Development and evaluation of HIV-۱ diagnostic kits based on recombinant reversetranscription loop-mediated isothermal amplification assay

The identification of HIV-۱ at the point-of-care or within resource-constrained settings presents asignificant challenge due to limitations in time and cost. Currently, PCR and real-time PCR tests stand as the soledependable method for diagnosing recent infections during the window period following infection and prior toseroconversion. Nonetheless, these tests are highly time-consuming and costly, necessitating expensivematerials, a thermal cycler, gel electrophoresis, or computational and data analysis software, all of which areexclusively suitable for well-equipped laboratory environments. Given the gravity of the disease, there exists anurgent need for a straightforward, economical, rapid, and accessible diagnostic approach. The ReverseTranscription Loop-Mediated Isothermal Amplification (RT-LAMP) technique offers a distinct molecular and costeffectivealternative method with exceptional specificity and sensitivity for the creation of a swift nucleic acidamplification test (NAAT). This amplification is conducted in a tube containing a blend of primers, reversetranscriptase enzyme, and Bst DNA polymerase at a temperature of ۶۰ °C for ۶۰ minutes. Furthermore, a basicwater bath or heat block suffices for the amplification of the LAMP products, culminating in direct visualdetection facilitated by the addition of a slight amount of Sybr green I fluorescent dye to the microtube underUV light. In this research, we initially fine-tuned the expression of recombinant Bst polymerase and subsequentlycarried out the RT-LAMP technique using six specific primers targeting the IN coding region of the POL gene torecognize different HIV-۱ subtypes. As a result, we foresee that the outcomes of this study will pave the way forprogress towards the development and production of an HIV-۱ diagnostic kit in Iran in the near future.

کلیدواژه ها:

HIV-۱ ، RT-LAMP ، Recombinant Bst DNA Polymerase