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Australian Researchers Use Isothermal Amplification Method to Detect Salmonella Subtypes

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NEW YORK – A new method developed at the University of New South Wales for detecting and differentiating Salmonella enterica serotypes holds promise for the creation of highly sensitive and specific clinical assays to diagnose illness caused by the foodborne bacteria. 

In a study published in the Journal of Molecular Diagnostics in May, researchers from the university used an isothermal amplification technique called multiple cross-displacement amplification (MCDA) to detect and differentiate the five most common Salmonella serovars in Australia — Typhimurium, Enteritidis, Virchow, Saintpaul, and Infantis — from pure culture. 

Ruiting Lan, lead author of the study and a professor at University of New South Wales' School of Biotechnology and Biomolecular Sciences, emphasized there are two new ideas in the paper. One is related to the use of the gene markers used for phenotyping salmonella. Traditionally, phenotyping salmonella relies on three antigens, the O antigen and the H1 and H2 antigens. The problem with that, Lan said, is that those antigens are present in a lot of subtypes and the difference is specifically in the sequence variation.  

For this study, researchers used genome data to find specific gene markers called proxy markers. "The cool thing about those proxy markers is they're very specific to a phenotype, so if they're present, you know that's the phenotype," Lan said.  

The other new idea raised by the study is the use of the MCDA method, Lan said. PCR can be used to detect the gene markers, but the MCDA method is much faster and more sensitive than PCR and doesn't require a thermocycler to perform the test due to its incubation at a constant temperature of about 63° C, he added.  

Kuppuswamy Kasturi, a microbiologist with the US Food and Drug Administration who was not involved with the study, noted that there are significant benefits to using an isothermal amplification method for salmonella detection.   

"It's very simple … you don't need a lot of mixture," Katsuri said. "You don’t need a machine to read the fluorescence, you can even visually see it."  

Katsuri noted, however, that the interpretation of results is "subjective, rather than objective" when not using an instrument to read the fluorescence. Individuals can interpret results differently, and there is always the chance for error.   

Another difference between MCDA and PCR, Lan said, is that the researchers used 10 primers for MCDA, rather than two primers for PCR, to detect 10 distinct regions of the gene. The increase in regions enhances specificity and sensitivity. Multiple molecular reactions are occurring at once during MCDA and, Lan said, "You can view it as more than one PCR happening in the same reaction."   

The researchers didn't create the MCDA method, but adopted it for their own testing, Lan clarified. The method was originally described in 2015 in Scientific Reports by Chinese researchers. Lan and colleagues, however, modified the method to target the Salmonella genes they previously identified in comparative genomic analyses. The method could be expanded to detect as many as 100 phenotypes based on previously discovered gene markers. Lan noted that salmonella has more than 2,000 different phenotypes but said that many of those are rarely transmitted to humans so are less of a concern.  

One of the most-used molecular methods for salmonella phenotyping is by detecting the two genes signaling the presence O, H1, and H2 antigens, but these genes are present in every type of salmonella, so researchers must look for specific sequences, which is significantly harder.  

For the newly published study, the researchers looked specifically at the sensitivity of their MCDA assays in pure culture. Sensitivity and specificity ranged from 93 percent to 100 percent and the limit of detection was 10 copies of DNA, with positive results detected in as few as eight minutes. However, Lan noted that the lower the limit of detection, the longer results take, so it generally takes around 30 minutes.  

The MCDA method could be used clinically to garner additional information about the Salmonella that's causing disease and assist with strain determination. For patient care, it's less important to know what salmonella phenotype someone is infected with and more important to just know whether they have salmonella or not by testing a fecal sample, Lan said. But for outbreak investigations, once it's determined that a sample has salmonella, strain identification is important for further public health purposes, he continued.

Clinical laboratories are continuing to move away from using culture for diagnosing salmonella, making culture-independent techniques like MCDA, as well as PCR and loop-mediated isothermal amplification, more useful. "The genomic signatures that were targeted by the MCDA assays can provide more useful serovar identification than traditional serotyping, especially for those strains with the same serovar but different evolutionary history," the study's authors noted. 

The MCDA method and assays also have clear applications in the food industry and food safety, since determining the specific phenotype of salmonella can help regulators identify potential sources of outbreak and stop them before they occur, cutting down on food recalls and improving early detection.  

Lan said the researchers are seeking additional funding to further develop the MCDA assays, but noted they haven't been patented, so anyone could use the biomarkers and develop tests for their own use or for commercialization.  

Although this study only investigates five phenotypes, Lan said reducing infections from those strains can still have a major impact.  

"If we can really reduce the infections from the top phenotypes causing the majority of outbreaks, it will help all the way from the farm level to the clinical level to reduce salmonella infections" Lan said.  

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