Development of CRISPR/LAMP-based microfluidic platform for human Mpox diagnosis

Abstract

Human Mpox is an infectious and zoonotic disease caused by the Mpox virus (MPX). It has recently become a global public health threat, determined by the World Health Organization (WHO). It possess an epidemic risk, as cases have been reported in numerous non-endemic countries worldwide in recent years. Although the number of cases in Turkey remains low, the high number of cases in countries with strong social and commercial ties to Turkey pose a threat to the country. To prevent a potential outbreak and ensure an effective public health response, rapid, accurate, and accessible diagnostic tools are essential. This thesis aimed to develop a microfluidic chip with high specificity and accuracy for rapid Mpox diagnosis in the field, using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (CRISPR-Cas) technology combined with loop-mediated isothermal amplification (LAMP), referred to as CRISPR/LAMP. CRISPR and LAMP were combined because together they offer high accuracy and specificity and can be designed to be pathogen-specific. The microfluidic chip was developed to minimize contamination risk and reduce reliance on specialist personnel and laboratory infrastructure, limitations commonly introduced by the multiple manual steps required in conventional diagnostic workflows. With the scope of the thesis, we developed a microfluidic chip, lyophilized the reaction components directly on the chip, developed an optical reading system for fluorescent detection, and performed LAMP on-chip and CRISPR/LAMP reactions on the chip to monitor results. The ultimate goal was to produce a user-friendly, rapid, reliable, and cost-effective diagnostic platform that enables early detection of Mpox infections without the need for laboratory infrastruc-ture. It represents a first-of-its-kind diagnostic approach combining CRISPR/LAMP within a microfluidic system and has the potential to make a meaningful impact on outbreak preparedness and response.