Analytical validation and clinical evaluation of digital PCR for absolute quantification of Crimean-Congo Hemorrhagic fever virus
Journal of Microbiological Methods, cilt.248, 2026 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 248
- Basım Tarihi: 2026
- Doi Numarası: 10.1016/j.mimet.2026.107649
- Dergi Adı: Journal of Microbiological Methods
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, EMBASE, Environment Index, MEDLINE, Academic Search Ultimate (EBSCO)
- Anahtar Kelimeler: Absolute quantification, Crimean-Congo hemorrhagic fever virus, Digital PCR, Molecular diagnostics, Tick-borne diseases, Viral load
- İstanbul Üniversitesi-Cerrahpaşa Adresli: Evet
Özet
Crimean-Congo hemorrhagic fever virus (CCHFV) is a high-consequence tick-borne pathogen requiring accurate molecular tools for reliable viral load assessment. This study evaluated and compared the performance of a laboratory-optimized quantitative real-time RT-PCR (qRT-PCR) assay with a digital PCR (dPCR) platform for the detection and absolute quantification of CCHFV RNA. Analytical validation was performed using a characterized viral isolate propagated in Huh7 cells, followed by serial dilution analysis to assess sensitivity, linearity, and dynamic range. Clinical performance was further evaluated using 27 human plasma samples obtained from suspected or confirmed Crimean-Congo hemorrhagic fever (CCHF) cases. All samples were analyzed in parallel using both qRT-PCR and dPCR platforms. qRT-PCR demonstrated strong linearity across a wide dynamic range (R2 = 0.992); however, amplification efficiency exceeded optimal limits (104.8%, slope: −3.211), with increased variability observed near the detection threshold. In contrast, dPCR provided absolute quantification independent of standard curves, exhibiting high analytical precision and a functional limit of detection of approximately 30 copies/μL. Clinical analysis revealed that the majority of samples possessed low viral loads (Ct ≥ 37), where qRT-PCR showed increased variability, while dPCR maintained stable quantification across all concentration ranges. Overall, a significant correlation was observed between the two methods (R2=0.7121, p<0.0001), although dPCR demonstrated enhanced sensitivity in low viral load conditions. These findings suggest that dPCR provides valuable diagnostic clarity for precise quantification of CCHFV RNA, particularly in low-titer samples near the detection threshold, highlighting its potential utility in supporting molecular diagnostics and epidemiological surveillance.