Development and validation of a duplex real-time PCR for the rapid detection and quantitation of HTLV-1

• Published in: Virology journal 2023-01, Vol.20 (1), p.9-9, Article 9
• Main Authors: Ji, Huimin, Chang, Le, Yan, Ying, Wang, Lunan
• Format: Article
• Language: English
• Subjects: Development and progression; Diagnosis; HTLV-I (Virus); HTLV-I infections; HTLV-I Infections - epidemiology; Human T-lymphotropic virus 1 - genetics; Human T-lymphotropic virus 2 - genetics; Human T-lymphotropic virus-1; Humans; Identification and classification; Methodology; Polymerase chain reaction; Proviral load value; Proviruses - genetics; Real-Time Polymerase Chain Reaction - methods; Real-time quantitative PCR; Reproducibility of Results; Risk factors; Testing; Viral Load
• ISSN: 1743-422X; 1743-422X
• DOI: 10.1186/s12985-023-01970-y

The HTLV-1 prevalence in China varies geographically, while HTLV-2 infection has rarely been found so far. Proviral load is one of the determining factors of pathogenesis and progression of HTLV-1 related diseases. However, neither molecular assays nor commercial kits are available for HTLV-1 diagnosis in China. The objective of the present study was to develop and validate a TaqMan qPCR assay for HTLV-1 proviral load quantification. A plasmid containing both the HTLV-1 of interest and a fragment of the RNase P (RPPH1) gene was constructed and used to establish the standard curves. The assay has a wide dynamic range (2.5 × 10 copies/reaction ~ 25 copies/reaction) and sensitive to 1 copy for HTLV-1 and RPPH1. The limit of detection for Hut102 cell concentration was 0.0218% (95% confidence interval 0.0179-0.0298%). The assay gave coefficient of variation (CV) for both the HTLV-1 and RPPH1 Ct values. All of the HTLV-1 sero-negative samples and MOT cell line (infected with HTLV-2) amplified only the RPPH1 gene by our method, presenting 100% specificity. 85 Samples confirmed positive or indeterminate by LIA were performed by established qPCR assay and WB. 90.0% (27/30) of LIA-HTLV-1-positive, 33% (2/6) of LIA-untypeable and 2% (1/49) of LIA-indeterminate samples were defined as qPCR-positive. The median PVL of LIA-positive samples (n = 27, 1.780 copies/100 cells) was much higher than that of LIA-untypeable and (n = 2, 0.271 copies/100 cells) indeterminate samples (n = 1, 0.017 copies/ 100 cells). Additionally, compared to WB, the duplex qPCR verified more positive samples, demonstrating a better sensitivity. The duplex qPCR developed here with high sensitivity, good specificity and reproducibility could accurately and quantitatively detect the HTLV-1 PVLs, which can be used to confirm the initial reactive samples for an improved cost/benefit ratio as well as to monitor the clinical progression and efficacy of therapy in patients with HTLV-1 related disease.