J Transl Med. 2025 Jun 11;23(1):646. doi: 10.1186/s12967-025-06642-9.
ABSTRACT
BACKGROUND: Human T-cell lymphotropic virus-1(HTLV-1) infection may lead to adult T-cell leukemia, HTLV-associated myelopathy/tropical spastic paraplegia, and various neurological diseases. Therefore, developing a rapid and accurate detecting method is crucial for preventing and controlling HTLV-1 infection. This study aims to develop and validate a detection assay for HTLV-1 proviral DNA by combining multienzyme isothermal rapid amplification(MIRA) and lateral flow dipstick(LFD) techniques.
METHODS: Primers and probes were designed based on the conserved sequence of the HTLV-1 Tax gene, and the MIRA-LFD method was established and optimized. Different concentrations of HTLV-1 plasmids were tested by the MIRA-LFD assay to verify the limit of detection(LOD) of the method. To evaluate the cross-reactivity, viral pathogens were detected by this assay, including hepatitis B virus(HBV), hepatitis C virus(HCV), hepatitis E virus(HEV), cytomegalovirus(CMV), herpes simplex virus-1/2(HSV-1/2), Epstein-Barr virus(EBV), Parvovirus B19(B19V), and human T-cell lymphotropic virus-2(HTLV-2). 500 clinical samples were simultaneously detected using real-time PCR(qPCR) and MIRA-LFD methods, and the consistency between the two methods was statistically analyzed. The qPCR was used as the reference method to determine the diagnostic sensitivity and specificity of the MIRA-LFD method.
RESULTS: The MIRA-LFD method can detect HTLV-1 within 20 min at 37℃. The LOD for HTLV-1 using this method was 200 copies/µL. This method had no cross-reaction with the other eight viruses, with good specificity. Using qPCR as the standard, the diagnostic sensitivity and specificity of the MIRA-LFD method for 500 clinical samples were 100%. The MIRA-LFD and qPCR methods had 100% consistency(kappa value = 1.00).
CONCLUSIONS: This study established a method based on MIRA-LFD for detecting HTLV-1 proviral DNA, which has the advantages of fast, accurate, good sensitivity, strong specificity, simplicity, and portability. This method meet the needs of rapid on-site detection and is easy to promote and use in grassroots medical institutions or blood stations.
PMID:40500737 | DOI:10.1186/s12967-025-06642-9
