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Rapid Methods and On-Farm Bacterial Testing of Foods

Each year, an estimated 47.8 million people in the U.S. will become ill from eating contaminated foods.[1] A study by the U.S. Centers for Disease Control and Prevention has recently concluded that leafy greens are responsible for almost half of these foodborne illnesses.[2] Foodborne outbreaks associated with produce have increased significantly, from 0.7 percent in the 1970s to 13 percent between 1990 and 2005.[3] From 1990 to 2005, there have been 713 recorded produce-related outbreaks and approximately 34,000 cases of illness associated with produce contamination.[3]

While demand has been growing for the consumption of fresh produce for better health and nutrition, at present, a pragmatic nonthermal process to reduce pathogenic risk in produce has not been put into practice. Food safety has continued to grow in importance, and the climate is changing to demand that stronger food safety programs are instituted throughout the food chain from farm to fork. Under the newly established Food Safety Modernization Act (FSMA), the U.S. Food and Drug Administration (FDA) will now have regulations for produce.[4] These regulations emphasize employee training, health and hygiene, agricultural water, biological soil amendments of animal origin, domesticated and wild animals, equipment, tools and buildings.[5]

Farmers will soon be responsible for validating the food safety of their on-farm water and soil. Part of the proposed ruling is to have agricultural water tested routinely to ensure that the water source is safe for its intended on-farm use. If the tested water fails the declared compliance, certain actions must be taken to make it safe (proposed sections 112.44 and 112.45). While these activities are intended to support the reduction of foodborne pathogens within produce, they will have a significant impact on many farmers’ methods of growing produce.

Current microbiological methods traditionally take between 24 and 72 hours to complete, plus the time it takes to send the sample to the laboratory for testing. In the operational process of produce, harvested product is shipped to distribution centers or sent directly to stores within 1 to 3 days to ensure the best quality and maintain that quality for at least 7 to 10 days. Adding more time to account for microbiological testing could be detrimental to overall product quality. Besides the time it takes to conduct testing, there are significant costs associated as well. The general cost to outsource a 100-mL water sample for an Escherichia coli/coliform assay to a microbiological lab ranges from $15 to $28 per sample. Due to the complexity of the test, currently this is the only option available for farmers.

During one of the most recent Q&A calls for the Produce Safety Alliance in regard to the Produce Safety Rules, many farmers expressed great concern about the proposed rulings for agricultural water. Farmers are very worried about how they will manage product if their water sampling results return out of regulatory compliance. One farmer pointed out that if he pulls a water sample on Monday, he would likely not get the results until Friday. The proposed rules require that you treat the water to ensure that the source is deemed safe; however, they don’t provide guidance on how to manage the produce that has been exposed to the contaminated water between Monday and Thursday. Although it is prudent that all contributors be involved in food safety, this issue illustrates a need for better on-farm tools to meet the upcoming food safety expectations.

While the current laws exempt small farms from mandated food safety plans such as a Good Agricultural Practices (GAP) certification, the climate is changing. Many wholesale and chain grocery buyers, such as Hannaford and Price Chopper, are requiring that their buyers have a GAP certification to reduce their business liability. Therefore, exempt farms that are not compliant to GAP certification may be at a significant competitive disadvantage if they do not initiate a food safety plan.

Testing on a Farm

A closer examination of the resources and requirements for on-farm testing reveals a significant difference from those of professional food labs. If rapid assays are to be used on a farm, either the on-farm resources would need to be upgraded or the assays would have to be modified to be amenable to this unique environment. Given the diversity in farm environments and cultures, the better approach would be improved design of the assays for use in nonlaboratory environments.

Rather than compare the testing environment on a farm with a processing plant or third-party testing laboratory, a more fitting model for diagnostic testing is in low-resource settings (LRS), such as rural India or sub-Saharan Africa. Scientists have been designing new strategies to aid in the diagnosis of chronic and infectious diseases in these challenging settings. The challenge is to develop a rapid assay for bacteria that does not include expensive external equipment, is low cost and requires little user training.[6] Given the resources and level of training on farms of all sizes, we could consider farms to be LRS.

In 2002, the U.S. Department of Agriculture’s Agricultural Research Service published an article entitled “On-Farm Testing for Pathogens on the Horizon.” The article outlines a detection method using fluorescent real-time polymerase chain reaction to detect pathogens. Although the detection was possible in 30 to 45 minutes, the instrumentation and cost make systems like this less practical for routine testing by a farmer. To design assays to be conducted on a farm, one needs to account for the farming environment and resources.

Diagnostic assays for on-farm use have different constraints compared with those used in traditional laboratory environment. These tools used on-farm must be rapid, low-cost, produce little waste and easy to use. In addition, pre-enrichment of pathogens should be avoided due to a lack of disposal options and increased risk of contamination. Currently, there are few pathogen diagnostic tools truly compatible with on-farm testing. The justification to provide on-farm testing seems to be growing.

Disposable, stand-alone, kit-based assays are ideal for testing in LRS. This type of system does not typically require expensive external diagnostic readers. The reliance on a single piece of equipment for testing increases maintenance requirements and often raises the initial cost of testing beyond what many farmers are willing to spend. Additionally, if the instrument breaks down and a service appointment is weeks away, the farmer must seek alternative means to test. The use of stand-alone kits therefore provides better reliability in these settings. As farmers cannot be expected to clean testing glassware on the farm, ideally all components of the kit would be disposable.

Nonprofit organizations such as Diagnostics for All, based in Cambridge, MA, and PATH in Seattle, WA, have been designing tests that can be used in LRS. Much of the funding for these projects has come from the Bill & Melinda Gates Foundation. Recently, Diagnostics for All established a group to aid small farmers in sub-Saharan Africa. These tests include bovine reproduction tests, milk spoilage tests and aflatoxin detection in maize. The goal of the projects is to allow farmers to maximize the price they receive for their commodities. The goals and requirements of these tests are clearly applicable to farms of all sizes in the United States as well.

Lateral Flow Shows Potential

Currently, several companies are manufacturing lateral flow assays to be used on the farm. These tests are easy to use, relatively inexpensive and very reliable. They are typically immunoassays utilizing colloidal gold for visual detection. Future requirements that include bacterial counts at low concentrations may require a new generation of rapid testing that is amenable to the farm environment.

Lateral flow tests are reliable enough that they have been used for in-home testing for decades (home pregnancy tests are probably the most familiar). FDA approval demonstrates confidence in this decades-old technology as a test that can be performed reliably outside of a laboratory setting. Until recently, lateral flow tests have been limited by relatively poor sensitivity and used almost strictly as a qualitative test. The increasing interest in diagnostics for LRS has encouraged research that addresses the current limitations of the traditional lateral flow assay. Although most of these research projects target infectious disease diagnostics in LRS, the technology can be seen as a potential benefit for farms everywhere.

The familiar red or blue line in a lateral flow assay has proven ideal for situations requiring positive or negative results. Unfortunately, for situations where a quantitative result is required, such as FSMA’s generic E. coli standards for agricultural water, a simple positive or negative is insufficient. The ability to reliably quantitate color reactions on test paper has been proposed using smartphones.[7] Most smartphones and tablets now come equipped with powerful cameras that can be used to quantify colorimetric results. The popularity of these devices has placed potential analytical tools in the hands of many farmers. Test results could be instantly electronically logged with a date and location, allowing the farmer to maintain accurate records with minimal effort. Smartphones can even be used to quantify fluorescent or chemiluminescent assays.[8] The use of chemiluminescent and fluorescent detection in place of visual colorimetric assays can reduce the limit of detection that is orders of magnitude lower.[9, 10] As the limit of detection continues to decrease, we get closer to the ability to detect low numbers of bacteria without the need to pre-enrich or perform genetic amplification.

When developing assays to be used on-farm, below are some of the questions that should be considered:

•    What is the true cost of the test? This includes labor, initial equipment costs and space requirements.

•    Does the assay require a clean environment such as a biosafety cabinet?

•    Can the assay be run by someone who was trained in approximately an hour?

•    Does the assay result in any waste that requires special handling?

•    Does the assay require specialized storage beyond a standard refrigerator?

•    Can the results be easily interpreted?

•    What is the total assay time from sampling to results?

•    How does the test perform when compared with the current standard?

•    Does the assay require timed steps by the user? How critical is the timing?

•    How temperature sensitive is the assay?

•    Does the assay require pre-enrichment of pathogens?


While federal funds for this area of research are limited, it is possible that future testing requirements will create a market attractive enough for additional companies to look toward this technology. Many researchers have aimed for years at the ability to rapidly detect foodborne pathogens. Unfortunately, the instrumentation used in a food testing lab typically cannot be used on a farm where there is no laboratory. The solution must be pragmatic and low cost. A look at the research being performed to bring low-cost diagnostics to LRS such as sub-Saharan Africa suggests that on-farm applicable diagnostics may be on their way. For now, farmers can continue sending out samples and waiting for results. Hopefully, in the near future, technology may empower the farmer to conduct rapid microbiological testing on the farm to better ensure a safe product.

Amanda Kinchla, M.Sc., is an assistant professor and food safety specialist at the University of Massachusetts, Amherst. Professor Kinchla researches both pre- and postharvest food safety practices.

Sam Nugen, Ph.D., is an assistant professor at the University of Massachusetts, Amherst. He specializes in the development of low-cost diagnostic assays for low-resource settings.

1. Morris, J.G. Jr. 2011. How safe is our food? Emerg Infect Dis 17:126–128.
2. Painter, J.A., R.M. Hoekstra, T. Ayers, R.V. Tauxe, C.R. Braden, F.J. Angulo and P.M. Griffin. 2013. Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, United States, 1998–2008. Emerg Infect Dis 19:407–415.
3. DeWaal, C.S. and F. Bhuiya. 2007. Outbreak alert! Closing the gaps in our federal food safety net. Washington, DC: Center for Science in the Public Interest.
5. LeBerre, V., E. Trevisiol, A. Dagkessamanskaia, S. Sokol, A.M. Caminade, J.P. Majoral, B. Meunier and J. Francois. 2003. Dendrimeric coating of glass slides for sensitive DNA microarrays analysis. Nucleic Acids Res 31:e88.
6. Yager, P., G.J. Domingo and J. Gerdes. 2008. Point-of-care diagnostics for global health. Annu Rev Biomed Eng 10:107–144.
7. Shen, L. J.A. Hagen and I. Papautsky. 2012. Point-of-care colorimetric detection with a smartphone. Lab Chip 12:4240–4243.
8. O’Driscoll, S., B.D. MacCraith and C.S. Burke. 2013. A novel camera phone-based platform for quantitative fluorescence sensing. Anal Methods 5:1904–1908.
9. Wang, Y. and S.R. Nugen. 2013. Development of fluorescent nanoparticle-labeled lateral flow assay for the detection of nucleic acids. Biomed Microdevices 10.1007/s10544-013-9760-1.
10. Wang, Y., C. Fill and S.R. Nugen. 2012. Development of chemiluminescent lateral flow assay for the detection of nucleic acids. Biosensors 2:32–42.

Source: Food Safety Magazine

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