Development of a Highly Multiplexed RT-LAMP Assay for Coverage of Genetic Sequence Diversity

Nucleic acid amplification tests (NAATs) can achieve high accuracy for diagnosing infectious diseases by targeting conserved genetic sequences specific to the target organism. Isothermal NAATs, such as reverse-transcription loop mediated isothermal amplification (RT-LAMP), simplify instrumentation requirements, facilitating point-of-care testing. However, sequence variation due to genetic variability can cause false negative results. Single-pot multiplex testing can improve sequence coverage, but RT-LAMP is complicated by requiring many primers for even a single assay, which can lead to nonspecific amplification. We implemented a process that leveraged manual primer design to develop a highly multiplexed RT-LAMP assay (Chain LAMP) targeting 7 adjacent genomic target regions of HIV, one of the most diverse clinically relevant pathogens. This process departed from standards for RT-LAMP design, including the omission of bumper primers whose activity was replaced by cooperative neighboring assays. The Chain LAMP is, to our knowledge, the highest order single-pot multiplexed RT-LAMP assay published. The assay has an analytical limit of detection of 25 copies of RNA/reaction without detectable nonspecific amplification, translating to 1000 copies of HIV/mL of plasma from a fingerstick sample, aligning with WHO standards for HIV viral load monitoring. When evaluated using 24 clinical RNA samples representative of global HIV diversity, Chain LAMP demonstrated robust coverage of sequence diversity, amplifying all samples with minimal sensitivity variation. We performed mechanistic analysis with Nanopore sequencing, identifying liftoff of multiple assay regions for each sample, indicating many initiation loci. The high level of multiplexing in the Chain LAMP effectively increases the coverage of HIV sequence diversity.