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Background <p>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.</p> Methods <p>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.</p> Results <p>The MIRA-LFD method can detect HTLV-1 within 20&#xa0;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).</p> Conclusions <p>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.</p>

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Development and validation of a multienzyme isothermal rapid amplification and lateral flow dipstick combination assay for HTLV-1 proviral DNA detection

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年6月11日

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  • Miaomiao Li,
  • Mengjiao Lin,
  • Yushan Xu,
  • Dawei Cui,
  • Jue Xie

摘要

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.