Making DNA and RNA Diagnostics More Amenable to Field-Based Testing

Point-of-care and field-based diagnostic testing using PCR is limited in that samples usually have to be shipped to a reference laboratory for analysis by highly trained personnel.  This can mean days to weeks before a result is available, reducing the ability for scientists to respond quickly to a potential disease outbreak.

Loop-mediated isothermal amplification, or LAMP,  is proving to be a game changer in point-of-care testing.  LAMP has the specificity and sensitivity of qPCR but with several distinct advantages that make it far more amenable to field testing.

At Varizymes, we are specialists in LAMP assay development.  In this post, I will review significant improvements we have made to LAMP, positioning this assay as the ideal way to perform diagnostics in the field.

1. Sample Preparation

Sample preparation must be as easy as possible for point-of-care testing.  Most nucleic acid tests (NATs) based on PCR need highly purified nucleic acid samples to perform well. Nucleic acid is typically purified from crude lysates by binding to silica matrices, followed by successive washing steps and then an elution step. These multiple steps are time consuming and hard to perform without access to centrifugation, vacuum filtration or expensive liquid handling instrumentation.

Varizymes' scientists have developed a lysis buffer for processing of vesicular and esophageal-pharyngeal fluid samples in the field. Briefly, the method involves adding sample to the lysis buffer followed by incubation at room temperature for 5 minutes. After incubation, sample is filtered with a handheld syringe, and lysate can be
used directly as template in the LAMP reaction without the need for any additional purification steps.  Varizymes' LAMP assays are unaffected by any inhibitors that may be present in rapid extraction samples.  Thus, our rapid sample prep method will be an asset to scientists testing samples in the field.

2. Test Kit Simplicity

The second way in which we have made field-based LAMP more accessible is in simplifying the assay itself. Varizymes' LAMP master reagent mixes are lyophilized, making them stable for months at ambient temperatures. Since refrigeration is not necessary, the assay is far more amenable to point-of-care use.

LAMP is ideal for field testing as it operates at a constant temperature.  Expensive thermal cycling equipment is not required.  A portable, battery-operated heat source and fluorometer provide real-time amplification data on site, eliminating lengthy time delays in shipping samples to a central reference lab.  Since results are available in minutes, scientists can respond much more quickly to a potential disease outbreak.

A key component to any LAMP assay is the strand-displacing DNA polymerase.  Standard LAMP polymerases can only amplify DNA targets.  For RNA targets, additional reverse transcriptase must be added.  This means multiple master mixes, complicating the assay.  At Varizymes, we have engineered a proprietary Bst-like strand-displacing polymerase to have high reverse transcriptase activity.  Thus, our neoBolt™ Bst DNA Polymerase is target-agnostic, simplifying the LAMP assay even further.

Unlike qPCR, very little training is necessary to run a LAMP assay.  This makes the technology far more accessible and easier to implement for veterinarians, farm producers, and other non-molecular biologists.

3. The Need for Speed

To be useful, a field-based diagnostic assay needs to be fast. While qPCR is effective, it needs to be run in a laboratory environment by highly trained personnel. This means samples need to be shipped to the lab, significantly delaying time-to-result. Unlike qPCR, LAMP is fully portable, and testing can be done easily, on-site.

Additionally, qPCR assays can take a significantly longer time to run than LAMP assays. Very often, a LAMP assay can be performed in under 30 minutes, including sample prep time, much faster than a qPCR assay.

4. Multiplexing—The Final Piece of the Puzzle

One advantage of qPCR is its multiplexing capability, whereby several targets can be assayed simultaneously.  This is trickier to do with a LAMP assay as primer design becomes more and more complicated as plex increases.

At Varizymes, we have solved the multiplexing problem, and now routinely perform sensitive and accurate multiplex LAMP (mLAMP) assays in-house.  For example, our duplex SARS-CoV-2 assay tests for an internal control and one SARS-CoV-2 target.  Our quadruplex TORCH™ mLAMP kit being developed for catfish pathogens tests for a control and 3 pathogens: Aeromonas spp., Edwardsiella spp., and Flavobacterium spp.

Figure 1:  TORCH mLAMP takes less than 30 minutes, including sample prep time.

Other TORCH mLAMP kits are in development, including kits for tilapia pathogens, Foot-and-Mouth Disease Virus, Hepatitis C Virus, Candida auris and more.  We are eager to find early-access users for these kits, so please reach out if you are interested.

5. Final Thoughts

Varizymes' TORCH mLAMP kits are an ideal solution for field-based and point-of-care diagnostics testing. Like qPCR, TORCH mLAMP is highly sensitive and very specific. Unlike qPCR, TORCH mLAMP assays are simple, fast, inexpensive, fully portable and provide results within minutes, on-site. In addition, we are now able to multiplex, enabling simultaneous detection of up to four targets in one test.