An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification

Background: Koi Herpesvirus (KHV) affects both juvenile and adult
common carp and koi, and is especially lethal to fry. The high
mortalities caused by the disease have had a negative impact on the
international koi trade. Different diagnostic techniques have been
used to detect KHV, including: isolation of the virus in cell
culture, electron microscopy, several PCR tests, ELISA and in situ
hybridisation. All of these methods are time consuming, laborious
and require specialised equipment. Results: A rapid field diagnosis
of KHV in common and koi carp was developed using loop-mediated
isothermal amplification (LAMP). The LAMP reaction rapidly
amplified nucleic acid with high specificity and efficiency under
isothermal conditions using a simple water bath. Two methods of
extracting DNA from host tissue were compared: extraction by
boiling and by using a commercial extraction kit. A set of six
primers - two inner primers, two outer primers and two loop primers
- was designed from a KHV amplicon. The reaction conditions were
optimised for detection of KHV in 60 min at 65 degrees C using Bst
(Bacillus stearothermophilus) DNA polymerase. When visualised by
gel electrophoresis, the products of the KHV LAMP assay appeared as
a ladder pattern, with many bands of different sizes from 50
base-pairs (bp) up to the loading well. The KHV LAMP product could
also be simply detected visually by adding SYBR Green I to the
reaction tube and observing a colour change from orange to green.
All samples positive for KHV by visual detection were confirmed
positive by gel electrophoresis. The KHV LAMP had the same
sensitivity as a standard PCR assay for the detection of KHV.
Conclusion: This paper describes an accelerated LAMP assay for
diagnosis of KHV. The entire procedure took only 90 minutes to
produce a result: 15 minutes for DNA extraction; 60 min for the
LAMP reaction; 2 min for visual detection using SYBR Green I. The
test can be used under field conditions because the only equipment
it requires is a water bath.