LAMP lights the way: UP grad students develop faster, more accurate Salmonella detector

By Andre DP Encarnacion on September 5, 2024

*Researchers Rance Derrick Pavon and Khristine Balaga hold the fruits of their work at UP Diliman's Institute of Biology. Photo by Jonathan Madrid, UP MPRO.*

For weeks, graduate students Rance Derrick Pavon and Khristine Balaga of the University of the Philippines Diliman Institute of Biology (UPD IB) woke up before the crack of dawn to go to the busiest wet markets in Metro Manila. As members of the IB's Pathogen-Host-Environment Interaction Laboratory, their mission was to get as many randomly selected raw samples as they could of beef, chicken, and pork from stalls.

"We wanted to simulate how a so-called normal consumer would buy meat," said Pavon. "So we didn't really pick."

The meat, however, was definitely not for eating. The pair instead allowed the meat to rot over the weekend, braving the smell just to isolate and grow one of humankind's oldest bacterial tormentors: Salmonella. This most underestimated of microbes was still responsible, according to the Philippine News Agency (PNA), for more than 13,000 illnesses in 2023, part of the 83 million or so recorded globally.

"I know people overlook Salmonella," Pavon said, "because it's just bacteria which just needs antibiotics. But it is responsible for millions of cases every year, even in developed countries. And these can result in hundreds of thousands of deaths."

In fact, a 2019 study by Azanza et al. identified Salmonella to be the leading cause of food poisoning in the country. The problem is, however, as Pavon, Balaga and their colleagues bemoan, that effective surveillance of farms and markets is extremely difficult due to the processes and technologies either taking too much time or being too expensive for day-to-day use. Both of them knew the nation needed something new.

LAMP lights the way

*Graduate Student Khristine B. Balaga works under the hood at the Pathogen-Host-Environment Interactions Research Laboratory. Photo by Jonathan Madrid, UP MPRO.*

To provide a context, Balaga said that the current gold standard for Salmonella detection that government agencies use involves culturing and biochemical detection. It requires numerous biological and serological tests that take five to seven days to complete. Conventional polymerase chain reaction (PCR), on the other hand, a technique that uses enzymes and several temperature changes called 'cycles' to create copies of a specific region of DNA (in this case, of bacteria), is faster and more sensitive. However, the reagents, not to mention the machines needed to do PCR, are too expensive for most to contemplate.

Clearly, something needed to be done. So, building on research previously done by senior scientists in their lab, the pair decided to try out methods that could be used out in the field and in routine laboratories. To be considered an improvement, the solution had to be highly sensitive like PCR, but simple and affordable enough to be highly teachable and usable. That's when they decided on the LAMP assay.

Put simply, the LAMP assay is an isothermal amplification method that allows one to detect nucleic acids from DNA without sophisticated equipment, the invA gene in Salmonella specifically. The key word here is isothermal. Unlike PCR with its different cycles of temperature, LAMP just requires a heat block—or any simple heating equipment like a pot or a stove—kept to a steady 65 degrees Celsius to replicate a region of DNA. And it can finish the step in just around 30 minutes, Balaga said.

Moreover, while LAMP still requires trained personnel to prevent contamination, it is relatively simple to do and its use, to teach. "You just need to add all of the reagents and the DNA," clarified Balaga. Also, unlike PCR, which requires a process called agarose gel electrophoresis to determine a true positive, LAMP uses a metal indicator dye called calcein, which changes color depending on which chemical element around it, magnesium or manganese, is more plentiful.

Reactions involving an enzyme called Bst polymerase used in LAMP releases magnesium, which binds to calcein and turns the former green, indicating a positive result. Otherwise, calcein binds to the more plentiful manganese, which visualizes as brown or orange and signifies a negative result. A relatively simple principle to visualize and interpret.

Everything and More

*The UP Diliman Institute of Biology's Rance Derrick Pavon holds up positive samples for salmonella, the leading cause of food poisoning in the country. Photo by Jonathan Madrid, UP MPRO.*

Even if the method were less expensive and easier to use, however, it would mean little if it were not sensitive enough in Salmonella detection when compared to current methods. So, what Pavon and Balaga did was to compare the sensitivity of their optimized LAMP in detecting Salmonella with conventional PCR, using the same upstream processes.

To ensure that they were pitting their LAMP assay against the best the country has to offer, the pair used the specific PCR protocol developed by their adviser, Dr. Windell L. Rivera. This protocol was validated against the current gold standard and was found to be superior. "It was able to detect everything that the culture method could and more," Balaga explained.

The first test of sensitivity involved diluting the DNA tenfold with a liquid buffer. While they were familiar with reports of LAMP possibly being more sensitive than PCR, the results surprised even the researchers. LAMP proved to be one thousandfold more sensitive than PCR under these conditions. Next, rather than just DNA, they took the bacteria itself and diluted it, effectively comparing both methods using bacterial culture. In this head-to-head test, LAMP proved a hundredfold more sensitive, as reported by Pavon.

Lastly, they tested both LAMP and PCR on 341 raw meat samples that they managed to collect, to see how LAMP's specificity and sensitivity would hold up. While the differences here were not statistically significant, the pair managed a 99.6% detection rate with PCR but a 100% Salmonella detection rate with LAMP. "So, to us that means our method is a good alternative to PCR, if not complementary to it," Pavon said.

The Way Forward

With the publication of their results, Pavon and Balaga know that the official adoption of their calcein-based LAMP assay for Salmonella will take time; but they are willing to do the work to get there. "Now that we've seen the benefits, we want to completely standardize the protocol, the mixes, so our components can be turned into some sort of test kit," Pavon said.

Beyond that, Pavon knows that LAMP is very flexible and can be used for much more than just Salmonella detection. You can use it to detect many different kinds of pathogens provided you have the appropriate primers for them. Currently, Pavon is doing a LAMP-related thesis that involves different serovars (distinct variants) of Salmonella. He hopes his work will make detecting different Salmonella serovars and other pathogens in meat more efficient. "I know you can also use different methods and dyes to make it all easier," he said.

On the other hand, since the publication of their article in the Journal of Microbiological Methods last May, Balaga has since left the lab to work in the private sector; but he remains affiliated to it as a graduate student. She hopes that their protocol can be improved to apply beyond chicken, beef, and pork to include things like seafood, and meat products for export. She also hopes that government partners like the Bureau of Animal Industry (BAI) can eventually use it to make their work easier.

"Maybe for now, since thermal cyclers (for PCR) are still expensive, it can replace some of the tests that they do and lighten the workload of doing culture and biochemical tests," she said. "If the end goal is just to detect salmonella, not to detect serovars or anything like that, they can shorten their one-week workflow to two or three days."

Better still is the fact that these government agencies have access to all the samples they need and more, since meat samples are voluntarily sent to them. "That means they can further improve what we developed if they apply it. Since the Department of Agriculture gave us funds to develop technologies the government can use, we hope this just doesn't end with us. The ball is in their court, so to speak."