Integrated Single-Step Duplex CRISPR-Based System for Point-of-Care Detection of Human Immunodeficiency Virus and Treponema pallidum Directly from Whole Blood

Abstract Acquired immunodeficiency syndrome (AIDS) and syphilis are two major sexually transmitted diseases that pose serious threats to public health, underscoring the urgent need for fast and sensitive diagnosis for human immunodeficiency virus (HIV) and Treponema pallidum (TP). Here, we developed a single-step duplex CRISPR assay that integrated reverse transcription, recombinase polymerase amplification, in vitro transcription, and CRISPR/Cas12a and Cas13a detection for simultaneous HIV and TP nucleic acid testing. We further engineered a fully integrated, low-cost microfluidic chip that consolidated whole-blood sample processing, nucleic acid release, and single-step duplex CRISPR detection. The chip featured a rapid plasma separator with a press-actuated design that removed inhibitors and extracted plasma within minutes without external equipment. The entire process was coupled with a homemade smartphone-based portable device that provided thermal incubation, dual-channel fluorescence excitation, and automated image analysis via a dedicated mobile application. This integrated system enables “sample-to-answer” detection within 30 min directly from whole blood, achieving a limit of detection of 2.5 copies/μL of both HIV and TP and demonstrating 100% sensitivity and specificity concordant with the real-time PCR in 51 cases of clinical samples. This work demonstrates a transformative point-of-care diagnostic platform that combines minimal user steps, equipment independence, and robust multiplex detection for decentralized pathogen testing.