Development of RT-LAMP assay for variants of HCV in Pakistan.

Hepatitis C Virus (HCV) elimination requires improved diagnostic methods, particularly in regions with high screening demands like Pakistan. Rapid and accurate detection is crucial, yet commercially available RT-qPCR assays often have limitations in sensitivity and specificity, reducing their reliability across diverse populations. Additionally, RT-qPCR is time-consuming, costly, and requires skilled personnel. In contrast, Re-verse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) offers a robust, cost-effective alternative with superior sensitivity and specificity. In the present study, for the first time, we reported a novel RT-LAMP for HCV diagnosis in Pakistan. We designed and optimized an RT-LAMP assay using degenerate primers targeting the 5’ UTR of HCV, incorporating sequences from reference and emerging genotypes, including Pakistani isolates. Key assay parameters, including reaction time, volume, primer concentration, and temperature, were optimized. Triple confirmation of HCV clinical samples using different sets of primers for conventional PCR, commercial lab RT-qPCR and RT-LAMP provided a unique validation approach. It was demonstrated that RT-LAMP detected HCV at a limit of detection of 131 copies, outperforming commercial RT-qPCR by identifying false negatives (71%) and achieving 100% sensitivity and specificity, compared to 82% sensitivity of RT-qPCR. The assay’s processing time was 60 min, supporting large-scale diagnostics. BLAST analysis confirmed specificity, with no significant alignment to related Flaviviridae genomes. RT-LAMP represents a significant advancement in HCV detection, offering high sensitivity, specificity, and ease of use, making it ideal for effective prevention and control strategies.