Rapid Testing Developments and Regulation

Gold Popcorn-Shaped Nanomaterials Gang Up on Salmonella

A new test that uses gold nanoparticles can identify Salmonella in five minutes.

The gold nanoparticles are bits of gold so tiny that more than 25,000 would fit across the width of a human hair. The Jackson State University researchers, led by Paresh Ray, attached antibodies—molecules similar to the ones that help the immune system find and fight infections with Salmonella—to the nanoparticles. Viewed under a microscope, the gold nanoparticles look somewhat like individual pieces of popcorn.

When the antibodies encounter Salmonella bacteria, they attach to its outer surface, carrying along their cargo of gold popcorn-shaped nanoparticles. “If the color changes from pink to bluish, that signals the presence of Salmonella,” Ray said. Because it doesn’t take a trained laboratory technician to perform the test or read the results, it is suitable for use in farm fields and in remote areas of the developing world, he explained.

The scientists described the method at a meeting of the American Chemical Society earlier this year, explaining that the test requires only a small sample of a lettuce leaf to quickly detect Salmonella, especially the multiple-drug resistant (MDR) strains that cause the most serious disease in food and drinking water.

The approach also has potential for killing the MDR Salmonella, Ray said. “When you shine the right wavelength of light into contaminated water, for instance, the gold nanoparticles absorb that light and heat up,” he explained. “Those hot particles burn through the outer membrane of the Salmonella bacteria, killing the bacteria.” The group has also used the nanoparticles to detect other microbes, such as E. coli.

Ray said the technology can be commercialized, and a patent is pending. With concerns about the potential health and environmental effects of many kinds of nanoparticles, Ray’s team is investigating the effects of gold nanoparticles remaining in purified water, for instance. So far, they have found no short-term toxicity and will be checking on any potential long-term toxicity.

Nanotech Regulation. This concern is also being addressed by the FDA, which issued two draft guidance documents in April, addressing the use of nanotechnology by the food industry. The draft guidance describes the factors manufacturers should consider when determining whether changes in manufacturing processes, including those involving nanotechnology, create a significant change that may affect the identity, safety of use, or regulatory status of the food substance or warrant a regulatory submission to FDA.

The guidance also encourages manufacturers to consult with the agency before taking their products to market. Such consultation can help FDA experts address questions related to the safety or other attributes of nanotechnology products, or answer questions about their regulatory status. FDA is also investing in a nanotechnology-regulatory science program to further enhance its capabilities, including development of necessary data and tools to identify properties of nanomaterials and assess the impact they may have on products.
 

Latex Beads Form Basis of USDA ARS E. Coli Test Method

While one USDA service was announcing a new regulation for the testing of beef manufacturing trimmings for the six non-O157 Shiga toxin–producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121), another service of the agency was announcing the development of a technique to test for the same.

As of June 4, USDA’s Food Safety and Inspection Service (FSIS) began testing for the six non-O157 STECs in beef trimmings. FSIS had announced its intent to declare these as adulterants in non-intact raw beef products and product components in a 2011 Federal Register notice of proposed rules.

Just prior to the date of enactment of the regulation, a new method for the detection of the six non-O157 STECs, based on latex agglutination assays utilizing polyclonal antibodies and developed by USDA’s Agricultural Research Service (ARS), was published in the May 2012 issue of Journal of Food Protection (JFP).

Latex Agglutination. Led by ARS Chemist Marjorie Medina, the team, including ARS associates from the Biosciences Research Laboratory in Fargo, N.D.; FSIS in Athens, Ga.; and Pennsylvania State University, developed the method to make a latex chemical agent that clumps bacterial cells in the presence of antibodies. In testing for E. coli, the latex agglutination results are obtained in about a minute.

To produce antibodies against the STECs, researchers inject dead bacteria into laboratory animals, which activates the immune response, resulting in antibody production. The immunoglobulins (IgGs) are purified from the antiserum collected from the immunized animals and linked to latex particles, resulting in a latex-IgG complex. The presence of the STEC strains is observed instantly after mixing the colonies onto latex-IgG that is placed on a slide. Clumping indicates a positive result and flaking, or suspension, indicates a negative result.  

Latex beads are formed from polymers and are similar to a latex (water-based) paint. If results are positive, the beads coated with antibody will clump (agglutinate). The agglutination assays can be used for identification of presumptive non-O157 STEC colonies from agar media. In addition, the techniques used to prepare the latex reagents can be used to test other STEC serogroups, other E. coli serotypes, or other pathogens.

The Study. In the ARS study, more than 100 target and non-target strains were tested in more than 3,000 test replicates. All target organisms produced positive results, but three antisera (anti-O26, anti-O103, and anti-O145) slightly cross-reacted with some other STECs.

Although latex agglutination reagents for E. coli O157:H7 are commercially available, these had been lacking for the top six non-O157 STEC serogroups. Thus, the objective of the current study was to use polyclonal antisera to develop latex agglutination reagents specific to the top six non-O157 STEC serogroups and to attach the immunoglobulins (IgGs) covalently to the latex particles.

The latex agglutination assay reagents can easily be prepared, and results indicate reliable recognition of target STECs, stated the study’s authors, adding that although cross-reactions were observed, these can be overcome by modification of antibody production or production of monoclonal antibodies for the cross-reacting IgG reagents. Additionally, the latex reagents will be useful for identification of presumptive non-O157 STEC colonies picked from various types of agar media before or after PCR assays. The reagents were stable for more than 12 months when stored at 39°F.