Development and Validation of Rapid Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification for Detection of Rift Valley Fever Virus

Rift Valley fever virus (RVFV) is a high-priority zoonotic pathogen with the ability to cause massive loss during its outbreak within a very short period of time. Lack of a highly sensitive, instant reading diagnostic method for RVFV, which is more suitable for on-site testing, is a big gap that needs to be addressed. The aim of this study was to develop a novel one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the rapid detection of RVFV. To achieve this, the selected RVFV M segment nucleotide sequences were aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE) software in MEGA11 version 11.0.11 program to identify conserved regions. A 211 pb sequence was identified and six different primers to amplify it were designed using NEB LAMP Primer design tool version 1.1.0. The specificity of the designed primers was tested using primer BLAST, and a primer set, specific to RVFV and able to form a loop, was selected. In this study, we developed a single-tube test based on calorimetric RT-LAMP that enabled the visual detection of RVFV within 30 minutes at 65°C. Diagnostic sensitivity and specificity of the newly developed kit were compared with RVFV qRT-PCR, using total RNA samples extracted from 118 blood samples. The colorimetric RT-LAMP assay had a sensitivity of 98.36% and a specificity of 96.49%. The developed RT-LAMP was found to be tenfold more sensitive compared to the RVFV qRT-PCR assay commonly used in the confirmatory diagnosis of RVFV.


Introduction
Rift Valley fever (RVF) is a transmissible zoonotic disease caused by the Rift Valley fever Virus (RVFV) [1]. During an outbreak, the disease spreads rapidly irrespective of the national border, causing serious socioeconomic and public health consequences [2,3]. Te infection is characterized by massive abortions in pregnant animals and death among the animal population with mortality rates as high as 90% in young animals and 30% in adults [4]. Tis leads to a drastic loss of herds and focks, resulting in food insecurity and loss of revenue not only to the farmers but also to the traders, butchers, and the afected country [5]. Te disease also causes death among the naive human population with everincreasing severity with a mortality rate of up to 28% [6].
Te RVFV belongs to the Phlebovirus genus in the family Phenuviridae [7]. It is an enveloped, tri-segmented, and negative-stranded RNA [8]. Its genome consists of three segments, large (L), medium (M), and small (S), based on their molecular size [9,10]. Te L segment encodes a viral RNAdependent RNA polymerase (Large protein) [11]. Te M segment encodes two glycoproteins, Gn and Gc that form the viral envelope, and two nonstructural proteins, NSm and Gn/ NSm fusion proteins [8,12]. Finally, the S segment encodes a nucleoprotein gene, NC, and a nonstructural protein gene, NSs, in sense and antisense orientations, respectively [13].
Laboratory diagnosis of RVFV currently relies on enzyme-linked immunosorbent assay (ELISA), viral neutralization tests (VNT), reverse transcriptase polymerase chain reaction (RT-PCR), and real-time RT-PCR (qRT-PCR) [14]. However, besides being cumbersome, the ELISA and VNT have shown cross-reactivity with antibodies of cocirculating members of other closely related phleboviruses, and they also require biocontainment or laboratory level 3 facilities and highly qualifed personnel to carry out [15]. On the other hand, molecular assays, like RT-PCR and qRT-PCR, require sophisticated and well-equipped laboratories with well-trained personnel [16]. Tese restrict their application in terms of resources and capabilities in many developing countries, with Kenya being no exception.
Tis study exploited LAMP technology in the development and evaluation of the sensitivity and specifcity of a rapid colorimetric RVFV RT-LAMP assay targeting the M RNA segment of RVFV. Te LAMP technology has been successfully employed in the development of diagnostic kits against dengue virus, Indian citrus ringspot virus, fowl adenovirus, Japanese encephalitis virus, flarial parasites, Talaromyces favus species among others [17][18][19][20][21][22]. Tis is because the RT-LAMP method ofers an inexpensive and simple-to-use point-of-care simple diagnostic assay that is highly accurate, sensitive, fexible, and easy to scale up during an outbreak [23][24][25].

Study Area.
Tis study was conducted at the KALRO Biotechnology Research Center, Kabete, Nairobi, Kenya.

Primer Design.
Twenty-three complete sequences of the RVFV M segment from the NCBI database were aligned as previously described using Multiple Sequence Comparison by Log-Expectation (MUSCLE) software in MEGA11 version 11.0.11 program to identify conserved regions [26] ( Tamura et al., 2021). Te potential target region of 211 bp corresponding to the RVFV M segment genome sequence position 2584-2794 of the RVFV prototype ZH-501 (accession number M11157.1) was used as the template against which the RT-LAMP primers were designed. A total of six primers were designed, two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB) (Figure 1). Te primers targeted eight distinct positions of the template and were designed using NEB LAMP Primer design tool version 1.1.0 (New England Biolabs) under default settings. Te specifcity of the designed primers against RVFV was determined using the nucleotide-based Basic Local Alignment Search Tool (BLASTn). Only primer sets that were highly specifc to the RVFV strain, able to form a loop, and with melting temperatures below 65°C were selected.  [27]. Tis was carried out by slight modifcation to include RPMI 1640 growth medium instead of DMEM medium. Te cells were observed daily for confuence and contamination. On the fourth day, the cells formed over 90% monolayer confuence and were used for viral replication.

. Infection of Vero Cells with RVFV
Te frozen RVFV seed stocks labeled RVF VIR 1983, RVF VIR G64, RVF VIR 66, RVF 5335, and OVI vaccine were used to infect a confuent monolayer of Vero cells as previously described by Smith et al. [28]. Te RPMI suspension medium was used instead of DMEM medium as previously described [28]. Two negative controls, media-only and Vero cell-only fasks, were included to check for contamination and comparison of cytopathic efect (CPE). Te fasks were observed daily for four days, and the fasks showing more than 90% CPE characteristic of Rift Valley fever virus were selected for total RNA extraction.

Extraction and Purifcation of RNA from the RVFV Strain.
Total RNA was extracted from the infected cell monolayer using the TRIzol Reagent method as described by the manufacturer (Cat. No. 15596026, Invitrogen Life Technologies, Waltham, USA). Briefy, the suspension medium was carefully decanted, and the Vero cell monolayers from both infected and uninfected fasks were used for total RNA extraction. Te cells were lysed by adding 4 mL of TRIzol Reagent to each fask on ice. Lysis was achieved by pipetting up and down until the monolayer was fully broken down. 1 mL of the lysate was aseptically transferred into clearly labeled 1.5 mL Eppendorf tubes and incubated at room temperature for 5 min to allow complete dissociation of the nucleoprotein complex. Phase separation was realized by the addition of 0.2 ml chloroform to each tube containing the lysate and mixing vigorously for 15 seconds. Tis was followed by incubation at 25°C for 3 minutes and spinning at 12000 rpm for 15 min at +4°C to separate the mixture into three phases, the aqueous upper phase, the interphase, and the lower organic phase. Te upper phase, carrying total RNA, was carefully transferred to a sterile 1.5 mL Eppendorf tube without disturbing the interphase. Tereafter, 0.5 mL of cold isopropyl alcohol was added and the tubes were incubated at −20°C overnight to allow precipitation of the total RNA. Te tubes were then centrifuged at 12000 rpm for 10 min at +4°C, the supernatant discarded, and the pellet 2 Advances in Virology washed by the addition of 70% ethanol. Washing was achieved by a brief suspension of the pellet in wash bufer (70% ethanol) followed by spinning at 7500 rpm for 5 min. Te supernatant was discarded, and the pellet was air-dried for 10 min. Te pellet was fnally eluted in 50 µL of molecular-grade water. Te purity of the extracted total RNA was determined by the ratio A260/A280, and the concentration was determined by measuring the OD at A260 using a Nanodrop 2000c spectrophotometer (Cat. No. ND-2000, TermoFisher Scientifc, Waltham, USA). Te extracted RNA from both the RVFV-infected and -uninfected cells was stored at −40°C.

Standard Curve for RVFV Using qRT-PCR.
Te efciency of the qRT-PCR as a reference diagnostic test was established by determining the values of R 2 and the slope of the RVFV standard curve as described in the QuantStudio ™ version 5 user manual. Briefy, a positive sample with a known concentration of RVFV total RNA was diluted to fve dilution points at a ratio of 1 : 10. Te concentration of total RNA was expressed in pg/µL starting from the highest to the lowest concentration. Te master mix for qRT-PCR was prepared according to the protocol previously described by Bird et al. [29]. Briefy, master mixes for 15 standard samples and 3 negative control samples were prepared by mixing 237.5 µL of 2X RTPCR bufer, 19 µL of RVFVL F/R primers, 9.5 µL of the RVFVL probe, 19 µL of 25X RTPCR enzyme, and 171 µL of molecular-gradenuclease-free water and mixed by pipetting up and down. Te 24 µL of the master mix was dispensed into each of the 0.2 mL sterile PCR tubes and 1 µL of each sample was added accordingly. Te samples were spun briefy to collect the content at the bottom of the PCR tubes. For accuracy and reproducibility, this test was carried out in triplicate. Te standard curve protocol was set up as described by the QuantStudio ™ version 5 thermocycler manufacturer (Cat. No. 437305, TermoFisher Scientifc, Waltham, USA) using default settings and the cycling conditions.

Monitoring of Colorimetric-RVFV-UDG-RT-LAMP
Amplifcation. Successful colorimetric RVFV-UDG-RT-LAMP reactions were monitored through naked eye visualization and confrmed through agarose gel electrophoresis analysis. Naked-eye visualization was achieved in two ways, mainly through color change from pink to yellow by the inclusion of 0.1 µL phenol red as a pH indicator to RT-LAMP reaction reagents or by observation of a white precipitate after a brief spin of the fnal product after RT-LAMP assay. As a confrmation, agarose gel electrophoresis analysis was carried out by running 5 µL of the RVFV-UDGRT-LAMP products on 1% agarose gel stained with ethidium bromide in 1% Tris-borate bufer (TBE) and visualized UV in a gel Doc ™ EZ imager version 5.1 (Cat. No. 735BR06006, Bio-Rad, California, USA).

Specifcity of the Colorimetric-RVFV-UDG-RT-LAMP
Assay. To test the specifcity, total RNA extracted from Vero cells infected with diferent strains of RVFV (G66, 1983, 5335, and G64) as well as from two other major pathogens of small ruminants (Peste des petits ruminants (PPR) and Capripox viruses) were used. Te RVFV-UDG-RT-LAMP reaction was performed using 1 ng of the total RNA extracted as the template under optimized conditions for both temperature and duration, as described earlier in this study. Te results were visualized by observation of color changes and confrmed through agarose gel electrophoresis as previously described.

Evaluation of Analytical Sensitivity of the Colorimetric-RVFV-UDG-RT-LAMP Assay.
Te analytical sensitivity of the RT-LAMP assay was assessed by comparing the optimized RVFV-UDG-RT-LAMP assay with real-time RT-PCR by performing a tenfold serial dilution of the known concentration of RVFV total RNA with an initial concentration of 1.85 × 10 6 pg/µL as determined using a Nanodrop 2000c spectrophotometer ((Cat. No. ND-2000, TermoFisher Scientifc, Waltham, USA). Nontemplate control and total RNA from noninfected Vero cells were used as negative controls. Te colorimetric-RVFV-UDG-RT-LAMP assay protocol was performed on all dilutions under optimized conditions and its outcomes were compared with those of qRT-PCR. Successful RT-LAMP assay amplifcation was analyzed visually based on color changes and/or by agarose gel electrophoresis, while the threshold cycle (Ct) value between 8 and 35 was regarded as positive by qRT-PCR. Te sensitivity for the colorimetric RVFV-RT-LAMP assay and qRT-PCR was defned as the fnal dilution that yielded positive amplifcation.

Validation of the Colorimetric-RVFV-UDG-RT-LAMP
Assay Using Clinical Samples. Te newly developed colorimetric-RVFV-UDG-RT-LAMP assay was validated under optimized conditions (63°C for 30 min) using total RNA extracted from 118 reference feld samples suspected to be infected with RVFV obtained with permission from the Directorate of Veterinary Services (DVS). Te 118 feld samples were purposefully selected after initial screening using IgM capture ELISA (61 IgM positive blood samples and 57 IgM negative blood samples) as previously described by Ellis et al. [30]. Total RNA was extracted from each sample using MagMAX ™ -96 Viral RNA Isolation Kit according to the manufacturer's protocol (Cat. No. AM1836, TermoFisher Scientifc, Waltham, USA). Briefy, 50 µL of blood sample was dispensed into each well on the processing plate and clearly labeled. Ten, 20 µL of bead mix and 130 µL Lysis/Binding Solution was added to each well-containing sample and mixed by shaking at maximum speed for 3 min to allow lysis of viral cells and binding of RNA to the binding beads. Te binding beads carrying the total RNA were magnetically captured by allowing the 96-well processing plate to stand for 1 min on 96-WellMagnetic-Ring Stands. Te supernatant was carefully discarded without disturbing the beads by aspiration. Te processing plate was removed from the magnetic stand and washed twice using 150 µL of wash solution 1. Tis was followed by washing with 150 µL wash solution 2 twice. Te RNA binding beads with RNA were then recaptured on a magnetic stand as in the previous step and the supernatant was aspirated. Te total RNA was eluted by the addition of 50 µL of elution bufer to each sample and shaking for 3 min at maximum speed. Te RNA binding beads were captured as outlined in the previous steps and the supernatant containing the RNA was carefully transferred to a clearly labeled RNase-free Applied Biosystems microamp 96 PCR plate (Cat. No 10124183, TermoFisher Scientifc, Waltham, USA) and stored at −20°C. Te extracted RNA was then subjected to RVFV qRT-PCR and colorimetric-RVFV-UDG-RT-LAMP assays as described previously, and the results were compared. Te results were termed positive when the Ct value was between 8 and 35 for the qRT-PCR and the color change from pink to yellow for the colorimetric-RVFV-UDG-RT-LAMP assay.

Data Analysis
Mega 11 version 11.0.11 was used to align selected M segment sequences of RVFV strains to determine highly conserved regions. A one-way ANOVA was used to determine whether there was a statistically signifcant diference between the means of three independent groups of total RNA extracted using TRIzol Reagent using IBM SPSS statistics version 28.0.0.0 (IBM corporation). For statistically significant data, the Tukey post-hoc test was used to compare the mean between each pairwise combination of groups to determine which groups were diferent from each other. For statistically insignifcant data, the group means and standard deviation (SD) were recorded.

Results
Te RVF viral strains were successfully replicated in Vero cells. Te rounding up of more than 90% Vero cells on the fourth day after RVFV infection compared with noninfected Vero cells (negative control) was a clear indication of RVF viral replication (Figure 2). Infection of Vero cells was performed in a replica of three and total RNA from both infected and noninfected Vero cells was extracted using TRIzol Reagent. Tere was no statistically signifcant difference in the concentration and purity of total RNA extracted from the three replica groups as determined using one-way ANOVA (F (2, 15) � 0.000187, p � 0.999). Te mean ± standard deviation of the concentration and purity of total RNA extracted from the RVFV-infected and noninfected Vero cells were recorded (Table 1 and Figure 3).
Te twenty-three RVFV M segment sequences whose genetic diversity is shown in Figure 4 were aligned and a highly conserved region of 211 bp was identifed (Figure 1). Six sets of primers were designed and their locations on the template sequence are indicated in Figure 1 and Table 2. Te primers were found to be specifc to diferent strains of RVFV around the world. Te relationship of the primers to one another is schematically shown in Figure 5.
To optimize the amplifcation temperature for the RVFV RT-LAMP assay, the total RNA from RVFV-infected Vero cells was used as the template at diferent temperatures based on previous studies (50, 55, 60, and 65°C) for 60 min. Te results revealed that the optimum amplifcation temperature was achieved at 65°C (Figure 6(a)). To determine the optimum amplifcation time, the RNA template was amplifed for diferent durations (30,45, and 60 minutes) at 65°C. Te optimum amplifcation time was 60 minutes (Figure 6(b)). Te determination of the fnal amplifcation products for the RVFV-UDG-RT-LAMP was carried out by visualization using the naked eye through the observation of a white precipitate, color change from pink to yellow, and ladderlike bands on 1% agarose gel electrophoresis (Figures 6-10).
Te impact of the loop primers on the sensitivity of the designed kit was determined by the amplifcation of the template at diferent durations (30,45, and 60 minutes) at 65°C using one set with loop primers, while the other set was determined without loop primers. Te loop primers could reduce the amplifcation duration by 30 min (Figure 7(a)).
Te specifcity of the designed RT-LAMP kit was determined using RVFV strains commonly used in our laboratory compared with PPR and capripox viruses. Te results showed that the kit was specifc to the RVFV strain (Figure 7(b)). Te limit of detection for the new kit was          (c) determined by analyzing the tenfold dilution of the reference RNA sample and comparing it with qRT-PCR ( Figure 9). Te efciency of the qRT-PCR was established by generating a standard curve for the dilutions. Te R 2 and the slope were tabulated as 0.984 and −3.761, respectively (Figure 9(b)). Te sensitivity of the new RVFV RT-LAMP assay was tenfold higher than that of the qRT-PCR used as a confrmatory molecular assay (Figures 9(a) and 9(c)). Field applicability of the new colorimetric-RVFV-UDG-RT-LAMP was validated using naked eye visualization of the color change, using a colorimetric RT-LAMP following incubation at 65°C for 30 min in a water bath. For this purpose, 120 reference samples were used. Te diagnostic sensitivity (DSe) and specifcity (DSp) of the new kit compared to qRT-PCR and IgM ELISA were found to be 98.36% and 96.49%, respectively ( Figure 10). Additionally, the positive predictive value (PPV) and negative predictive value (NPV) were found to be 96.77% and 98.21%, respectively ( Figure 10).

Discussion
Rift Valley fever virus (RVFV) is a high-priority zoonotic pathogen with the ability to cause massive loss during its outbreak within a very short period of time. Te choice of the RVFV M segment as the template for the development of the colorimetric-RVFV-UDG-RT-LAMP was based on the fact that the segment encodes mainly glycoproteins that are the major structural antigens and the most detectable part of the RVFV envelope [1,31]. Te relationship among the twentythree RVFV M segment sequences used during alignment is shown in Figure 4. In this study, six sets of primers were successfully and skillfully designed using the NEB LAMP Primer design tool to target 211pb of the highly conserved region of the template (Table 2 and Figure 1). Te selection of the NEB LAMP Primer design tool for the design of the RT-LAMP primers was based on its fexibility, precision, and worldwide acceptability [19,32,33]. Te choice of the primer set for this study was determined by twofold factors, specifcity and the ability to form a loop, as previously described [33]. Te size, positions, and locations of the six designed primers are shown in (Table 2, Figures 1, and 5).
Te quality and integrity of the template used in the development of any diagnostic kit are as good as the kit itself. Terefore, the emphasis on reliance on a high-quality template cannot be underestimated [34]. In this study, total RNA was extracted from Vero cells infected with RVFV showing high levels of CPE (Figure 2). Te total RNA was extracted using the TRIzol reagent method. Te selection of the TRIzol reagent method was based on previous studies that intimated that the TRIzol reagent method was superior to the current column-based RNA extraction kits in the generation of high-quality RNA [33,35,36].
Te absorbance at wavelengths 260 nm, 280 nm, and 230 nm was used to determine the concentration and purity of the extracted total RNA (Table 1 and Figure 3). Since nucleic acid maximumly absorbs light at A260 nm, the concentration of total RNA extracted was automatically calculated by multiplying the absorbance at A260 nm wavelength with an extinction coefcient factor of 40 [37]. A pure RNA sample must have A260/A280 and A260/A230 ratios at 1.8-2.2 and > 1.7, respectively [36]. During this study, all values of total extracted RNA were within the recommended ratio ranges of 1.91-2.01 and 1.86-2.00 for A260/A280 and A260/A230 ratios, respectively (Table 1). Tese fndings agree with the fndings of Wang et al [36], in terms of purity. However, the concentration of extracted total RNA was found to be averagely higher compared to that extracted by Wang et al [36]. Te purity and integrity of the extracted total RNA were key to downstream processing [38]. Te colorimetric RVFV-UDG-RT-LAMP assay developed in this study involved four main stages: synthesis of cDNA from the RNA template, production of the starting material for the LAMP reaction, cyclic amplifcation, and fnal elongation combined with recyclization, as previously described by Sahni et al. [18]. Reverse transcription (RT) was initiated by the binding of BIP primers at the 3′ end of the RNA template in the presence of RTx reverse transcriptase present in the 2x warmstart multipurpose LAMP/RT-LAMP reaction mixed with UDG at 65°C. Te B3 primer annealed to the complementary RNA sequence (B3c) at the 3′ end outside the BIP primer to initiate strand displacement DNA synthesis in the presence of bst 2.0 polymerase. Tis process released the BIP-linked cDNA strand. Since the sequences of B1 and B1c are complementary to one another, a loop-out structure was formed at the 5′ end. Tis single-stranded DNA served as a template for FIP-initiated cDNA synthesis and subsequent F3-primed strand displacement DNA synthesis. Tis results in two products, a double-stranded DNA segment and a single-stranded DNA sequence with F1 and F1c complementary sequences. Te two sequences hybridize to form another loop at the 3′ end, forming a dumbbell-shaped structure. Te dumbbell-shaped structure then served as the starting material for the loopmediated isothermic amplifcation (LAMP) [17]. Te third and fourth stages involve exponential and rapid amplifcation and reamplifcation of the self-priming DNA template with concomitant replacement of strands, resulting in a mixture of stem-looped DNA products of varying lengths, as shown on agarose gel electrophoresis (Figures 7-9). Te fndings of this study were similar to those of previous studies [17,22,23,39].
Te optimal amplifcation conditions for the colorimetric RVFV-UDG-RT-LAMP without loop primers were found to be 65°C for 60 min ( Figure 6). Dao Ti et al. [39] reported fndings similar to ours, contrary to the fndings by Kokane et al. [22] who had established that the optimal time was 75 min. Te addition of loop primers that are complementary to the loops on the dumbbell-like DNA template reduced the amplifcation time by 50%, from one hour to only 30 min (Figure 7(a)). Tese fndings agreed with those of previous studies [40][41][42]. Lee et al. [40] and Soroka et al. [41] argued that loop primers increased the "starting points" during the LAMP reaction up to a total of eight amplifed DNA sequences, thereby improving the specifcity, sensitivity, and efciency of the reaction.
In this study, the colorimetric RVFV-UDG-RT-LAMP assay developed was able to detect as low as 1.85 pg/µl of total RNA extracted from the RVFV-infected cells within 30 min at 65°C (Figure 7(a)). Te inclusion of uracil-DNA glycosylase (UDG) enzyme in the RT-LAMP reaction mix prevented carry-over contamination by the termination of any nonspecifc amplifcation during the RT-LAMP reaction [43,44]. Tis was based on previous studies that established that UDG hydrolyzes the N-glycosidic bond between deoxyribose sugar and uracil in DNA containing deoxyuridine in place of thymidine by initiating the base excision repair (BER) pathway [44]. Tis process removes uracil resulting from spontaneous deamination of cytosine or incorporation of dUMP during DNA synthesis [44]. However, since UDG could not remove preexisting contamination from standard dTTP-containing PCR products, good laboratory practices were employed and the RT-LAMP reagents were prepared in a separate sterile room from where the samples were kept and/or added to the reaction mix [43].
Te RVFV-UDG-RT-LAMP amplifcation products were detected through agarose gel electrophoresis and turbidimetric and colorimetric analyses (Figures 6-9). In this study, gel electrophoresis was only limited to the optimization of the amplifcation parameters due to the limitations associated with the time of preparing the gel and electrophoresis process and the inclusion of a visualization dye, ethidium bromide, which is carcinogenic [41]. Unlike the agarose gel images showing only one distinct band during gel electrophoresis of PCR for positive results, in the RT-LAMP reaction, bands of diferent lengths were observed (Figures 6, 7, and 9). Silva et al. [45], attributed this to the fact that there are diferent start points and hence different lengths of DNA template products.
Turbidimetric analysis was achieved by the observation of a white precipitate after a brief spin (Figure 8). Te white deposit was magnesium pyrophosphate (Mg 2 P 2 O 7 ), a byproduct of the LAMP reaction [46,47]. During RT-LAMP reaction, pyrophosphate is formed, which in turn, reacts with the magnesium ions present in the reaction bufer, creating Mg 2 P 2 O 7, a white sediment that is visible after a brief centrifugation [48]. Yuan et al. [21] and Silva et al. [45], reported similar fndings in their previous studies. However, this is highly dependent on individual judgment, making it unreliable in cases where the product is low in quantity.
Colorimetric analysis of the RT-LAMP assay products was achieved by the addition of phenol red in the Warmstart LAMP 2x master mix with UDG. Tis made it easier to identify positive amplifcation by observing a color change because of the change in pH during amplifcation from basic to acidic [49]. Phenol red has a red color in the basic environment and turns yellow in the acidic environment. Terefore, tubes whose reaction content color turned yellow were termed positive while those that remained red were termed negative (Figures 7 and 9). Color changes correlated well with the agarose gel electrophoresis results, providing a quicker and safer way of identifying the RT-LAMP reaction results (Figures 7(b) and 9(c)). Tese fndings agree with the fndings of previous studies by Poole et al. [19] and Zhang et al. [33].

Advances in Virology
In this study, samples selected for validation of the new kit were frst subjected to commercial IgM capture ELISA. Both ELISA positive and negative samples were selected and subjected to qRT-PCR and colorimetric-RVFV-UDG-RT-LAMP. Tis is because IgM antibodies to RVFV last only for six to eight weeks after the initial exposure to the RVFV and their presence indicates recent or current RVFV infection [50,51]. Te colorimetric-RVFV-UDG-RT assay was found to be ten-fold more sensitive compared to the qRT-PCR (Figures 9(a) and 9(c)). Tese fndings agreed with the fndings of Han et al. [14], Poole et al. [19], and Marino et al. [52], who established that the RT-LAMP assays are more sensitive to the detection of lower concentrations of target organisms, making them ideal for feld/point-of-care diagnostics. Te diagnostic sensitivity (DSe) and specifcity (DSp) of the designed kit were found to be 98.36% and 96.49%, respectively. Additionally, the PPV and NPV were found to be 96.77% and 98.21%, respectively ( Figure 10). Similar fndings were previously reported [39,44,49]. However, the DSe and DSp found in this study were slightly lower than those established by Asih et al. [53]. Te DSe and DSp were used to assess the ability of the designed kit to correctly classify a sample as positive and negative, respectfully [44]. Since the DSe and DSp may be misleading due to the false positive and false negative, it was paramount to establish the positive predictive value (PPV) and negative predictive value NPV [54]. Te PPV and NPV take into account the false positives and false negatives and demonstrated the practical usefulness of the new kit in screening the RVFV samples in the feld [55].

Conclusions and Recommendations
Te colorimetric RVFV-UDG-RT-LAMP assay developed could positively detect RVFV in RNA extracted by MagMAX viral RNA isolation kit. Both blood and serum samples could be used as samples. Te new kit would take a minimum of 30 min at 65°C to obtain the results. Tere was a 100% correlation between color change and positive amplifcation. Importantly, our colorimetric RVFV-UDG-RT-LAMP assay yielded comparable diagnostic results to qRT-PCR when directly applied to RNA samples. Further studies need to be carried out to develop a lateral fow assay based on this RT-LAMP principle to improve the efciency and usability of the new kit for the diagnosis of RVFV.

Data Availability
All data and material that support the fndings of this study are included in this manuscript.

Conflicts of Interest
Te authors declare that there are no conficts of interest.