Primer Payload System for Higher-Order Multiplex LAMP: Design and Development of Unit Processes

Design and Development of Platforms for the Application of Loop-mediated Isothermal Nucleic Acid Amplification, LAMP, in the Diagnosis of Polymicrobial Diseases Tochukwu Dubem Anyaduba, Travis Schlappi (PI) For the past two decades, several isothermal nucleic acid amplification technologies have emerged. These are mostly in response to the need for robust molecular diagnostic tools amenable to point-of-care and limited-resource settings. Of these, loop-mediated isothermal amplification, LAMP, stands out as a highly specific and rapid alternative to the polymerase chain reaction, PCR. One of LAMP\u27s significant characteristics involves using four essential and two loop (rate increasing) primers to recognize six to eight (6 – 8) distinctive regions in a target DNA sequence. While the assortment of primers makes LAMP highly specific, it also poses a challenge to its exploitation in multiplex molecular diagnostics. Several published methods present means of adopting LAMP in multiplexing; however, only very few can detect up to four targets within the same sample stream. Our research\u27s overall goal is to develop platforms capable of exploiting LAMP\u27s high degree of specificity in identifying 9+ pathogens within the same sample stream using rapid prototyping/ simplistic technologies. This goal is fundamental in diagnosing polymicrobial diseases such as urinary tract infections, diarrheal diseases, respiratory tract infections, and other diseases whose attendant symptoms compel uninformed prescriptions. To meet this need, we designed multiple methods and developed unit processes toward achieving a more promising platform, A Primer Payload Platform (P3). The P3 is borne out of the ideology that the isolation of LAMP primer sets and their specific target DNA moieties [AH1] in micro-reaction vesicles from a single reaction mix could aid the differential detection of multiple targets without the limitations attendant to current multiplexing systems. Thus, as a first step toward achieving the P3, we adopted methods for a multifaceted use of beads (as pathogen identity signatures, primer-delivery machinery, and specific target DNA carriers). Secondly, we employed simplistic rapid prototyping methodologies to develop microfluidic cartridges to generate highly monodisperse picoliter-scale droplets. As these droplets are digital-LAMP and digital PCR-ready, we further applied them to detect and quantify Escherichia coli and Lactobacillus acidophilus genomic DNA. Finally, we developed a mechanism for the encapsulation of the beads in picoliter-scale droplets. By unifying the droplet formation and bead introduction rates at the flow-focusing junction, we recorded more single-bead-carrying droplets than predicted by Poisson statistics. While we do not have a perfect system for single-particle encapsulation, we achieved higher single bead encapsulation than ever reported in systems using rapid prototyping for microfluidics or dense bead manipulation. Our vision is to fully integrate these unit processes into a unified microfluidics-based platform for polymicrobial diseases molecular diagnostics at the point of care. We believe this platform will enable timely and precise healthcare interventions within patients\u27 first clinic visits