Development of a Disposable dLAMP Chip for Respiratory Syncytial Virus RNA Quantification
YUHUI GUO, Deepak Sapkota, Zhenpeng Qin, Hui Ouyang, The University of Texas at Dallas
Abstract Number: 367
Working Group: Bioaerosols
Abstract
Nucleic acid testing is widely used in the bioaerosol field to identify and quantify airborne pathogens collected by bioaerosol samplers. PCR is the most commonly used method for detecting viral RNA; however, it typically requires several hours to complete, regardless of the type, RT-PCR, qPCR, or digital PCR. Digital LAMP (Loop-Mediated Isothermal Amplification) offers a faster alternative, completing reactions within 60 minutes and detecting RNA concentrations as low as 10 copies/µL. However, the chip used in dLAMP is expensive and has currently been discontinued. In this study, a membrane-based chip for dLAMP RNA quantification has been developed that eliminates the need for complex fabrication or specialized equipment. A track-etched polycarbonate (PCTE) membrane with uniform pore size and cylindrical structure was purchased from STERLITECH, costing less than $1 per membrane. Each pore serves as an individual nanoreactor for DNA or RNA amplification. The total number of pores, averaging 6,340 ± 20, was verified using a ZEISS Axio Observer microscope. The membrane was pretreated to remove polyvinylpyrrolidone. The final chip configuration consists, from bottom to top, of a glass slide, the membrane, a polycarbonate spacer ring, heat-resistant double-sided tape, and another glass slide. Mineral oil is used to prevent sample evaporation. Calibration was performed using serial dilutions of RSV A2 quantitative RNA (ATCC VR-1540DQ, from 106 to 100 copies/µL), yielding a linear response with R² = 0.97. This approach can be adapted for quantitative nucleic acid testing of a wide range of airborne viral and bacterial targets, providing a rapid and accurate method for bioaerosol analysis.