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A Two-Step RT-LAMP Provides Improved Sensitivity for Point of Care Detection of ArbovirusesAuthors: Jason Benzine, James Koelbl, Tony Rockweiler and Dipankar Manna
Info: Lucigen Corporation, 2905 Parmenter Street, Middleton, WI-53562, USA
Lucigen is registered to the ISO13485 quality management standard for medical devices through BSI, Inc. For research or investigational use only.
IntroductionArboviruses (ARthropod BOrne VIRUSES) are a group of viruses transmitted by insect vectors (e.g. mosquitos
and ticks), and include Zika (ZIKV), dengue (DENV), and chikungunya (CHIKV). Symptoms that arise from
ZIKV, DENV, or CHIKV infection (fever, rash, and joint pain) are similar and often benign, but may cause
encephalitis or viral hemorrhagic fever, and many can lead to sequelae, including long-term physical and
cognitive impairment, or early death [1]. The recent outbreak of Zika in Brazil revealed a link between the virus
and severe fetal abnormalities, drawing attention to the increasing global prevalence of arbovirus infections,
and highlighting the need for rapid, low-cost diagnostics. The past 30 years have seen a dramatic resurgence
or reemergence of epidemic arboviral diseases affecting humans and domestic animals. Due to the wide
geographic range arboviral vectors inhabit, large human populations are at risk. Globally, arboviral infections
are rising due to several factors, including climate change, increased human travel and international
commerce, increased industrial activities, and the effects of (un)employment and income [2-3]. Recent
increase in vector populations and an expansion of their geographic range have also increased the risk of
outbreaks. The greatest risks for emergence of arboviral diseases are extensive tropical urbanization and
territorial expansion of Aedes aegypti, a highly anthropophilic mosquito. These factors have led to the
permanent cycle of outbreaks in endemic areas, as well as proliferation into newer areas. Examples of
increasing arboviral diseases include dengue virus (DENV) infections, now estimated to cause ~ 400 million
clinical cases per year [4], chikungunya virus (CHIKV), with recent outbreaks in the Indian and African
continents that spread to Southeast Asia, Europe, and most recently to the Americas [5-7], and Zika virus
(ZIKV), now rapidly spreading in the Americas associated with thousands of cases of microcephaly [8,9]. Here
we present an improved “two-step” RT-LAMP (Reverse-Transcription Loop-mediated isothermal
Amplification) method for highly sensitive, specific and rapid detection of ZIKV, DENV types 1-4, and
CHIKV.
Materials/MethodsLAMP primers were designed for ZIKV, DENV types 1-4, and CHIKV using web-based primer design
software, Primer Explorer V4 (http://primerexplorer.jp/e). Optimal reaction conditions and temperatures
were determined for each LAMP target/design. Conventional “one-step” RT-LAMP reaction is carried
out using a combination of strand displacing DNA polymerase and a reverse transcriptase but suffers
from poor sensitivity. The two-step RT-LAMP, developed here, utilized a single thermostable DNA
polymerase with strand displacement and reverse transcriptase activities (LavaLAMP™ enzyme,
Lucigen). In the first step, RNA target is converted to cDNA with a single primer, and in the second
step, a proprietary enzyme aids RNA strand displacement, and the resulting cDNA is amplified through
LAMP. Lyophilized reagents for the two-step LAMP contained all necessary primers, enzymes and
fluorescent dye for real time monitoring of amplification. Assays were carried out at constant
temperature, and reactions that yielded fluorescence signal above a set threshold within 30 minutes
were called positive. Assay sensitivity was determined using serial dilutions of target RNA or virus
while assay specificity was determined against a panel of viral pathogens.
LavaLAMP DNA Polymerase is the best RT Enzyme for 2 Step RT-LAMPUsing LavaLAMP DNA polymerase as the RT Enzyme for 2 Step RT-LAMP gives superior TTR (Time To Result) performance and sensitivity than other RT enzymes tested in the 2 Step RT-LAMP. Example is Arbovirus Control MS2 RT-LAMP using several RT Enzymes for 2 Step RT-LAMP versus the One Step (RNA Direct) Method.
Assay SpecificityAssay specificity was measured by assaying each Arbovirus test against a set of related flavivirus family species at ~10(6) copies per reaction. Zika virus example below.
Lucigen’s LAMP based assays can be integrated with the cartridge and instrument to complete a simple, easy to use solution. This configuration may be suitable for the development of point-of-care diagnostic tests.
Conclusion• The 2 Step RT-LAMP method greatly increases the sensitivity and time to result for all
Arbovirus and Control RT-LAMP tests, compared to the conventional 1 step method, and to within a log of qPCR.
• The Arbovirus RT-LAMP tests are very specific to the intended virus only.• When performed in our cartridge with the Lucigen instrument (not available), the 2 step
method can be performed without manual addition of step 2 reagents.
LAMP TechnologyNon-Cyclic Isothermal Amplification generation of stem loop DNA with dumbbell-shaped structure at both ends,
and Amplification involving self priming and concatemer formation with the help of Loop primers.
Improved Sensitivity of 2 Step Arbovirus RT-LAMPTo demonstrate the efficacy of the 2-step method, we compared assay performance to conventional “one-step” RT-LAMP using serial dilutions of commercially available ZIKV (NATtrol™ Zika Virus Range Verification Panel, Zeptometrix), DENV types 1-4 (culture fluids, Zeptometrix), and CHIKV (Quantitative Synthetic Chikungunya virus RNA, ATCC® VR-3246SD™) arboviral controls. The 2 Step RT-LAMP Sensitivity is within a log of qPCR, using a cutoff of 30 minutes for RT-LAMP and 40 cycles for qPCR.
References1. Weaver SC and Reisen WK (2010) Present and future arboviral threats. Antiviral Res 85(2):328.2. Chastel C, (2002) Impact of global climate changes on arboviruses transmitted to humans by mosquitoes and ticks. Bull Acad Natl Med 186(1):89-100.3. Bram RA, et al. (2002) Threat of foreign arthropod-borne pathogens to livestock in the United States. J Med Entomol 39(3):405-416.4. Bhatt S, et al. (2013) The global distribution and burden of dengue. Nature 496(7446):504-507.5. Leparc-Goffart I, et al. (2014) Chikungunya in the Americas. Lancet 383(9916):514.6. Weaver SC (2014) Arrival of chikungunya virus in the new world: prospects for spread and impact on public health. PLoS Negl Trop Dis. 2014. 8:e2921.7. Weaver SC and Lecuit M (2015) Chikungunya virus and the global spread of a mosquito borne disease. N Engl J Med. 2015. 372:1231-1239.8. Dyer O (2015) Zika virus spreads across Americas as concerns mount over birth defects. BMJ 351:h6983.9. Gatherer D, and Kohl A (2015) Zika virus: a previously slow pandemic spreads rapidly through Americas. J Gen Virol PMID:26684466 (Epub ahead of print).