware program. Resulting rapid methodology will be be suitable for use by industry to prevent recalls. With leafy vegetables such as spinach and lettuce 250g of seeded eaves (held at 4 C overnight) will be vigorously rinsed with 200 ml of 0.025% SDS. Samples will then be subjected to differential centrifugation and steps 3 to 7 performed.

Progress 10/01/12 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Project terminated early due to P.I.retirement

Publications


    Progress 10/01/12 to 09/30/13

    Outputs
    Target Audience: Academicians, industry technical staff, research institution staff and administrators, and graduate students at various universities. This work has been widely disseminated via publication in major journals of the field of food microbiology. As a result, I have received many e-mails requesting details of the methodology and reprints from graduate students, postdoctoral scientists, and other scientists from various research institutions and universities. Changes/Problems: We have not encountered any problems or delays regarding progress and results. The only unexpected outcome has been the extraordinary success of our efforts. We are now able to reproducibly detect 1 CFU of Salmonella per gram of ground beef (in a 25 gram sample) by real-time PCR without enrichment and involving a total assay time of 4.5 hrs. This has ben found to work with ground beef containing 7, 15, and 25% fat. What opportunities for training and professional development has the project provided? One graduate (M.S.) student was trained, and a second is further advancing the technology. One postdoctoral scientist is developing modifications of this technology to detect 1 CFU of Salmonella per gram /g of ground beef. How have the results been disseminated to communities of interest? Results were disseminated world-wide via publication in leading peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? We are presently documenting the ability of this technology with certain modifications to detect 1 CFU of Salmonella per gram of ground beef utilizing soluble starch to remove fat prior to real-time PCR.

    Impacts
    What was accomplished under these goals? This is the only laboratory globally that has developed methodology for the PCR detection of 3 cells (CFU) per gram of Salmonella in ground beef within 4.5 hrs. without enrichment cultivation utilizing coated activated charcoal fore removal of PCR inhibitors and beta-cyclodextrin to remove fat.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Opet, N., Levin, R. 2013. Efficacy of coating activated carbon with milk proteins to prevent binding of bacterial cells from foods for PCR detection. J. Microbiol. Meth. 94:69-72.
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Opet, N., Levin, R. 2013. Use of ?-cyclodextrin and activated carbon for quantification of Salmonella enterica ser. Enteritidis from ground beef by conventional PCR without enrichment. Food Microbiol. 38:75-79.