Page 303 - The Veterinary Laboratory and Field Manual 3rd Edition

272 Susan C. Cork and Roy Halliwell

by enzymes so that complex temperature cycling
is not required; only a constant temperature
for nucleic acid amplification is required. The
LAMP technique was originally developed by
Eiken Chemical Co. (Tokyo, Japan) and can be
employed for the amplification of the genome
of DNA viruses using four primers recogniz-
ing six sequences of the DNA template at 61°C.
The NASBA technique relies on two primers
and three enzymes employed for the amplifica-
tion of target nucleic acid of RNA viruses at a
constant temperature of 41°C. Both versions of
isothermal amplification result in approximately
10 –10 copies of the target genome within 1 h
9
10
of assay time.
There are number of endpoint and real-time
monitoring systems available for the detection
of amplicon produced by LAMP technique. The
optimized endpoint detection systems for the
detection of LAMP products include visualiza-
tion of the amplification product using the naked
eye (Figure 4.34b), turbidometry or fluorimetry.
The more sensitive real-time monitoring sys-
tem using fluorochrome or turbidity also has
been developed. Detection of amplicons in the
NASBA technique is based on endpoint estima-
tion of products via hybridization analysis using
an electrochemiluminescent detection system or
real-time monitoring using fluorophore-tagged
oligonucleotide probes.
This molecular technique of viral genome
amplification is increasingly being recognized as
a diagnostic tool particularly in resource-poor sit-
uations or field situations due to (1) simplicity,
(2) speed, (3) specificity and sensitivity and (4)
cost effectiveness. Once total nucleic acids are
Figure 4.34 (A) Isothermal amplification requires extracted from the sample, either assay requires
only a simple heating block to maintain constant only simple equipment which is, a heating block
temperature up to an hour. Photo: Dr Regula to maintain constant temperature at 61°C or
Waeckerlin, University of Calgary, Canada. (B) LAMP 41°C, primers and/or enzymes. The main dis-
isothermal amplification products can be visualized advantage of the technique is the extra effort
using naked eye due to the accumulation of PCR required for the design of the four to six primers
by product magnesium pyrophospahte (cloudy) or needed for developing one assay. Multiple prim-
colour change using SYBR green. See also Plate 19. ers are necessary for increasing sensitivity and a

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