Determination of Human T-Cell Leukemia Virus-1 Using a Real-Time Loop-Mediated Isothermal Amplification Assay

Abstract

Background: Accurate and rapid detection of human T-cell lymphotropic virus type 1 (HTLV-1) is a critical aspect in monitoring and developing prevention strategies to reduce its transmission rates in blood donors. However, there are two challenges, the expensive reagents and labor-intensive process, faced by the development of HTLV-1 testing. Thus, a real-time loop-mediated isothermal amplification (RT-LAMP)-based method was used for the rapid detection of HTLV-1 in this article. Methods: The PX gene (GenBank: L36905.1) of HTLV-1 was cloned into Escherichia coli DH5a plasmid pUC57 to construct a reference strain for HTLV-1 detection. RT-LAMP primers were designed for the PX gene, the experimental reaction system was optimized, and the specificity and sensitivity of the method were investigated. Results: The optimal reaction system of 25 μL RT-LAMP contained 0.2 μM each of primers outer forward (F3) and backward (B3) primers, 1.6 μM each of forward inner primers (FIP) and backward inner primers (BIP), 0.8 μM each of loop forward (LF) and loop back (LB) primers, 1.2 mM deoxynucleotide triphosphates (dNTPs), 1.0 M betaine, 6 mM MgSO₄, and 8 U Bst 3.0 DNA polymerase. Conclusions: The HTLV-1 LAMP method was sensitive, specific, simple-to-operate, and inexpensive, and could be a new method for the rapid detection of HTLV-1.