20 for 20: Day 5 : From the “LAMP”-post to Cair Paravel on the Eastern sea

Once in a while, a diagnostic technique comes along that is everything you ever dreamed about, and wonderful like a Narnian summer. It is highly sensitive, highly specific, costs very little, does not require bulky equipment or highly trained personnel. One that has been hailed that way is the Loop mediated isothermal AMPlification assay or LAMP for short.

LAMP results in the amplification of nucleic acid sequences at a single, constant temperature (hence, isothermal). A special strand displacement polymerase, such as Bst polymerase from Bacillus stearothermophilus is used, along with 6 primers that confer specificity to the assay. Amplicons are later visualized using a fluorescent dye (increase in fluorescence indicates amplification), on an agarose gel or using a magnesium pyrophosphate (precipitation of the salt implies amplification). 

The LAMP assay at first glance seems to be all that it promises to be. It is specific and sensitive, costs less than conventional PCR, requires only a heat block or water bath at a constant temperature, and monkeys can probably be trained to do it. However, despite the hundreds of original research articles that have been published for parasite detection, no one seems to be routinely using it in diagnostics.

A little digging into the details of the assay brings the disadvantages to light.

(1) The LAMP assay is an end-point assay. One can measure the turbidity or fluorescence and try to correlate that to parasite number, but the amplicons themselves cannot be used for further cloning or sequencing, ruling out its use in the research lab.

(2) Designing primers for the LAMP may not be the easiest thing in the world, despite the availability of softwares. Since non-specificity at low annealing temperatures is always an issue when designing nucleic acid primers, designing 6 primers on a short sequence that all anneal at one temperature is pretty hard.

(3) Contaminants in the template inhibit amplification. If you have ever tried to amplify sequences from feces directly, you know what I am talking about. There are (probably) a million (I exaggerate) inhibitors in feces that impede conventional PCR even with a robust polymerase. These same inhibitors also impede the sensitive Bst polymerase as well. If you have to purify DNA using a kit, you might as well do conventional PCR/qPCR, and sequence the amplicons.

(4) Multiplexing is hard because of the numbers of primer pairs one will have to design. For example, duplexing the assay requires one to design 12 primers. A triplex assay requires 18 primers and so on.

The utility of the LAMP assay depends on what it is going to add to clinical diagnosis. For example, a veterinarian handling a sheep flock with trichostongylosis is probably going to help the farmer more by doing a FECRT than LAMP assays separately for Haemonchus, Cooperia, Ostertagia, Trichostrongylus, Nematodirus and any other common nematodes in the area.

Therefore, while the LAMP is a great assay to design, read about and think through, it’s practical value in veterinary parasitology may be pretty limited at present.

This post is part of a goal to write for 20 mins for 20 days.