Development of an Integrated Sample Amplification Control for Point-of-Care Pathogen Testing

Introduction:: The COVID-19 pandemic has led to an increase in point-of-care (POC) and home-based tests available, but this also raised concerns about the accuracy and effectiveness of the tests, as well as the future of at-home diagnostics for other infectious diseases. While many emerging publications on POC nucleic acid amplification tests (NAATs) detecting SARS-CoV-2 show remarkable analytical sensitivity in the lab, clinical accuracy associated with false-negative and false-positive results is often overlooked. False tests can occur due to inappropriate sample collection, degradation of analysts in samples, and inhibitors in human specimens. An internal amplification control (IAC) is commonly run in parallel to NAATs pathogen targets in the lab, in order to ensure clinical accuracy. However, the few publications on POC NAATs that do include IACs have run the IAC separately from the test for viruses. Here, we developed a one-pot duplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2 RNA and IAC target -human 18s ribosomal RNA- in saliva samples. This assay was designed to run in an amplification chamber on our remodeled Microfluidic rapid and autonomous analytical device (MicroRAAD) [1], a fully automated device that integrates RT-LAMP and a lateral flow immunoassay (LFIA) readout (Fig. 1A).


Materials and Methods:: The optimized RT-LAMP reactions were performed according to the New England Biolab standard by using WarmStart RT-LAMP 2X Master Mix and two sets of 10x Primer Mix targeting the SARS-CoV-2 Orf1ab gene and human 18s rRNA. The loop primers of SARS-CoV-2 primer were labeled with biotin and fluorescein, respectively, whereas the loop primers of IAC were conjugated to biotin and digoxigenin, allowing result readout on 3-line LFIA strips. To determine the limit of detection (LOD) or analytical sensitivity of the assay, various concentrations of SARS-CoV-2 viral particles (vps) ranging from 0 to 5000 SARS-CoV-2 vps/µL were spiked into saliva. 5 µL of sample and 20 µL of mastermix were incubated at 65°C for 30 minutes. The duplex RT-LAMP assay was then blinded validated against 30 de-identified patient samples obtained from the Indiana Biobank, and directly amplified without prior RNA extraction. Clinical sensitivity and specificity calculations were evaluated from the results of triplicate reactions on each sample.


Results, Conclusions, and Discussions:: The duplex reaction demonstrated the multiplexing capabilities of 18s rRNA and the SARS-CoV-2 RNA in saliva samples. The successful amplification of SARS-CoV-2 spiked water, saliva without viral particles and SARS-CoV-2 spiked saliva were shown on the gel electrophoresis (Fig. 1B left). The LFIAs demonstrated differentiation between saliva (with 18s rRNA) and water matrix (without 18s rRNA) and SARS-CoV-2 spiked into each matrix. The two lines (flow control and IAC lines) represent valid negative results and three lines (flow control, IAC, and test lines) represent valid positive results (Fig. 1B, right). The optimized assay enabled the rapid and sensitive detection of SARS-CoV-2 down to 100 viral copies per mL of saliva within 30 minutes without RNA extraction (Fig. C). The developed assay correctly identified 20 of the 21 RT-qPCR–positive samples and accurately detected all 9 SARS-CoV-2 negative specimens.

We demonstrated the integration of sample collection control for the POC test and the target pathogen by developing the duplex RT-LAMP for SARS-CoV-2 and IAC. The duplex assay was successfully amplified in a one-pot reaction without cross-reactivity and exhibited the analytical sensitivity of 100 vps/µL. The assay achieves 95% clinical sensitivity and 100% clinical specificity. Further, the assay successfully amplified using our self-regulating positive temperature coefficient (PTC) microheaters demonstrating compatibility with our remodeled microRAAD.


Acknowledgements (Optional): : Funded in part by NIH NIDA DP2DA051910, Moore Inventor Fellowship #9687.


References (Optional): : [1] E.A. Phillips, T. Moehling, K.F.K. Ejendal, et al. Lab on a Chip 19, 3375-3386 (2019).