a. Zika viruses
Three Asian ZIKV strains (P6-740, PRVABC59 and H/PF/2013) [44-46] and one African ZIKV strain (MR766)  were used. Strain P6-740 was obtained from Dr. Robert Tesh (World Reference Center for Emerging Viruses and Arboviruses, The University of Texas Medical Branch, Galveston, USA). Strain PRVABC59 was maintained in the laboratory by K. Zandi (author). Strain H/PF/2013 and MR766 were provided by Dr. Li-Sze Lim (Medical Innovation Ventures Pte. Ltd., Malaysia). The viruses were propagated in Vero cells using Dulbecco’s modified Eagle medium (DMEM, Gibco, NY, USA) supplemented with 2% heat-inactivated fetal bovine serum (FBS collected in South America, Capricorn, Germany), 0.1 mM non-essential amino acids (NEAA) and 2 mM L-glutamine. The infected cells were incubated at 37°C in 5% CO2 atmosphere for 7 days and the infected cell culture supernatants were then harvested and stored at -80°C until further use.
b. Simulated clinical samples
The study was approved by the UM Institutional Biosafety and Biosecurity Committee (Approval Number: UMIBBC/NOI/R/TNCPNI/TIDREC-007/22072020) and the UMMC Medical Ethics Committee (Ethics Committee/IRB Reference Number: 908.11). All ZIKV-positive simulated clinical samples were prepared by spiking the viral culture supernatant into actual human saliva, urine and serum at final viral titres ranging from 10-3 to 103 PFU/ml. The human saliva, urine and serum without spiked ZIKV were used as ZIKV-negative simulated clinical samples. A total of 24 simulated clinical samples consisting 8 saliva, 8 urine and 8 serum samples were prepared. The saliva, urine, and serum samples were obtained from healthy donors. Only Asian ZIKV strains P6-740 was used for the preparation of simulated clinical samples.
c. Viral plaque assay
The viral plaque assay was performed to determine the infectious titer of the ZIKV used for the preparation of simulated clinical samples. Briefly, the Vero cells were seeded in 24-wells plate and allowed to grow overnight until more than 80% confluency in DMEM supplemented with 10% FBS, 0.1 mM NEAA and 2 mM L-glutamine. The overnight culture media was replaced with a 10-fold serially diluted virus stock in the serum-free media (200μl of each virus dilution). The plate was placed on a rocker and gently rocked for 1 hour for virus adsorption. Subsequently, the virus supernatants were removed each well and overlaid with 1 ml of DMEM supplemented with 2% FBS, 0.8% carboxymethylcellulose (CMC, Sigma-Aldrich, USA), 0.1 mM NEAA and 2 mM L-glutamine. The plate was incubated at 37°C in 5% CO2 atmosphere for 5 days. After 5 days of incubation, the overlaid media was removed and cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) for 30 min at room temperature. The cells were then washed using 1×PBS for three times. Finally, 0.5% crystal violet in 20% ethanol (Sigma-Aldrich, USA) was used to stain the cells for 15 min in order to enable the visibility of virus plaques. The virus plaques were counted using the SMZ 1000 stereomicroscope (Nikon, Tokyo, Japan) and the virus infectious titer was expressed in plaque forming unit/ml (PFU/ml).
d. RNA extraction
The infected cell culture supernatant and simulated clinical serum samples (140 µl) were subjected to RNA extraction using QIAamp Viral RNA Mini Kit (Qiagen, Germany) following the manufacturer’s protocol. The eluted RNA (60 µl) was stored at -80°C until further use.
e. Design of ZIKV-specific RT-LAMP assay primers
The virus genomes of both Asian and African lineages were retrieved from GenBank (Table S1). The genomes of other flaviviruses were also included for comparison. The sequences were aligned using Clustal X 2.0 . The RT-LAMP primers were designed following the criteria previously described . Conserved regions among flaviviruses were identified and excluded from the primer sequences. The RT-LAMP primers designed were exhaustically compared with an alignment of ZIKV genomes retrieved from GenBank. The five nucleotides at the 3’ end of F3, B3, FLP and BLP primers as well as the five nucleotides at both 3’ and 5’ end of FIP and BIP primers were considered critical sites for priming and amplification. The coverage of the primers was further validated by assessing the assay using both Asian and African ZIKV strains.
f. RT-LAMP assay
The RT-LAMP reaction was prepared using the Loopamp RNA Amplification Kit (Eiken Chemical Co. Ltd., Japan). Each RT-LAMP reaction (25 µl) was added with the inner primers (20 pmol each), outer primers (2.5 pmol each), loop primer (20 pmol each), Fluorescent Detection Reagent (Eiken Chemical Co. Ltd., Japan; 1 µl), and the eluted RNA (5 µl). The RT-LAMP were performed using LA-500 Loopamp real-time turbidimeter (Eiken Chemical Co. Ltd., Japan) according to the following conditions: 90 min at 63°C followed by 5 min of assay inactivation at 80°C. The turbidity of RT-LAMP reaction was measured at 650 nm every 6 s. The threshold time (Tt) value was recorded when the turbidity crossed the threshold cut-off value at 0.07 absorbance units [48, 49].
g. Cross-reactivity of RT-LAMP assay
The cross-reactivity of the ZIKV RT-LAMP primers was evaluated against other arboviruses including dengue virus type 1 (DENV-1), DENV-2, DENV-3, DENV-4, Japanese encephalitis virus (JEV), Langat virus (LGTV), Sindbis virus (SINV), Chikungunya virus (CHIKV), and Getah virus (GETV). All these viruses were attained from the TIDREC viral repository [48-52]. Nuclease-free water was used as the negative control.
h. Detection limit of RT-LAMP assay
The detection limit of the RT-LAMP assay was assessed by using a 10-fold serially diluted ZIKV RNA (ranged from 1 to 103 RNA copies) extracted from the virus culture supernatant. The initial ZIKV RNA copy number in the culture supernatant stock used were quantitated using the qRT-PCR assay. The detection limit test of RT-LAMP was repeated four times. Nuclease-free water was used as the negative control and as the diluent for the preparation of serially diluted viral RNA.
i. Evaluation of RT-LAMP assay
The feasibility of the RT-LAMP assay for detection of ZIKV RNA in clinical setting was assessed using the simulated clinical samples as described above. The qRT-PCR was used as a reference assay for the detection of ZIKV RNA in the samples. Results from the RT-LAMP and qRT-PCR assays were compared.
j. Real-Time qRT-PCR assay (reference assay)
The viral RNA and samples used were quantitated using the Genesig Real-Time qRT-PCR ZIKV Detection Kit (PrimerDesign Ltd., UK). The Genesig qRT-PCR assay standard plot was prepared using a 10-fold serially diluted synthetic ZIKV RNA template with known copy number (ranged from 10 to 106 RNA copies). The qRT-PCR reaction consists of the real time master mix (10 µl), probe/primer mix (1 µl), nuclease-free water (4 µ), and the extracted RNA (5 µl), in a 20 µl final reaction volume. The qRT-PCR were performed according to the following conditions: 10 min at 55°C, 8 min at 95°C followed by 50 cycles of 10 s at 95°C and 60 s at 60°C using StepOnePlus real time PCR system (Applied Biosystems, USA). The threshold cycles (Ct) obtained were used to determine the ZIKV RNA copy number in the samples based on standard curve from qRT-PCR using StepOne Software v2.2.1.
k. Statistical Analysis
A probit analysis (P ≤ 0.05) was performed to determine the detection limit of the RT-LAMP assay. The degree of agreement [kappa value (), P < 0.001] between RT-LAMP and qRT-PCR test results was measured. The degree of agreement and probit analyses were performed using IBM SPSS Statistics, version 21 (IBM Corporation, New York, United States). The diagnostic performance of RT-LAMP compared to qRT-PCR was determined using web-based EBM Diagnostic Test Calculator (http://ebm-tools.knowledgetranslation.net/calculator/diagnostic/).