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Published on: November 10, 2023
Development and validation of a duplex real-time PCR for the rapid detection and quantitation of HTLV-1
Huimin Ji1,2, Le Chang1,2, Ying Yan1,2
1National Center for Clinical Laboratories, Institute of Geriatric Medicine, Beijing Hospital, National Center of Gerontology, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
Background:
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.
Results:
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 × 108 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.
Conclusion:
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.
