Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) is a novel bunyavirus that has emerged as a significant public health threat in East Asia, particularly in China, Japan, and Korea [1], [2], [3]. SFTSV infection is characterized by high fever, thrombocytopenia, leukopenia, and multi-organ dysfunction, potentially leading to severe outcomes and high mortality rates [4], [5]. The SFTSV is a single-stranded RNA virus belonging to the genus Bandavirus, family Phenuiviridae, and the viral genomes consist of three segments, large (L), medium (M), and small (S) [6].

Higher viral load, as estimated by cycle threshold (Ct) values from reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses, has been shown to be an independent prognostic mortality marker in several SFTS studies [7], [8], [9]. Furthermore, viral load also represents a crucial criterion for the stratification of case enrollment and the assessment of antiviral efficacy in clinical trials [10]. However, different assays, platforms, and protocols of RT-qPCR used for viral load detection could reduce the comparability between different institutions and yield inconsistent results [10]. Additionally, various substances in clinical samples could interfere with the RT-qPCR efficiency [11]. The absolute concentration of viral load in peripheral blood was highly variable, from 1.91 × 103 to 9.80 × 107 copies per mL, after calibration from the Ct value of RT-qPCR using a standard curve [12], [13], [14].

Digital PCR (dPCR) is an advanced molecular technique that enables highly sensitive and precise quantification of specific nucleic acids [15]. Compared to the RT-qPCR method, dPCR offers superior accuracy in measuring low-abundance viral genomes and the absolute concentration [16]. Moreover, dPCR allows for direct quantification without calibration curves and improves the comparability of results between different centers and laboratories [17], making it an ideal tool for assessing viral load in clinical samples [18].

In this study, we developed a method for viral load quantification using dPCR with primers and probes combination targeting the S segment of SFTSV. Then, we evaluated and validated its feasibility and relevant parameters based on viral cultures. Subsequently, a prospective and observational study involving 166 suspected SFTS cases was conducted to assess its applicability in a clinical setting in Shandong Province.