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