Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya

Virus propagation and mosquito infection studies were performed at BSL-3 facility of the Florida Medical Entomology Laboratory in Vero Beach, FL. RT-LAMP experiments were performed in the BSL-2 laboratory shared by FfAME and Firebird Biomolecular Sciences LLC in Alachua, FL. Patient samples (whole blood collected in EDTA) suspected with chikungunya or dengue were centrifuged to separate plasma in GenePath Dx facility (Causeway Healthcare, Pune, India). All samples that were used for RT-LAMP have previously been tested positive by quantitative PCR.

Primer design was performed using in-house software, OligArch v2 (FfAME, Alachua, FL), designed to create primer sets that account for the evolutionary variation within the genomes of viral targets. Viral sequences for dengue-1 were downloaded from the Broad Institute [20], while those for other targets were downloaded from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21]. Multiple sequence alignments (MSAs) were created for these sequences using MUSCLE v3.8.31 [22]. The resulting MSAs were used as input to OligArch, which searches for primer sets that are conserved within a target of interest while avoiding unintended targets also included within the MSA (allowing, for example, distinction between dengue subtypes).

Rules for LAMP design were followed using criteria from the Eiken Genome website [23]. Designed LAMP sets were compared to the NCBI RNA virus database using NCBI BLAST [24] to eliminate sets that would cross-react. Sets were further compared, using in-house software PrimerCompare v1 (FfAME, Alachua, FL), to eliminate sets with primers that would dimerize in a multiplexed assay to produce the final sets of LAMP primers.

LAMP primers and strand displaceable probes were purchased from Integrated DNA Technologies (IDT, Coralville, IA) (Table 1). Strand-displaceable probes were 5′-labeled with FAM, HEX, TAMRA and TET for Zika, chikungunya, dengue-1 and mitochondrial DNA (positive control), respectively. Quencher probes which partially complementary to the fluorescently labeled probes was 3′-labeled with IowaBlack-FQ. Alternatively, probes targeting Ae. aegypti small subunit ribosomal RNA (SSU rRNA) were 5′-labeled with IowaBlack-FQ and 3′-labeled with FAM. Double strand portion of the probes were screened against any viral genome sequence and mosquito genomic sequence (see Additional file 1: Table S1 for additional primers and probes as positive controls).

Virus isolates included the following: Zika virus (Puerto Rico), two chikungunya viruses (La Réunion and British Virgin Islands) and dengue-1 (Key West, FL). All viruses were passaged 1-3 times in African green monkey kidney (Vero) cells and viral titers for Zika and dengue-1 were determined by plaque assay. Chikungunya viral RNA was quantitated using the Superscript III One-Step qRT-PCR with Platinum® Taq kit by Invitrogen (Invitrogen, Carlsbad, CA) as described previously [25] with the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) (Table 2).

Infection of Ae. aegypti females with Zika and chikungunya viruses was explained in detail in Additional file 1. Following infection, mosquito legs were separated from the body to confirm the infection. The legs were placed in a centrifuge tube with 1 mL media, two zinc beads, and homogenized at 25 Hz for 3 min (TissueLyser: Qiagen, Inc., Valencia, CA). The homogenate was then clarified by centrifugation for 10 min at 4 °C. RNA was then extracted from an aliquot of the mosquito leg homogenate (160 μL) using the QIAamp viral RNA mini kit (Qiagen, Valencia, CA) and eluted in TE buffer (50 μL) according to the manufacturer’s protocol. Viral RNA was detected using the Superscript III One-Step qRT-PCR with Platinum® Taq kit by Invitrogen (Invitrogen, Carlsbad, CA) with primers and probes specific to each virus (Integrated DNA Technologies) (Additional file 1: Table S2). Programs used for qRT-PCR were described elsewhere for Zika [2], chikungunya [26]. Leg viral titers were then determined by plaque assay (Table 3).

Reaction mixtures (50 μL total volume) contained a 10X primer set (5 μL, 16 μM FIP and BIP, 2 μM F3 and B3, 5 μM LF (or LB for chikungunya), 2 μM LB (or LF for chikungunya), 4 μM LF quencher probe, and 3 μM LB-fluorescent probe (or LF probe for chikungunya)), deoxynucleoside triphosphates (dNTPs, 1.4 mM of each), Tris-HCl buffer (20 mM, pH 8.8), KCl (50 mM), (NH₄)₂SO₄ (10 mM,) MgSO₄ (8 mM), Tween® 20 (0.1%), DTT (1 mM), Bst 2.0 WarmStart® DNA Polymerase (16 U, NEB, Ipswich, MA), WarmStart® RTx Reverse Transcriptase (15 U, NEB, Ipswich, MA), and RNaseOUT™ recombinant ribonuclease inhibitor (80 U, Thermo Fisher Scientific, Waltham, MA). To this mixture was added extracted viral RNAs (1 μL, Zika, chikungunya or dengue-1). Samples were incubated at 65 °C for 45 min, then analyzed by agarose gel electrophoresis (2.5%) in 1X TBE buffer, followed by ethidium bromide staining, using an appropriate DNA size marker (50 bp ladder; Promega, Madison, WI).

For multiplexed RT-LAMP, each 10X primer set (5 μL each, Zika, chikungunya and dengue-1) was added in the same manner to RT-LAMP mixture (total 50 μL volume).

Initially, varying concentrations of urine (50% to 0%) were tested in RT-LAMP reactions. Typically, viral RNA spiked urine was included in the reaction mixture to a 10% final concentration without any purification step. As a positive control, mitochondrial DNA targeting LAMP primers were designed for use in urine [27]. Similarly, 10% saliva and plasma samples were also tested for Zika detection.

For the real-time monitoring of RT-LAMP, the reactions were incubated at 65 °C for 60-90 min and the fluorescence signals from FAM-labeled probe for Zika (λ_ex/λ_em = 495 nm/520 nm (using filter 483-533 nm), HEX-labeled probe for chikungunya (λ_ex/λ_em = 538 nm/555 nm (using filter 523-568 nm), or TAMRA-labeled probe for dengue-1 (λ_ex/λ_em = 559 nm/583 nm (using filter 558-610 nm) were recorded every 30 s using Roche Light Cycler 480 (Roche Life Sciences, Indianapolis, IN). Initial real-time LAMP experiments contained only 80 nM of fluorescently labeled LB or LF probe instead of 300 nM. Final primer concentrations in this set-up were as follows: 1.6 μM FIP and BIP, 0.2 μM F3 and B3, 0.4 μM LF and LB, 0.08 μM LB-fluorescent probe (or LF for chikungunya) and 0.2 μM quencher probe. Final primer concentrations with 300 nM strand displacing probes were as follows; 1.6 μM FIP and BIP, 0.2 μM F3 and B3, 0.5 μM LF (or LB for chikungunya), 0.2 μM LB (or LF for chikungunya), 0.3 μM LB-fluorescent probe (or LF for chikungunya) and 0.4 μM quencher probe.

Additionally, images of fluorescence generated by strand displaceable probes, induced by blue LED light (470 nm) at room temperature, were recorded through an orange filter by a cell phone camera (e.g. iPhone 6 s).

Quaternary ammonium modified paper (Q-paper) was made by treating Whatman filter paper (Grade 1) with an NaOH solution, followed by washing with water and then treatment with glycidyltrimethyl ammonium chloride, following a literature procedure [28]. The Q-paper sheets were cut into small squares (~0.5 cm²). Aedes aegypti female mosquitoes were crushed on each paper square with a micro pestle. The crushed carcasses were treated with aqueous NH₃ (1 M, 100 μL, pH ≈ 12). The papers were washed once with 50% EtOH (100 μL) and once with ddH₂O (100 μL), and air-dried. The paper squares, with and without target virus, were then placed inside RT-LAMP mixture and incubated 65 °C for 45 min. Prior to testing viruses, a primer set (as positive control) targeting Ae. aegypti SSU rRNA was tested on Q-paper crushed non-infected mosquito samples.

Carryover contamination was prevented by incorporation of dUTP by Bst 2.0 WarmStart® DNA Polymerase (NEB, Ipswich, MA) during RT-LAMP, and Antarctic thermolabile UDG (NEB, Ipswich, MA) was used to destroy DNA containing dU. Reactions were run with a 50% inclusion of dUTP mixed with dTTP giving final 0.7 mM dTTP, 0.7 mM dUTP, and 1.4 mM each dATP, dCTP and dGTP. Antarctic thermolabile UDG (1 μL, 2 units) was added to RT-LAMP reaction mixture (50 μL). Samples were first incubated at 25 °C for 5 min and then heated to 65 °C for 20-45 min.

A mixture of Bst 2.0 WarmStart® DNA Polymerase (16 U), WarmStart® RTx Reverse Transcriptase (15 U), RNaseOUT™ (80 U) and Antarctic thermolabile UDG (2 U) in dialysis buffer (200 μL, 10 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA10 and 0.1% Triton X-100) was placed in an ultrafiltration membrane (10 kDA cut-off limit, Millipore, Billerica, MA). Samples were centrifuged at 14,000 x g for ca. 15 min to concentrate (down to ~5 μL) and to remove glycerol. 10X LAMP primers (5 μL), dNTPs (10 mM each, 7 μL) and glycerol free enzyme mix (5 μL) were combined and lyophilized and supplemented with 1.1X-LAMP rehydration buffer (22 mM Tris-HCl, pH 8.8, 55 mM KCl, 11 mM (NH₄)₂SO₄, 8.8 mM MgSO₄, 0.11% Tween® 20, 1.1 mM DTT). Plasma samples (5 μL) were mixed with 1.1X rehydration buffer (45 μL) and incubated at 65 °C for 45 min. The resulting fluorescence signal was observed by blue LED excitation (470 nm) through an orange filter.

Standard RT-LAMP architecture from Fig. 1a was modified to improve the signal detection and to support multiplexing. Here, an additional component was added in the form of a “strand displaceable probe”. This comprises two DNA strands that are complementary over part of their lengths. The first oligonucleotide strand has a quencher moiety at its 3′-end; the second DNA strand has a fluorophore covalently attached at its 5′-end. When the two strands are hybridized, the quencher and the fluorophore are brought into close proximity, and no fluorescence is observed. However, the 3′-portion of the second DNA strand is not covered by a hybridizing segment of the first DNA strand; left in a single stranded form, this is a priming sequence complementary to a segment of the loop region of the dumbbell structure created by the initial step of RT-LAMP, not on the target RNA itself.

Further, since the priming sequence hybridizes on the loop region, the signal is created only after the initial dumbbell is formed. Therefore, it cannot be created by any number of artifacts that are common in RT-LAMP. This duplex region is entirely under the control of the designer, and need not have any relation to any target sequence. Further, when multiplexing is applied, same sequence may be used with different fluorophore:quencher pairs.

During RT-LAMP, the priming region of the fluorescently tagged probe is extended by a strand-displacing polymerase (Fig. 1b ). Then, extension from the reverse primers reads through the primer on the fluorescently tagged probe, displacing the probe that bears the quencher moiety. This separates the fluorescently tagged oligonucleotide from the quencher tagged probe, allowing the fluorescence to be observed in real-time and measured from fluorescently tagged probe that has been incorporated into RT-LAMP products.

Prior to sealed tube analysis, the performance of the RT-LAMP primers (Table 1) with the various virus targets as well as positive controls for urine and mosquito samples was assessed by gel electrophoresis. The samples were total RNA extracted from viral stocks cultured in African green monkey kidney (Vero) cells. In one case (for chikungunya), the viral RNA used was extracted using a “total nucleic acids” preparation kit from virus-infected mosquitoes (Table 2). This sample included, of course, mosquito RNA and DNA. Figure 2a shows some representative results with RT-LAMP performed with these samples. In both singleplexed and multiplexed cases, the yields of LAMP products, appearing in a gel as a ladder of concatemers, were similar. Negative control samples gave only the bands for primers themselves, including the non-specific target control where total nucleic acid extracted from healthy Ae. aegypti female mosquito was used as the template. To optimize LAMP conditions, magnesium concentrations and operation temperatures were varied. Higher magnesium concentrations and lower LAMP temperatures generated nonspecific amplicons. Therefore, 8 mM magnesium and 65 °C were used in all-subsequent studies (Fig. 2b). Primer sets targeting mitochondrial DNA in urine, and SSU rRNA in Ae. aegypti mosquitoes; as positive controls, yielded similar pattern of LAMP amplicons in gel electrophoresis (Additional file 2: Figure S1 and Additional file 3: Figure S2).

Gel electrophoresis requires, of course, expert personnel not only to run the gel, but also to prevent forward contamination, arising if amplicons from an earlier assay contaminate later samples, leading to false positives. Here, the displaceable probe generates fluorescence that can be read through the wall of a sealed tube, which may remain sealed throughout the assay.

Figure 3a shows the results for the LOD for Zika using fluorescent probes targeting Zika RNA. Serial dilution study showed a clean sigmoidal amplification with 2.85 pfu per assay, with a signal rise largely complete at 20-25 min, both in cases where only Zika primers were present (1-plex) or when all three primer sets were present (3-plex). Slightly less sigmoidal curves were observed with 1.425 pfu, with signal generation being substantially complete after ~30 min. When diluted further to ~0.71 pfu, a fluorescent signal was observed only after ~40 min for a singleplexed assay, and after ~50 min for the 3-plexed mixture. In parallel, limits of detection were measured to be 37.8 copies and 1.22 pfu for chikungunya and dengue-1, respectively (Additional file 4: Figure S3).

Cross-reactivity was then tested using only Zika-targeting FAM-labeled probes in the presence of the two primer sets for chikungunya and dengue-1. Again, a strong signal was seen arising within 25-30 min, but only if Zika is present; chikungunya or dengue-1 viral RNAs gave no cross-reacting signals (Fig. 3b). Last, complete Zika virus was added to an authentic human urine sample at increasing concentrations (Fig. 3c). Presumably because of its electrolytes, the LAMP signal was delayed from 15 to 35 min at the highest ratio of urine:buffer (1:1), but not substantially at lower ratios.

Strand displacing probes were then introduced to RT-LAMP that allowed the three viruses to signal with different fluorescent species: fluorescein (FAM) for Zika, HEX for chikungunya, and TAMRA for dengue. Complete viral RNAs extracted from cell cultures were used as targets, and these were presented in 10% urine. Real time analysis of the emergence of fluorescence showed a substantial difference for each target (about 10 min delays) in the 1-plex curves (where the only primers were specific for the target virus) and the 3-plex curves (where primers for all target viruses are present). However, in all cases, signal generation was effectively complete by 30 min (Fig. 4a-c). The fluorescence was observed through an orange filter upon excitation by a blue LED emitting it 470 nm. This led to different signal strengths based on the different photophysics of the three fluorophores. Thus, the FAM signal was the strongest, as the 470 nm excitation light is closest to the maximum of the FAM excitation spectrum (Fig. 4d).

To visualize all three colors without changing the LED, the amounts of probes were increased from 80 nM to 300 nM. The results are shown in Fig. 5a-c. However, increase in the probe concentration resulted in ca. 20 min delay to obtain fluorescent signal in the 3-plexed format. In all cases, FAM-labeled probes for Zika were visualized as bright green, HEX-labeled probes for chikungunya were visualized as green-yellow, and TAMRA labeled probes for dengue-1 were visualized as orange when excited with blue LED (470 nm) and filtered through orange glass (Fig. 5d).

As a part of mosquito surveillance, a square of Q-paper carrying mosquito carcasses infected with Zika or chikungunya (Table 3), following washing with ammonia and ethanol, could be directly introduced into the LAMP mixture without negative effect (Fig. 6). Again, visual fluorescence signal was generated within 30 min (Additional file 5: Figure S4).