February 15-18, 2009 Washington State Convention Center Seattle ...

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RT-LAMP-IMMUNOCHROMATOGRAPHY (ICT) FOR DETECTION OF INFECTIOUS
MYONECROSIS VIRUS (IMNV) IN RESOURCE-POOR DIAGNOSTIC SETTINGS
Thales P. D. Andrade*, Kathy Tang and Donald V. Lightner
Department of Veterinary Science and Microbiology
University of Arizona
1117 E. Lowell St., Bldg.90, Rm 106
Tucson, Arizona 85721 USA
thpa@email.arizona.edu
Delays and misdiagnosis of infectious shrimp diseases are most common in marine shrimp culture zones because the equipment
necessary for pathogen detection is beyond what most diagnostic sites can afford. We report the development of an
IMNV RT-LAMP-immunochromatographic (ICT) diagnostic method for detection of Infectious myonecrosis virus (IMNV) in
resource-poor and point-of-care diagnostic settings. The RT-LAMP-ICT method combines simplified nucleic acid extraction, a
reverse-transcription isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified
sequences using a generic ICT qualitative detection test strip.
IMNV-specific RT-LAMP primers were designed for detecting the structural capsid protein gene of IMNV. For purpose of comparison,
efforts were made to design RT-LAMP primers that amplify the region between 365-568 bp, which encompass the region
amplified by the real-time RT-PCR (412-545bp) (Andrade et al., 2007). The sensitivity of RT-LAMP (using two and three
primer pairs) and nested RT-LAMP (using three primer pairs) was compared by Real-time RT-PCR using TaqMan probe. The
RT-LAMP using three primers pairs was 10-fold more sensitive than when using two primers pairs (10 -4 ), and the ladders 10 -3
and 10 -2 dilutions were more intensely amplified by RT-LAMP by using three primers pairs than two primer pairs. The addition
of loop primers (use of the third primers pair) to the reaction tube increased sensitivity and specificity, and this same pattern of
results was obtained in others assays. Even though the sensitivity of the nested RT-LAMP is equivalent to that of the Real-time
RT-PCR (10 -6 ), it was not selected for adaptation to ICT because of increased time processing and aberrant amplifications.
The detection of IMNV RT-LAMP products was accomplished by using an ICT test strip. The biotin-labeled IMNV RT-LAMP
amplicons were hybridized with an FITC-labeled probe specific for the IMNV capsid gene. The hybridization product was
dotted on the spot for sample application of the ICT strip, the IMNV RT-LAMP immune complexes were captured and a red
test band was generated. Labeled immune complexes not captured on the test line overflowed the control band and were immobilized
by species-specific antibodies resulting in an intense control band.
These results clearly demonstrate that the RT-LAMP-ICT method is specific, sensitive and user- friendly. The RT-LAMP-ICT
method also can shorten the time for analysis and has potential application for IMNV diagnosis in resource-poor and point-ofcare
diagnostic settings. Additional studies are under way to determine the feasibility for this method to become commercially
available.