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Article

Reverse Transcription Loop-Mediated Isothermal Amplification Assay Using Samples Directly: Point-of-Care Detection of Severe Fever with Thrombocytopenia Syndrome Virus

by
Marla Anggita
1,2,
Kyoko Hayashida
3,
Miyuka Nishizato
4,
Hiroshi Shimoda
1,4,5 and
Daisuke Hayasaka
1,4,5,*
1
Laboratory of Veterinary Microbiology, Joint Graduate School of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan
2
Laboratory of Microbiology, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta 55281, Indonesia
3
Division of Collaboration and Education, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020, Japan
4
Laboratory of Microbiology, Joint-Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan
5
Division of Pathogenic Microorganisms, Research Center for Thermotolerant Microbial Resources, Yamaguchi University, Yamaguchi 753-8515, Japan
*
Author to whom correspondence should be addressed.
Zoonotic Dis. 2025, 5(3), 19; https://doi.org/10.3390/zoonoticdis5030019
Submission received: 9 April 2025 / Revised: 4 July 2025 / Accepted: 8 July 2025 / Published: 11 July 2025
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Simple Summary

This study presents a simplified, field-portable dried RT-LAMP assay for detecting severe fever with thrombocytopenia syndrome virus (SFTSV). The assay enables direct testing from serum and plasma samples without RNA extraction by using heat treatment and a specialized buffer for viral inactivation and RNA release. The dried format of the RT-LAMP reaction mix enhances storage stability and ease of use, with the results being detectable via fluorescent readouts. While it is slightly less sensitive than RT-qPCR, the assay effectively identifies SFTSV in infected animals with high viral loads. This cost-effective and rapid diagnostic tool is suitable for point-of-care use, particularly in resource-limited settings where early SFTSV detection is essential.

Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by the SFTS virus (SFTSV). A rapid and cost-effective point-of-care testing detection system is important for the early diagnosis of SFTS. Herein, we developed a ready-to-use dried reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the direct detection of SFTSV in clinical samples. The assay enables simple, RNA-extraction-free detection using heat-treated serum or plasma, followed by a 30 min incubation at 65 °C. The results are visually interpreted through the color emitted, which can be observed under LED light. The established assay demonstrated detection sensitivity for SFTSV at 104 copies/µL and was effective in identifying infections in cats. Despite being less sensitive than real-time RT-PCR, this dried RT-LAMP method offers a rapid, cost-effective alternative suitable for point-of-care use, particularly in remote or resource-limited settings. The simplified workflow and visual readout make it a practical tool for the early detection and daily surveillance of SFTSV in animals.

1. Introduction

Severe fever with thrombocytopenia syndrome (SFTS) is a new emerging tick-borne infectious disease. SFTS was first identified in China in 2009 [1] and has since been reported in Japan, South Korea, Vietnam, Myanmar, Thailand, and Pakistan [2,3,4,5,6,7]. The causative agent of SFTS is the SFTS virus (SFTSV) [1], which is currently classified as a Bunyavirales, family Phenuiviridae, genus Bandavirus, and species Dabie bandavirus. SFTSV is a single-stranded, negative-sense RNA virus whose genome comprises three RNA segments: large (L), medium (M), and small (S). These encode an RNA-dependent RNA polymerase (RdRp), glycoproteins (Gn and Gc), and the nucleoprotein (N) and non-structural proteins (NSs), respectively. The primary clinical signs of SFTS are fever, thrombocytopenia, gastrointestinal disorders, and leukocytopenia, which can lead to multi-organ failure, with a fatality rate of 10–16% [1,8]. Currently, there are no specific treatment drugs or licensed vaccines available for SFTS, making it a significant medical problem. However, the detailed pathogenesis of the disease remains unknown [2]. SFTSV is transmitted primarily via ticks such as Haemaphysalis longicornis [3]. SFTSV infections have been identified in wild animals (wild boars and wild deer), livestock (cattle, pigs, and goats), and companion animals (cats, dogs, and captive cheetahs) [9,10,11,12,13,14]. In particular, many cat cases of SFTS have been reported in Japan [15,16,17], and transmission of SFTSV from animals to humans is considered to be an important public health concern [18,19,20].
The detection of SFTSV genes is a standard diagnostic method for SFTS in humans and animals [1,16]. Real-time reverse transcription polymerase chain reaction (RT-PCR) has been applied to detect SFTSV RNA in blood, serum, and oral swab samples taken from humans and animals [16,21]. RT-PCR requires advanced and high-throughput equipment and is primarily performed at specialized institutions such as hospital laboratories or institutes, and for small animal hospitals, it takes time to transport samples and receive diagnostic results. Therefore, a point-of-care testing (POCT) method other than RT-PCR is required for the rapid diagnosis of SFTSV infection.
Loop-mediated isothermal amplification (LAMP) is a DNA amplification technique that enables rapid and accurate gene detection. LAMP can be performed at a constant temperature for less than an hour, with high sensitivity and specificity. In addition, LAMP can be performed even in unprocessed samples, including whole blood, serum, urine, and stool samples, which may inhibit DNA amplification with PCR [22]. Owing to its simplicity and minimal equipment, the LAMP method can be used as an effective and cost-efficient molecular diagnostic method in areas with limited resources [23].
Previous studies have reported that the RT-LAMP system for SFTSV provides useful insights into the possibility of POCT for SFTS [24,25,26,27,28,29,30]. In these studies, the RT-LAMP reaction was evaluated using SFTSV RNA extracted from samples. Ishijima et al. (2023) directly demonstrated SFTSV RT-LAMP using clinical samples mixed with an RNA extraction reagent [29,30]. However, result determination is still dependent on an adequate machine, such as a real-time fluorometer.
Herein, we developed a diagnostic technique using a dried RT-LAMP system that can be performed with a single-tube, sample-in–answer-out format without requiring sophisticated equipment, with the final goal of creating a simpler method for detecting SFTS based on previous studies. Our approach allows the direct detection of serum or plasma samples without an RNA extraction step, for which the outcomes can be observed by a simple observation under an LED light. This dried-RT-LAMP system can be used to detect SFTSV in limited-resource clinics without requiring advanced equipment.

2. Materials and Methods

2.1. Viruses and Cells

The SFTSV YG-F33 and YG-F185 strains were propagated in Vero E6 cells in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies Corporation, Grand Island, NY, USA) supplemented with 2% fetal bovine serum (FBS) (Biosera, Cholet, France; BioNordika AS, Oslo, Norway). Vero E6 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). The supernatant of SFTSV-infected cells was stored at −80 °C as a viral stock. All experiments using infectious SFTSV were performed in a biosafety level 3 laboratory at Yamaguchi University.

2.2. Samples

Clinical serum and plasma samples from cats were provided by veterinary clinics in Japan and sent to our laboratory for the diagnosis of SFTS. Plasma samples of experimental mice were collected from SFTSV-infected IFNα/βR−/− (A129) mice at 0 to 4 days post-infection (dpi).

2.3. Viral RNA Extraction

SFTSV viral RNA was extracted from the viral stock using ISOGEN-LS (NIPPON GENE Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. A total of 60 µL of RNA was extracted from 140 µL of serum or plasma from cat patients and SFTSV-infected mice using a QIAamp® Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions.

2.4. Real-Time RT-PCR

Real-time RT-PCR was performed to measure the SFTSV copy number. A two-primer set, comprising 965F (5′-GCRAGGAGCAACAARCAAACATC-3′) and 1069R (5′-GCCTGAGTCGGTCTTGATGT C-3′), and probe FAM/5′-CTCCCRCCC-3′/ZEN/5′-TGGCTACCAAAGC-3′ (Integrated DNA Technologies) were used to amplify and detect the L segment of SFTSV. Real-time RT-PCR was performed using the CFX Connect™ Real-Time System (BIO-RAD) software (v. 2.3). Real-time reactions were carried out using a TaKaRa One Step PrimeScript™ RT-PCR (Perfect Real Time) Kit (Takara Bio Inc., Shiga, Japan), as follows: reverse transcription at 42 °C for 5 min, initial denaturation at 95 °C for 10 s, 40 cycles of denaturation at 95 °C for 5 s, and an annealing and extension step at 60 °C for 34 s. The viral copy numbers were determined as the ratio of the copy numbers to the standard control prepared from a SFTSV gene-cloned plasmid vector, as previously studied [31].

2.5. Preparation of the Dried RT-LAMP Assays in a PCR Tube

Dry-format LAMP reagents were prepared using an air-drying technique, as reported previously [32]. For the LAMP primer sets used to detect SFTSV, previously reported primer sets targeting the L segment were used [25]. Briefly, 3.85 µL of enzyme mix comprising 1.6 µL of 2 M trehalose (Fujifilm Wako Pure Chemical, Osaka, Japan), 1.4 µL of 25 mM dNTPs (Nippon Gene, Tokyo, Japan), 0.25 µL of WarmStart RTx reverse transcriptase (New England Biolabs Inc., Ipswich, MA, USA), 0.05 µL of Bst 2.0 WS DNA polymerase (120 U/µL) (New England Biolanbs Inc., Ipswich, MA, USA), 0.25 µL of Bst 2.0 WS DNA polymerase (8 U/µL), and 0.3 µL of RNase inhibitor (Takara Bio Inc., Shiga, Japan) were applied inside the flat tube lid of the 0.2 mL PCR tube (Watson, Kobe, Japan). Two different concentrations of Bst 2.0 WarmStart DNA polymerase were used to adjust the glycerol amount to maintain the effective drying time and stability of the enzyme in the assay. A total of 3 µL of LAMP primers mixed with colori-fluorometric indicator developed by Hayashida et al. [32], comprising 0.4 µL of 100 mM FIP and BIP (forward and backward inner primers), 0.2 µL of 100 mM LF and LB (loop primers), 0.05 µL of 100 mM F3 and B3 (outer primers), 0.7 µL of 2 M trehalose, and 1 µL of colori-fluorometric indicator (CFI; 3 mM hydroxyl-naphthol blue, DOJINDO LABORATORIES, Kumamoto, Japan and 0.35% v/v GelGreen (10,000X stock solution in DMSO), Biotium, CA, USA), were placed into the bottom of the tube. To dry the liquid reagents, the tubes were placed in a glove box filled with dried air produced by an ultralow dew point air dryer (QD20-50; IAC Co., Kawasaki, Japan) for 12 h. The in-house-dried LAMP reagent for the lids and tubes was stored separately in an aluminum bag with silica gel to avoid light exposure at room temperature until use.

2.6. RT-LAMP Reaction

For the LAMP reaction, 23 µL of LAMP reaction buffer, comprising 20 mM Tris-HCl (pH 10.0), 50 mM KCl, 6 mM MgSO4, 10 mM (NH4)2SO4, and 0.1% Triton X-100, was ap-plied into the tube with 2 µL of the reaction template RNA or lysate. Triton X-100 was added to lyse the viral membrane and enhance the detection sensitivity. The tube was then placed upside down for 2 min until the enzyme mix on the tube lid was reconstituted and then spun down. The LAMP reaction was performed using a real-time PCR machine (Rotorgene 6000; Corbett Life Science, Sydney, Australia) to monitor the reaction and experimental setup, allowing us to determine the reaction time threshold. For the experiment using viruses and clinical samples, a dried RT-LAMP reaction was performed by incubating the RT-LAMP tube at 65 °C for 30 min using a BI-516S Block Incubator (Astec Co., Ltd., Fukuoka, Japan). The LAMP products were detected directly by the naked eye and under LED light using a handmade, battery-operated LED illuminator [33]. DNA amplification was monitored by observing the fluorescence from the dsDNA-specific dye CFI contained in the dried RT-LAMP mixes. Staining enabled the detection of amplified DNA under an LED light with a wavelength of 500 nm. A portable, battery-operated handheld LED illuminator was built to facilitate result evaluation (Figure 1); hence, there was no need to use an additional advanced device to read the results from the RT-LAMP reaction. Under the naked eye, the positive tubes showed a light blue color, and the negative tubes showed a light purple color. A greenish-yellow color emitted from the RT-LAMP tube under LED light indicated a positive result, whereas an orange color indicated a negative result (Figure 1).

2.7. Heat Inactivation

Measures of 2 µL of the SFTSV stock samples (107 focus forming unit (ffu) per mL) were boiled with LAMP reaction buffer in 25 µL total reaction volume including 2 × 104 ffu of SFTSV at 99 °C for 10, 20, and 30 min. Non-heat-treated samples were used as the controls. All tests were performed in duplicate. Subsequently, 2 µL of the serum or plasma samples obtained from the cats or mice was mixed with 23 µL of LAMP reaction buffer in a 1.5 mL micro-centrifuge tube. The sample mix was treated with heat inactivation at 99 °C for 10 min. After heat inactivation, the tube was spun down briefly, and a total of 25 µL of reaction volume was transferred into the dried RT-LAMP tube. The dried RT-LAMP tubes were inverted several times until the dried reagent was reconstituted into the sample mix. The tubes were then placed upside down and incubated for 2 min at room temperature. The LAMP reaction was performed with a dry bath at 65 °C for 30 min. The SFTSV copy numbers in the serum and plasma samples were measured beforehand using a real-time RT-PCR assay, and the correlations between the Ct values, copy numbers, and RT-LAMP assay results were compared. Purified SFTSV RNA was used as a positive control, and RNase-free water was used as a non-template control.

3. Results

3.1. Subsection Sensitivity and Detection Limit Analysis of the SFTSV-Dried RT-LAMP Assay

Purified viral RNA prepared from the SFTSV samples was used to monitor amplification by light blue color change and fluorescence. RNA was successfully amplified, and a positive reaction was observed within 30 min. Subsequently, using a 10-fold serial dilution of purified SFTSV RNA, the detection limit of the dried RT-LAMP assay was evaluated. The detection limit of the dried RT-LAMP assay was 104 copies/µL, because no positive reaction was observed in 103 copies/µL (Figure 2).

3.2. Heat Inactivation Test

To establish a safe and rapid diagnostic test workflow, we tested the SFTSV stock samples inactivated with simple boiling for 10 min in the LAMP reaction buffer that included Triton X-100. In general, it seems that boiling for 10 min inactivates infectious viruses, and our unpublished data indicate that 99 °C for 10 min significantly inactivates infectious SFTSV. Positive reactions of heated SFTSV samples were clearly detected by color emissions under an LED light, whereas those light blue color changes were not distinctly observed (Figure 3), suggesting that direct naked eye observation is not suitable for SFTSV samples without RNA extraction. Interestingly, the heat-treated samples performed better than the unheated samples (Figure 3). Therefore, the simple boiling method is likely to be useful not only to minimize the risk of infection by heat inactivating but also to eliminate the RNA extraction step without additional reagents. For further studies, we conducted the LAMP assay for clinical samples using heat inactivation at 99 °C for 10 min and observed the positive reactions under an LED light.

3.3. Application of the Dried RT-LAMP Assay to Clinical Samples

To test the clinical samples, we first confirmed the RT-LAMP assay using plasma samples from SFTSV-infected mice. Copy numbers of the mouse samples were determined using real-time RT-PCR. Positive reactions were observed compared to the non-template control after the completion of the reaction.
Positive reactions with color emission under LED light were clearly detected in the sample numbers (Day 2—118; Day 2—123; Day 3—131; Day 3—134; Day 4—124; Day 4—133), corresponding to high copy numbers with more than 104 copies; however, those emissions were comparatively weak in other sample numbers: Day 2—124; Day 3—132 (Figure 4). Positive reactions of color emission under LED light were not observed in the other samples with fewer than 102 copies (Figure 4). From these results, it was suggested that, for the SFTSV-infected mouse samples, the dried RT-LAMP-positive results observed by color emission mostly appeared on days 2–4 post infection, showing a viral copy number above 104 copies/reaction.
Next, we examined the positive reactions of SFTSV-suspected cat samples with high copy number (more than 104 copies) confirmed by real-time RT-PCR. The dried RT-LAMP assay clearly detected SFTSV positive-confirmed samples with color emission via LED light (Figure 5). All the SFTS-negative samples did not show positive reactions of color emission under LED light (Figure 5). These observations suggest that the dried RT-LAMP assay under LED light is useful for SFTS diagnosis using cat samples as a POCT.

4. Discussion

In this study, we developed a dried RT-LAMP assay for SFTSV using serum and plasma samples for point-of-care testing without RNA extraction. The assay contains a liquid LAMP reaction buffer and dried reagents in the reaction tubes, which can be maintained at room temperature or 4 °C for extended periods of time. We further used an emission colorimetric indicator to simplify the interpretation of the results. Negative and positive results of cat samples can be easily distinguished under LED light with a simple, handmade, battery-operated handheld LED illuminator.
Interestingly, we found that the heat inactivation test at 99 °C for 10 min increased the sensitivity of the assay for SFTSV samples without RNA extraction. The LAMP reaction buffer contained Triton X-100, and previous research has suggested that a detergent containing 0.1% Triton X-100 in the buffer can accelerate viral membrane lysis during boiling [34,35]. Several studies have shown that boiling samples with endogenous RNase-reducing or lysis agents to inactivate endogenous RNase can improve the sensitivity and specificity of an assay [36,37]. In our unpublished study, which investigated the inactivation effects of various reagents with high temperature, we showed that a reagent including 0.1% Triton X-100 efficiently inactivated the infectious SFTSV in clinical samples from SFTSV-infected animals at 99 °C for 10 min. Therefore, it was considered that Triton X-100 in the LAMP reaction buffer would lyse the viral membrane and expose the viral genome to the LAMP reagent. Preheating the sample and LAMP reaction buffer containing Triton X-100 at a boiling temperature for 10 min provides a simple yet effective method for replacing RNA extraction that can be performed anywhere.
Compared to the previous LAMP SFTS, which detects purified RNA from a sample, the current approach eliminates the RNA extraction step, simplifying the method. Alter-native, non-commercial approaches to RNA isolation, such as those involving magnetic beads or a silica gel matrix, require specialist knowledge and are difficult to adopt for on-site screening [38]. Another method using the Loopamp viral RNA extraction solution with preheat treatment at 90 °C for 1 min has been used to simplify the RNA extraction step [29,31]. Although the procedure for RNA extraction using this reagent is simple and rapid, it is expensive.
Using a colorimetric RT-LAMP mixture, the LAMP results can be visually determined by the naked eye via a color change [25]. Based on a combination of one-pot colorimetric visualization and a pocket warmer reaction platform, the RT-LAMP system can detect approximately 10 viral genome copies of SFTSV [25]. The detection of SFTSV using one-step RT-LAMP with the fluorescent detection reagent SYBR Green I can be visualized under UV light [26]. However, these methods still require RNA extraction. Our dried RT-LAMP assay also clearly showed positive reactions by naked eye observation with light blue color changes for extracted viral RNA. However, the naked eye observation was not suitable for heated SFTSV samples without RNA extraction. Instead, those positive observations were clear under the LED illuminator that was handmade at a low cost, suggesting that it could be useful for clinical site. It is of note that the viral copy number of SFTSV that can be detected using the dried RT-LAMP assay is 104 copies/µL of sample or 2 × 104 copies per reaction. The sensitivity of our dried RT-LAMP assay using clinical samples was similar to that of the purified SFTS RNA.
In mice samples, several samples that tested positive using real-time RT-PCR exhibited negative interpretations despite the viral copy being above the detection limit, 104 copy/reaction. Meanwhile, in the cat samples, after confirmation by real-time RT-PCR, all the positive samples showed positive results, and all the negative samples showed negative results according to the RT-LAMP assay. These results show consistency within our dried RT-LAMP assay. In clinical samples, direct visual interpretation was compromised, as the colorimetric change was not clearly observed through naked eye despite positive results being distinctly evident under LED illumination. This result could be due to the presence of blood cell pellets within the samples, which interfered with visual color assessment using direct naked eye observation. Previous studies have demonstrated that, while blood components can impair naked eye color interpretation, they do not affect fluorescence detection [33]. For field applications, we suggest that the result interpretation should be performed by both the direct naked eye and fluorescence observations, given the variable quality of the clinical specimens that cannot be standardized in remote settings. Therefore, the handmade, battery-operated, handheld LED illuminator was made previously [33] to facilitate the detection in the field, enhancing the capability of field detection. This approach maintains diagnostic accuracy while minimizing the need for sophisticated laboratory equipment, making the assay suitable for resource-limited environments where advanced instrumentation may not be available.
SFTS-suspected samples should be sent to a recommended institution to confirm the diagnosis. Although the sensitivity and specificity of SFTSV RT-LAMP may not be high, the RT-LAMP assay could be very useful for rapid diagnosis in hospitals for primary diagnosis before confirmation with a laboratory diagnosis in an institution. Furthermore, a positive result reflects a high viral load in the sample, indicating the risk of transmission between humans and animals.
RT-LAMP has been used to detect many RNA viruses, including Dengue, Japanese encephalitis, SARS-CoV-2, MERS-CoV, tick-borne encephalitis virus, and influenza A [39,40,41,42,43,44]. The requirement of a facility to diagnose SFTS makes it difficult to acquire disease surveillance data, and the rapidity of diagnosis also has an impact on the medical care treatment for the patient [45,46]. Many clinics rely on large institutions with adequate facilities, since diagnosing SFTS in patients requires specialized diagnostic equipment. As such, a quick and simple one-pot diagnostic method that can be performed in clinics with minimal equipment is crucial for the early detection and diagnosis of SFTSV. Owing to its quick turnaround time, RT-LAMP is a promising alternative to RT-PCR for detecting SFTSV in clinical samples.

5. Conclusions

This study demonstrated that our dried RT-LAMP assay can serve as an alternative diagnostic tool for SFTSV detection using serum and plasma samples, providing a point-of-care testing (POCT) method. The dried RT-LAMP for the SFTSV assay requires only a heat block, a portable handmade LED illuminator, and a LAMP tube that has already been mixed with the dried reagents (substrate, primer, and enzyme) to carry out simultaneous RNA- and DNA-specific amplifications. We hope that this study will facilitate the implementation of an affordable diagnostic method using the RT-LAMP assay in settings where the use of commercial kits is not possible, such as in clinics with limited access to advanced facilities. As such, this approach could help address several issues associated with the accessibility of reliable, affordable, quick, and efficient diagnostic instruments for detecting SFTSV in animals, though we suggest that samples should be sent to recommended institutions to confirm diagnoses. Nonetheless, this assay will have significant utility in areas with limited resources, particularly when rapid diagnostic outcomes are required.

Author Contributions

Data curation, formal analysis, methodology, visualization, writing—original draft, review, and editing: M.A.; resources: M.N.; conceptualization and methodology: K.H.; formal analysis and resources: H.S.; conceptualization, methodology, supervision, funding acquisition, writing—original draft, review, and editing: D.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported financially by JSPS KAKENHI (grant numbers JP17H04661 and 24K01930); the Research Center for Thermotolerant Microbial Resources (RCTMR), Yamaguchi University (grant number 2021-11); and the Joint Usage/Research Center on Tropical Disease, Institute of Tropical Medicine, Nagasaki University (2022-Kyoten-03, 2023-Kyoten-02, and 2024-Kyoten-05).

Institutional Review Board Statement

The animal experimental protocols were approved by the Animal Care and Use Committee of Yamaguchi University (approval number 05-31 565).

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon request.

Acknowledgments

The authors would like to thank Yamashita Animal Hospital, Fukuda Dog and Cat Hospital, Shiranaga Animal Hospital, Saikyo no Mori Animal Hospital, Matsuda Veterinary Clinic, Fujishima Animal Hospital, Wakayama Animal Medical Center, Takegawa Dog and Cat Hospital, Yoshida Animal Hospital, Kato Animal Hospital, Koshodo Animal Hospital, Mori Animal Hospital, Tagawa Animal Clinic, Shimodoka Animal Hospital, Hashimoto Animal Hospital, Tatsuno Dog and Cat Hospital, Tanaka Animal Hospital, Ogigawa Animal Hospital, Niigata Prefecture, and Oji Animal Hospital for providing serum and plasma samples of SFTSV-infected animals.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Handmade-battery operated handheld LED illuminator. The handmade LED illuminator emits 500 nm wavelength light to detect the LAMP fluorescence reaction. The portable illuminator is easy to assemble and suitable for clinical site.
Figure 1. Handmade-battery operated handheld LED illuminator. The handmade LED illuminator emits 500 nm wavelength light to detect the LAMP fluorescence reaction. The portable illuminator is easy to assemble and suitable for clinical site.
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Figure 2. Analytical sensitivity of the dried SFSTV RT-LAMP assay. SFTSV viral stock was extracted for RNA, and viral copy number was measured using real-time RT-PCR. Ten-fold dilutions of SFTSV RNA were amplified via RT-LAMP and detected using a colorimetric indicator. The assay detected the SFTSV RNA up to 104 copies/µL (red arrow). The assay results could be observed with light blue color changes (upper panel) and greenish-yellow color emissions under an LED light (bottom panel).
Figure 2. Analytical sensitivity of the dried SFSTV RT-LAMP assay. SFTSV viral stock was extracted for RNA, and viral copy number was measured using real-time RT-PCR. Ten-fold dilutions of SFTSV RNA were amplified via RT-LAMP and detected using a colorimetric indicator. The assay detected the SFTSV RNA up to 104 copies/µL (red arrow). The assay results could be observed with light blue color changes (upper panel) and greenish-yellow color emissions under an LED light (bottom panel).
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Figure 3. Heat inactivation test of the RT-LAMP assay. The RT-LAMP results could be observed with the naked eye (upper panel) and under an LED light (bottom panel). After being treated with boiling at 99 °C for 10, 20, and 30 min, the SFTSV sample including 2 × 104 ffu of SFTSV was tested with dried RT-LAMP assay. The results observed under LED light showed that heat treatment of samples increased the sensitivity of the dried RT-LAMP assay compared to the unheated samples. SFTSV viral RNA and distilled water were used as positive control (PC) and negative control (NC), respectively.
Figure 3. Heat inactivation test of the RT-LAMP assay. The RT-LAMP results could be observed with the naked eye (upper panel) and under an LED light (bottom panel). After being treated with boiling at 99 °C for 10, 20, and 30 min, the SFTSV sample including 2 × 104 ffu of SFTSV was tested with dried RT-LAMP assay. The results observed under LED light showed that heat treatment of samples increased the sensitivity of the dried RT-LAMP assay compared to the unheated samples. SFTSV viral RNA and distilled water were used as positive control (PC) and negative control (NC), respectively.
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Figure 4. Evaluation of the SFTSV RT-LAMP assay in the SFTSV experimental mice plasma samples. Positive results (upper) indicated by greenish-yellow color under LED light and negative results (bottom) indicated as orange under LED light. The same samples were also measured for the viral copy number using real-time RT-PCR, and the numbers are shown in the figure. In the mouse samples, positive results were achieved in samples with high viral copy numbers. Samples with a copy number less than 2 × 104 copies/reaction provided negative results. SFTSV viral RNA and distilled water were used as positive control (PC) and negative control (NC), respectively.
Figure 4. Evaluation of the SFTSV RT-LAMP assay in the SFTSV experimental mice plasma samples. Positive results (upper) indicated by greenish-yellow color under LED light and negative results (bottom) indicated as orange under LED light. The same samples were also measured for the viral copy number using real-time RT-PCR, and the numbers are shown in the figure. In the mouse samples, positive results were achieved in samples with high viral copy numbers. Samples with a copy number less than 2 × 104 copies/reaction provided negative results. SFTSV viral RNA and distilled water were used as positive control (PC) and negative control (NC), respectively.
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Figure 5. Evaluation of the SFTSV RT-LAMP assay in the SFTS-suspected cat serum and plasma. Positive results indicated by a greenish-yellow color emission (upper panel) and negative results without emission (bottom panel) under LED light. The same samples were also measured for the viral copy number using real-time RT-PCR, and the numbers are shown in the figure. SFTSV viral RNA and distilled water were used as positive control (PC) and negative control (NC), respectively.
Figure 5. Evaluation of the SFTSV RT-LAMP assay in the SFTS-suspected cat serum and plasma. Positive results indicated by a greenish-yellow color emission (upper panel) and negative results without emission (bottom panel) under LED light. The same samples were also measured for the viral copy number using real-time RT-PCR, and the numbers are shown in the figure. SFTSV viral RNA and distilled water were used as positive control (PC) and negative control (NC), respectively.
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MDPI and ACS Style

Anggita, M.; Hayashida, K.; Nishizato, M.; Shimoda, H.; Hayasaka, D. Reverse Transcription Loop-Mediated Isothermal Amplification Assay Using Samples Directly: Point-of-Care Detection of Severe Fever with Thrombocytopenia Syndrome Virus. Zoonotic Dis. 2025, 5, 19. https://doi.org/10.3390/zoonoticdis5030019

AMA Style

Anggita M, Hayashida K, Nishizato M, Shimoda H, Hayasaka D. Reverse Transcription Loop-Mediated Isothermal Amplification Assay Using Samples Directly: Point-of-Care Detection of Severe Fever with Thrombocytopenia Syndrome Virus. Zoonotic Diseases. 2025; 5(3):19. https://doi.org/10.3390/zoonoticdis5030019

Chicago/Turabian Style

Anggita, Marla, Kyoko Hayashida, Miyuka Nishizato, Hiroshi Shimoda, and Daisuke Hayasaka. 2025. "Reverse Transcription Loop-Mediated Isothermal Amplification Assay Using Samples Directly: Point-of-Care Detection of Severe Fever with Thrombocytopenia Syndrome Virus" Zoonotic Diseases 5, no. 3: 19. https://doi.org/10.3390/zoonoticdis5030019

APA Style

Anggita, M., Hayashida, K., Nishizato, M., Shimoda, H., & Hayasaka, D. (2025). Reverse Transcription Loop-Mediated Isothermal Amplification Assay Using Samples Directly: Point-of-Care Detection of Severe Fever with Thrombocytopenia Syndrome Virus. Zoonotic Diseases, 5(3), 19. https://doi.org/10.3390/zoonoticdis5030019

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