DEVELOPMENT OF ACTIVATED CHARCOAL TO REMOVE PCR INHIBITORS FROM COMPLEX FOOD MATRICES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0209096
• Project Start Date: Oct 1, 2006
• Project End Date: Oct 1, 2012
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
FOOD SCIENCE

Non Technical Summary
Some meat products contain potent PCR inibitors that are not completely removed by commercial DNA extraction and purification kits. The purpose of these studies is to develop methodology to facilitate the rapid 3 hour detection and quantification of low numbers of pathogenic bacteria (1 to 10 per gram) in complex food matrices by PCR using specialy treated activated charcoal to remove the PCR inhbitors from food samples.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

pcr

polymerase chain reciton

foods

meats

pseudomonas fluorscens

bentonite

pcr inhibitors

activated carbon;

Goals / Objectives
PCR is one of the most powerful analytical techniques ever developed. It allows segments of minute amounts of double stranded DNA to be amplified several million-fold in 2 to 3 hr. Its most notable application to foods is for the detection of food borne pathogenic and toxigenic bacteria in a wide variety of food products in addition to confirming the identification of such organisms isolated from foods. However, one of the limitations in the application of the PCR for detection and enumeration of food borne disease organisms is the presence of PCR inhibitors in complex food matrices that usually makes it impossible to detect low numbers (1 to 100 per gram) of target bacteria in foods. Experience has shown that the various DNA purification resins, kits, and mini-spin columns available are not effective in completely removing such PCR inhibitors. This has resulted in the use of enrichment procedures that require 18 to 48 hrs. incubation. Such enrichments procedures and the incubation times involved negate the rapid time advantage of the PCR. This investigator has recently developed the use of activated charcoal for the complete elimination of soluble PCR inhibitors from complex food matrices containing very high levels of potent PCR inhibitors, resulting in the detection of as few as 5 target organisms per gram of food without enrichment culture. Our methodology involves coating activated carbon particles with the non infectious bacterium Pseudomonas fluorescens so as not to bnd infectious target bacteria. A limitation to this new activated carbon methodology is our observation that this technique works with certain target organisms but not with others. The objectives of these studies are therefore to (1) to identify bacterial species that are maximally suitable for blocking the surface of activated charcoal for use with a maximum number of food borne pathogenic bacterial species, (2) to identify alternate coating agents such as bentonite in place of Pseudomonas fluorescens, and (3 to develop a universal protocol for all food borne bacterial pathogenic bacteria for direct detection of low numbers (1 to 10 per gram of food) by PCR without enrichment utilizing coated charcoal.

Project Methods
Particles of activated charcoal have binding capacity on their surface for both charged organic molecules and bacterial cells. Hence bacterial cells will be bound tenaciously to the surface of charcoal particles. In addition, the majority of the binding capacity of activated charcoal resides in the submicroscopic pores. By coating (blocking) the binding sites on the surface of charcoal particles with intact bacterial cells, it is hoped that further binding of target bacterial cells will not occur. The submicroscopic pores will still be available for binding soluble organic molecules from foods that contain potent PCR inhibitors. We will use several approaches. (1) variation in pH from 5.0 to 8.0 when target pathogenic organisms (E. coli O157:H7, L. monocytogenes, Vibrio vulnificus, Vibrio parahaemolyticus, Campylobacter jejuni, Campylobacter coli, and various serotypes of Salmonella) are applied to charcoal coated with P. fluorescens, (2) coating the activated charcoal with additional non pathogenic organisms(Acetobacter aceti, Bacillus subtilis, Moraxella immobilis (Moraxealla cryophilus), Pediococcus cerevisiae, Micrococcus luteus, Lactococcus lactis, Sarcina lutea, and a commensulate strain of E.coli and (3) assessing the ability of activated charcoal coated with bentonite to not bind target pathogenic bacteria. The final stage of these studies will be to apply the optimized carbon blocking system to the enumeration by conventional and SYBR green mediated real-time PCR of the various food borne pathogens seeded into various foods such as hamburger, fish tissue, shellfish tissue, and soft cheeses. Conventional and real-time primers and protocols will be from the published literature.

Progress 10/01/06 to 10/01/12

Outputs
OUTPUTS: We have found that casein is a suitable replacement for bentonite as a coating for activated charcoal to be used to remove PCR inhibitors from aqueous extracts of complex food matrices such as ground beef. Casein has the advantages of being more easily prepared and results in more quantitative coating of activated charcoal. PARTICIPANTS: The data referred to was generated by an M.S. graduate student TARGET AUDIENCES: These result are applicable to U.S.D., FDA, and industry laboratories involved in rapidly detecting infectious bacteria from complex food matrices by PCR. PROJECT MODIFICATIONS: We have replaced bentonite satisfactorily with casein for the coating of activated charcoal so that the charcoal will no longer bind bacteria.

Impacts
Activated carbon coated with casein was found to yield a 90% recovery of Salmonella enteritica ser. Enteritidis in cell suspensions placed in agitated contact for 15 minutes with the coated carbon particles. In contrast, without coating of activated carbon with casein, 0% of the cells in suspension were recovered, indicating that all cells were bound undesirably by the uncoated activated carbon.

Publications

• No publications reported this period

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: A novel detection system has been developed for eliminating PCR inhibitors and increasing the recovery of E. coli O157:H7 in ground beef samples with the use of beta cyclodextrin and activated carbon coated with bentonite. The methodology allowed the direct detection and enumeration of 5 CFU/gram of ground beef in 4 hours without enrichment. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Commecrial laboratories, food processors, US.D.A. laboratories and personnel. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This methodology allows producers of ground meat to asses the presence of E. coli O157:H7 before shipment and distribution.

Publications

• No publications reported this period

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: The effectiveness of activated charcoal coated with bentonite for the removal of PCR inhibitors from foods without binding targeted bacterial cells has been found to persist for at least six weeks. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Target audiences consist of federal laboratories, commercial laboratories, large food processors with in-house laboratories. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The ability of activated charcoal coated with bentonite to be fully functional for at least six weeks after storage at room temperature will greatly facilitate the rapid detection of infectious bacteria in a wide variety of foods by the PCR without enrichment cultivation.

Publications

• Levin, R. E. 2009. Molecular Methods for Detecting and Discriminating Shigella Associated with Foods and Human Clinical Infections. A Review. Food Biotechnol. 23:214-228.
• Levin R. E. 2009. Molecular methods for detecting and discriminating virulent aeromonads associated with foods and human clinical infections-A mini review. Food Biotechnol. 23:1-7.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Methodology has been successfully developed for detecting 5 cells of E. coli O157:H7 per gram of hamburger using 25 g samples, and 3 cells per gram of lettuce using 250 g samples. This methodolgy has been extended to seafood. The methodology utilizes real-time PCR without enrichment and has a total assay time of 3 hours. I have presented by invitation a number of technical presentations regarding this technology to various industry groups during the past year since the methodology is recognized by industry as being the latest cutting edge technology for eliminating massive and costly recalls in the meat and produce industries. Several key papers in this area in have been published in refereed journals. This faculty member has just published a 540 page sole authored book (Rapid Detection and Characterization of Foodborne Pathogens by Molecular Techniques)encompassing the latest molecular techniques for rapid detection of all major food borne bacteria. PARTICIPANTS: During the past year technical presentations regarding this technology have been made to Infraegis Co., Dole Co, and Taylor products Co, in California and to Dupont and the Podesta group in Washington D.C. in addition to several Asian industry groups. Arrangements are presently underway for technical presentations to be made to white house personnel, members of congress, FDA and USDA via the Podesta group in Washington D.C. TARGET AUDIENCES: Food industry personnel, the white house, the US congress FDA, and USDA. PROJECT MODIFICATIONS: Methodology has been extended to seafood.

Impacts
The food industry is presently investing in excess of two billion dollars to implement this technology and is establishing a multimillion dollar research and development Biotechnology facility to further expand this technology.

Publications

• Gu, W. and Levin, R E 2008. Innovative Methods for Removal of PCR Inhibitors for Quantitative Detection of Plesiomonas shigelloides in Oysters by Real-Time PCR. Food Biotechnol. 22: 98 -113.
• Luan, C. and Levin, R. E. 2008. Use of activated carbon coated with bentonite for increasing the sensitivity of PCR detection of Escherichia coli O157:H7 in Canadian oyster (Crassostrea gigas) tissue. J. Microbiol. Meth. 72:67-72.

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Cells of a strain of Escherichia coli O157:H7 seeded into meat tissue homogenates were completely bound to untreated charcoal after an incubation period of 15 min at room temperature. In contrast, activated charcoal particles coated with cells of Pseudomonas fluorescens (1.5 x 1,000,000,000 cells/ml) resulted in 92.6% + 3.7 recovery of E. coli O157:H7. This allowed the successful use of the coated activated charcoal for the absorption of PCR inhibitors from seeded tissue samples. With coated charcoal, real-time PCR was able to detect 1 x 1000 CFU of E. coli 0157:H7/g of tissue which was equivalent to 50 genomic targets per real-time PCR. In contrast, without the use of treated charcoal, the real-time PCR failed to detect 10,000,000 CFU/g. A novel method for directly increasing the recovery of Escherichia coli O157:H7 and efficiently eliminating PCR inhibitors in meat tissue without preenrichment was developed with the use of activated carbon coated with bentonite. The recovery of Escherichia coli O157:H7 was significantly affected by the amount of bentonite used to coat the activated charcoal and the pH value of sample preparations. When 4.2 g of activated carbon were coated with 0.4 g of bentonite and seeded oyster samples were adjusted to a pH of 5.0, a high recovery of E. coli O157:H7 (91.6%) was obtained. Activated carbon, coated with bentonite, allowed the PCR detection of 150 CFU/g of oyster tissue which was equivalent to 30 genomic targets per PCR reaction. Without the use of activated carbon coated with bentonite, the minimum level of detection was 150,000 CFU/g of oyster tissue, which is equivalent to 30,000 genomic targets per PCR reaction. PARTICIPANTS: Ruth Witkowski was a technician on the prject. Chunguan Lee was a Ph.D studen on the project. TARGET AUDIENCES: Target audiences are: FDA personnel USDA personnel,and food industry personnel. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The goal of this project is to successfully detect low numbers of E. coli O157:H7 in meat products rapidly without enrichment. The procedures developed have at this point allowed further development of activated charcoal methodology as a means of selectively removing PCR inhibitors from foods.

Publications

• No publications reported this period

Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: A novel method for directly increasing the recovery of Escherichia coli O157:H7 and efficiently eliminating PCR inhibitors in oyster tissue without preenrichment was developed with the use of activated carbon coated with bentonite. The recovery of Escherichia coli O157:H7 was significantly affected by the amount of bentonite used to coat the activated charcoal and the pH value of sample preparations. When 4.2 g of activated carbon were coated with 0.4 g of bentonite and seeded oyster samples were adjusted to a pH of 5.0, a high recovery of E. coli O157:H7 (91%) was obtained. Activated carbon, coated with bentonite, allowed the PCR detection of 150 CFU/g of oyster tissue which was equivalent to 30 genomic targets per PCR reaction. Without the use of activated carbon coated with bentonite, the minimum level of detection was 150,000 CFU/g of oyster tissue, which is equivalent to 30,000 genomic targets per PCR reaction. Three commercial DNA purification systems were used for comparison. The limit of detection with the Wizard DNA Clean-Up System and the Chelex 100 Resin was 1,500 CFU/g of oyster tissue which was equivalent to 300 CFU/PCR reaction. The QIAamp DNA Mini Kit resulted in a detection limit of 500 CFU/g of oyster tissue which was equivalent to 500 genomic targets per PCR reaction. The use of activated carbon coated with bentonite is an inexpensive method for removal of PCR inhibitors from tissue samples prior to the release of DNA from target cells resulting in relatively low numbers of target cells detected without enrichment. PARTICIPANTS: Chunguan Luan, a Ph.D. student was funded on the project. TARGET AUDIENCES: Target audiences include FDA, USDA, and the food industry. PROJECT MODIFICATIONS: no changes in approach

Impacts
The methodology developed now make it possible to detect relatively low numbers of E. coli O157:H7 in various complex food matrices rapidly by PCR. The funds provided supported one graduate student who developed the key methodology.

Publications

• Abolmaaty, A., Gu, W., Witkowsky, R. and Levin, R. E. 2007. The use of activated charcoal for the removal of PCR inhibitors from oyster samples. J. Microbiol. Meth. 68: 349 - 352.