A Two-Step RT-LAMP Provides Improved Sensitivity for Point of Care Detection of ArbovirusesAuthors: Jason Benzine, James Koelbl, Tony Rockweiler and Dipankar Manna

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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.

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