Tick samples positive for TcTV-1, SGLV, YEZV, TBEV, SFTSV, and BJNV were stored in the laboratory at −80°C until use. In April 2025, a total of 50 questing Dermacentor marginatus ticks were collected from Xinjiang, China. RNA was extracted from homogenized tick samples and reverse-transcribed into complementary DNA (cDNA) as previously described (Gui et al., 2024).

The primers were designed based on the NP gene sequences downloaded from GenBank (Supplementary Table S1). To prevent cross-reactivity between TcTV-1 and the genomic sequences of other tick-borne pathogenic viruses, sequence alignment and comparison were performed using ESPript 3.0 (Supplementary Figure S1). The candidate primers were designed using Primer Explorer V5, with critical parameters optimized: GC content (40–60%), avoidance of non-specific binding, stability at both the 3’ and 5’ ends, melting temperature (Tm) range of 50–60°C, and absence of secondary structures such as self-dimers and cross-dimers (Figure 1) (Das et al., 2012; Panno et al., 2020). LAMP amplification efficiency was negatively correlated with cycle threshold; primer sets with lower thresholds were more effective. The optimal set was further confirmed in eight replicates to ensure stability.

The 297 bp amplification region targeted by the primers (Supplementary Figure S2) from the TcTV-1 JH16 strain (GenBank accession number MK554695) was commercially synthesized, digested with Sma I, and cloned into the pUC57 vector (Sangon Biotech, China). The recombinant plasmid was transformed into Escherichia coli TOP10 cells (Invitrogen), purified using a plasmid purification kit (Qiagen), and verified by Sanger sequencing. Plasmid DNA was then extracted using the TIANprep Mini Plasmid Kit (TIANGEN, China), quantified via NanoDrop 2000 spectrophotometry (ThermoFisher, USA), and copy numbers were calculated using the formula: copies/μL = [6.02 × 10²³ × concentration (g/μL)]/[average molecular weight per base pair (g/mol) × length (bp)]. The final plasmid solutions were adjusted to 1 × 10¹⁰ copies/μL and stored at -20°C for downstream applications.

The LAMP reaction system was performed as previously described (Gui et al., 2024). Briefly, the reaction mixture contained 2.5 μL of 10× isothermal amplification buffer, 1.5 μL of MgSO₄ (100 mM), and 3.5 μL of dNTP Mix (10 mM). Amplification was initiated by adding 1 μL of Bst 2.0 WarmStart DNA polymerase (320 U/mL). Primer sets were added at final concentrations of 1.6 μM (FIP and BIP), 0.2 μM (F3 and B3), and 0.4 μM (LF and LB), with 1 μL of each primer used. The mixture was supplemented with 1 μL of EvaGreen dye, 1 μL of hydroxynaphthol blue (HNB), and 1 μL of cDNA template, and the volume was brought to 25 μL with 8.5 μL of ultrapure water. Ultrapure water was used in place of the cDNA template as a negative control.

To determine optimal conditions, amplification was performed across 63–68°C for 60 min by the Real-Time Fluorescent Quantitative PCR System (Dragonlab, China). A color change from violet to azure or a fluorescence amplification curve indicated a positive TcTV-1 result. The optimal reaction temperature is defined as the temperature at which amplification efficiency is maximized and the reaction time is minimized.

The SYBR green RT-qPCR was performed as the gold-standard assay, utilizing primers and reaction conditions detailed in the previous study (Liu et al., 2020). Briefly, 1 μL of each NP gene-specific primer (forward: 5′-AGAGAATGATGCTATGATTGC-3′; reverse: 5′-GAGTCCTCGTTCAACCAT-3′), 2 μL cDNA template, and 6 μL RNase-free water were added to 10 μL of 2×TB Green Premix DimerEraser (TaKaRa) to yield a final reaction volume of 20 μL. Amplification was carried out as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s. Viral copy numbers were calculated using the equation Y=-3.3477x+37.214 derived from the amplification plot and standard curve (Supplementary Figure S3).

Recombinant plasmids were serially diluted tenfold, spanning concentrations from 10⁵ to 10–¹ copies/µL, and utilized to determine the assay’s sensitivity. Each dilution was tested in triplicate. In addition, cDNA derived from other tick-borne pathogenic viruses (SGLV, BJNV, YEZV, SFTSV, and TBEV) was tested alongside TcTV-1 to evaluate potential cross-reactivity. The LAMP reaction was performed under isothermal amplification conditions at 65°C (the optimal temperature) for 60 minutes using the Real-Time Fluorescent Quantitative PCR System (Dragonlab, China), with eight replicates. Nuclease-free ultrapure water was incorporated as a no-template control to replace cDNA in all reaction sets, enabling systematic monitoring of exogenous contamination and non-specific amplification events.

The field-collected questing ticks from Xinjiang, China were tested individually using both the established TcTV-1-specific LAMP assay and the reference SYBR Green RT-qPCR method developed in a previous study (Liu et al., 2020). The diagnostic performance metrics, including prevalence, sensitivity, specificity, PPV, NPV, and accuracy, along with their 95% confidence intervals (CI), were calculated for the LAMP assay in comparison to SYBR Green RT-qPCR using an online tool, the “Diagnostic Test Evaluation Calculator” (https://www.medcalc.org/calc/diagnostic_test.php).

Of the three designed LAMP primer pairs (Supplementary Table S2), the first set was selected for its optimal amplification kinetics and demonstrated reliable detection of the recombinant plasmid at a concentration of 1×10⁶ copies/μL (Figure 2A). The consistent results across eight replicate tests confirm the stability of this primer pair (Supplementary Figure S4). A reaction temperature of 65°C proved optimal for minimizing the detection cycle and enhancing fluorescence of TcTV-1 (Figure 2B, Supplementary Figure S5).

The detection limit for the LAMP assay was 1×10–¹ copies/μL, with all positive results detected within 35 minutes (Figure 3A). All diluted samples exhibited a color shift from violet to azure, apart from the negative control (Figure 3B).

The LAMP assay demonstrated high specificity, detecting only the targeted TcTV-1 with no cross-reaction to other tick-borne pathogenic viruses (SGLV, BJNV, YEZV, SFTSV, and TBEV) (Figure 4A). Visual detection confirmed TcTV-1 via a violet-to-azure transition in its tube, whereas non-target viruses maintained purple, confirming the LAMP assay’s accurate recognition of TcTV-1 (Figure 4B).

A total of eight tick (16.0%; 95% CI: 7.2–29.1) individuals tested positive for TcTV-1 using the LAMP assay, with amplification times ranging from 7 to 29 (Figure 5A, Supplementary Table S3). A clear color change from violet to azure was observed in the positive samples (Supplementary Figure S6). Furthermore, these eight tick samples were confirmed as TcTV-1 positive by SYBR Green RT-qPCR, yielding Ct values between 24 and 36 (Figure 5B, Supplementary Table S3). The sensitivity, specificity, PPV, NPV, and accuracy of the established LAMP assay, when compared to RT-qPCR, were 100% (95% CI: 63.1–100.0), 100% (95% CI: 91.6–100.0), 100% (95% CI: 63.1–100.0), 100% (95% CI: 91.6–100.0), and 100% (95% CI: 92.9–100.0), respectively (Supplementary Table S4).