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NY DOH Develops Multiplex Real-Time PCR to Combat Staph Food Poisoning, Bioterrorism

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NEW YORK (GenomeWeb) – Researchers at the New York State Department of Health's Wadsworth Center have developed a multiplex real-time PCR assay to detect the enterotoxin genes of various Staphylococcus aureus strains and enable rapid outbreak detection and control.

The assay, along with a singleplex positive extraction control to detect inhibition, was published online in Molecular and Cellular Probes.

The protocol also includes a modification of the MagNA Pure Compact automated extraction method from Roche that allows improved S. aureus detection from stool samples.

Staphylococcal contamination caused 570 outbreaks of food poisoning between 1998 and 2010, and Staphlococcus aureus type B is considered a potential bioterrorism agent. Wadsworth Laboratory is New York State's reference lab tasked with responding to these types of public health threats.

"It's important for us to be able to quickly determine the cause of an outbreak of illness so that it doesn't spread throughout the public," Christina Egan, corresponding author on the study and director of the Wadsworth Center Biodefense Laboratory, told GenomeWeb in an interview.

"Our previous methods were based on culturing the agent from different food sources — which could take days — and we really wanted to be able to more rapidly identify a causative agent," she said.

In the literature there are a few assays for S. aureus in food, but Egan believes this is the first that combines multiplex real-time PCR with the automated extraction process needed to process larger numbers of samples.

As described, the assay focuses on clinical patient stool samples, but Egan said her group has also validated it in several different types of food matrices.

The assay detects staphylococcal enterotoxins A through E as well as toxic shock syndrome toxin production genes.

It also uses a novel enzyme-based pre-lysis extraction paired with Roche MagNA Pure Compact.

"That's really key because we are testing directly from patient specimens and foods and we don't have an incubation step, so we really want to be able to have as efficient a reaction as possible to detect any nucleic acid that's present and has a toxin sequence," Egan said.

The researchers tested the assay on 47 strains of microorganism, and found 100 percent specificity and a sensitivity of 8 to 20 colony forming units per real time PCR in clinical stool matrix.

The researchers are not planning to pursue patent protection for this particular assay, Egan said.

"We've published it and made it available so that other public health labs and clinical laboratories can utilize it to improve their diagnostic capability," she said.

However, she noted that the lab is glad to work with companies that are developing diagnostics in this area. "We've done that in the past and that's something that we're always willing to do."

S. aureus is not represented in the multiplex GI panels that have recently become commercially available, possibly because it is so self-limited, but Egan noted that "it would be great to develop a panel that includes staph enterotoxin."

Wadsworth houses several regulatory programs, including one that grants permits to both environmental and clinical laboratories, including CLIA certification. It is also involved in development of molecular diagnostics, particularly ones aimed at "testing more agents with less specimen," Egan said.

For example, Wadsworth has collaborated with Columbia University and Northrup Grumman to develop a biothreat agent diagnostic using mass spectrometry. With Akonni Biosystems, Wadsworth has worked on diagnostic viral arrays, including ones for meningitis/encephalitis and influenza, as well as a tuberculosis assay, as previously reported.

Mass food poisoning and bioterror

The Molecular and Cellular Probes study also notes that the S. aureus assay was used to detect an outbreak in New York State.

In that case, attendees of a large event at Chuang Yen Buddhist Monastery in Putnam County consumed tainted food.

After eating at the event, one large contingent from the New York City area then boarded several buses for a visit to the Monroe-Woodbury outlet mall.

"By the time they got to the mall, many of them were really ill," Egan said. The presence of such a large number of sick people, some of whom could hardly walk according to news coverage from the time, triggered an alert and the county health department was then able to retrieve food from the event. All told, about 150 people became ill, according to another report

"What was unfortunate was that many of the [food vendors] had traveled from the tri-state area and we think that the food was stored under conditions that allowed Staph enterotoxins to be produced," Egan said.

Deliberate mass food poisoning is also a biothreat that Wadsworth vigilantly monitors, and Staph enterotoxin type B is on the US Centers for Disease Control and Prevention's Bioterrorism agents list.

S. aureus enterotoxin illness is self-limiting and usually non-fatal, but it could potentially be used to disrupt the food supply, a situation which one study estimates could cost $30 billion to remedy.

Although there are a number of known non-terrorist biocrime cases, there have only been two confirmed incidences of terrorist use of biological weapons since 1945.

Egan mentioned that one was the 1984 case of mass food poisoning in Oregon perpetrated by a cult, referred to as the Rajneeshee bioterror attack.

In that incident, salmonella was cultured and then used to contaminate food and condiments at 10 restaurant salad bars. The attack ultimately sickened 751 people, but failed to exert the desired political effect of disrupting a local election.

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