Developing Rapid and Sensitive Pathogen Detection Systems for Food Safety and Biosecurity

Linda Benedict  |  8/22/2007 11:22:58 PM

Figure 2. Positive samples (bright fluorescent green) and negative samples (dark, non-fluorescent) under UV. (Photo by John Wozniak)

Photo By: John Wozniak

Figure 1. Positive samples (green to greenish-yellow color) and negative samples (orange color) under normal light. (Photo by John Wozniak)

Photo By: John Wozniak

A dry heating block that maintains a constant temperature for performing the isothermal detection assay. (Photo by John Wozniak)

Photo By: John Wozniak

Figure 3. Different clinical and environmental strains of Vibrio vulnificus. (Photo by Reshani Senevirathne)

Beilei Ge, Marlene E. Janes, Feifei Han, Reshani Senevirathne and Janet Simonson

America boasts one of the safest and most plentiful food supplies in the world. Unfortunately, food by nature or by accident is vulnerable to contamination by harmful microbes at any point from the farm to the table.

According to the U.S. Centers for Disease Control and Prevention in Atlanta, each year 76 million Americans get sick from something they eat. Of these, 325,000 are hospitalized and 5,000 die. In the fall of 2006, E. coli-tainted spinach led to one of the largest and deadliest foodborne outbreaks in recent years. Shortly after, peanut butter contaminated with Salmonella sickened more than 600 people in 47 states, heightening growing public concerns over food safety. Along with food safety concerns, the intentional contamination of our food supply – referred to as agro-bioterrorism – can cause graver social and ecological damage, underscoring the urgency and significance of research to minimize such risks.

The ability to quickly and accurately detect the presence of even low levels of harmful microbes is essential for food safety and biosecurity. An ideal detection method is above all rapid, sensitive, specific and cost-effective. Currently, foodborne pathogen detection relies heavily on conventional microbiological culturing techniques, which are labor-intensive and timeconsuming. Although newly developed molecular techniques have improved performance, they still lack sensitivity, take a long time for analysis or require expensive equipment.

LSU AgCenter researchers are improving methods to detect these foodborne pathogens. In particular, they are focusing on detecting and counting a deadly foodborne pathogen that has been the foremost concern to the Louisiana oyster industry for the past few decades – Vibrio vulnificus.

This particularly troublesome microorganism is salt-loving and favors warm coastal and estuarine waters. The Gulf of Mexico is an ideal natural habitat for V. vulnificus. In summer months, the prevalence rate can be as high as 100 percent, and 95 percent of seafood-related deaths – about 40 annually – are due to eating raw oysters containing this pathogen. Fortunately, V. vulnificus is a pathogen that causes illness only in certain atrisk groups, which include people who have weakened immune systems, liver disease or alcoholism.

V. vulnificus detection is primarily based on culturing the bacteria followed by confirmation using biological and chemical reactions. Few rapid tests – including convenience-based (like a pregnancy test) or immunological-based (antigen-antibody recognition) methods – are available. Molecular-based methods, such as polymerase chain reaction (PCR), which amplify certain genes of the bacteria, are just starting to gain some momentum. The problems with PCR include expensive instrumentation and difficulty in field testing.

LSU AgCenter researchers are developing and evaluating a novel isothermal (one constant temperature) method to amplify – or extensively duplicate – certain genes in V. vulnificus. Similar to PCR, this technique starts with efficiently amplifying the target bacterial genes, duplicating the initial gene millions of times in about an hour. This assay is actually more sensitive and rapid than PCR. The accuracy of detection is ensured by targeting genes unique in V. vulnificus. When the assay gives a positive testing result, it will indicate the presence of V. vulnificus, not something else.

Another attractive feature of this technique is how the amplified genes are detected. After adding a few drops of a DNA dye, positive samples turn the tube green to greenish-yellow while negative samples remain orange (Figure 1). The signals are even stronger under ultraviolet light (Figure 2). Such color changes can be easily identified by the naked eye, so it is easy to tell the positives from negatives. This technique can also be used to quantify the number of V. vulnificus cells in an oyster sample. The researchers are enhancing this detection system and will evaluate it for field applications.

LSU AgCenter researchers have also developed a V. vulnificus antibody-based detection method that is more user-friendly than U.S. Food and Drug Administration recommended methods. The LSU AgCenter method involves growing bacterial colonies on agar plates, transferring the colonies to membranes, treating the membranes with antibodies for 1 hour and washing the membranes three times. The membranes are then incubated with another chemical for 1 hour and washed three times. Finally, a color-development mixture is added for five minutes. Positive colonies produce a purple color. The test was positive for all V. vulnificus strains tested (Figure 3) and did not react with other Vibrio species.

This newly developed V. vulnificus test can consistently detect 100 V. vulnificus cells mixed with 10,000 cells of a related pathogen. The method was compared with two FDA recommended methods for counting naturally occurring V. vulnificus in oysters, and all three methods are comparable. The LSU AgCenter method exhibited better color development and was less time-consuming than the FDA methods. It can be completed in 3.5 hours while one FDA method takes 24 hours and the other method takes 50 hours to perform. The LSU AgCenter process for detecting V. vulnificus could be used as a rapid counting method by regulatory agencies or the seafood industry.

Both detection systems developed by LSU AgCenter researchers can be modified and applied in detecting other harmful microbes of food safety and bioterrorism concerns. Taken together, these research efforts will greatly advance the ability to fight against pathogens associated with foodborne illnesses and bioterrorism threats.

Beilei Ge, Assistant Professor, Marlene E. Janes, Assistant Professor, Feifei Han, Graduate Student, and Reshani Senevirathne, Graduate Student, Department of Food Science, LSU AgCenter, Baton Rouge, La.; and Janet Simonson, Instructor, Department of Agricultural Chemistry, LSU AgCenter, Baton Rouge, La.

(This article was published in the summer 2007 issue of Louisiana Agriculture.)
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