pH-EVD: A pH-Paper-Based Extraction and Visual Detection System for Instrument-Free SARS-CoV-2 Diagnostics

Abstract

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths worldwide. However, most SARS-CoV-2 detection methods depend on time-consuming sample preparation and large detection instruments. Herein, a method employing nonbleeding pH paper to achieve both RNA extraction and visual isothermal amplification is proposed, enabling rapid, instrument-free SARS-CoV-2 detection. By taking advantage of capillary forces, pH-paper-based RNA extraction can be accomplished within 1 min without need for any equipment. Further, the pH paper can mediate dye-free visual isothermal amplification detection. In less than a 46-min sample-to-answer time, pH-paper-based extraction and visual detection (termed pH-EVD) can consistently detect 1200 genome equivalents per microliter of SARS-CoV-2 in saliva, which is comparable to TaqMan probe-based quantitative reverse transcription PCR (RT-qPCR). Through coupling with a chemically heated incubator called a smart cup, the instrument-free, pH-EVD-based SARS-CoV-2 detection method on 30 nasopharyngeal swab samples and 33 contrived saliva samples is clinically validated. Thus, the pH-EVD method provides simple, rapid, reliable, low-cost, and instrument-free SARS-CoV-2 detection and has the potential to streamline onsite COVID-19 diagnostics.

Keywords: RNA extraction; SARS-CoV-2; instrument-free onsite diagnostics; nonbleeding pH paper; visual isothermal amplification detection.

Figure 1

Schematic of instrument‐free SARS‐CoV‐2 detection enabled by pH‐paper‐based RNA extraction and visual detection (termed pH‐EVD).

Figure 2

Development and characterization of the pH‐paper extraction system for rapid, instrument‐free nucleic acid preparation. A) Assembly of the pH‐paper extraction system in a 3D‐printed clamshell device. B) Time course of liquid transportation during three sequential liquid loadings. Red dye was added for visual purposes. C) Example SEM images of nonbleeding pH paper.

Figure 3

Visual detection of isothermal amplification by direct insertion of nonbleeding pH paper disc into the reaction solutions. A) Schematic of RT‐DAMP. B) Hydrogen ions produced during primer extension in isothermal amplification. C) Time course of the pH paper color change and the EvaGreen‐based real‐time fluorescence detection. Positive, reactions with 6.8 × 10⁴ copies of SARS‐CoV‐2 RNA. NTC, reactions without any template. D) Specificities of pH‐paper‐based visual detection and real‐time fluorescence detection. SARS‐CoV‐2 PC, SARS‐CoV control, MERS‐CoV control, and Hs_RPP30 PC are commercial plasmids with corresponding gene sequences (Integrated DNA Technologies). E) Sensitivities of pH‐paper‐based visual detection and real‐time fluorescence detection. Various copies of commercial SARS‐CoV‐2 RNA control from Twist Bioscience were used. The visual detection was conducted with a 40 min incubation. Each experiment involving real‐time fluorescence detection and tube‐based visual detection was repeated three times.

Figure 4

Optimization of pH‐paper‐based RNA extraction for visual isothermal amplification detection. A) Effect of various diameters of pH paper disc on extraction performance. In this experiment, 28 μL sample, 112 μL Buffer AVL, 112 μL absolute ethyl alcohol, 112 μL Buffer AW1, and 112 μL Buffer AW2 from the commercial QIAamp Viral RNA Mini Kit (QIAGEN) were used. Buffer AVL is a lysis buffer. Buffer AW1 and AW2 are two wash buffers. B) Effect of varying the amounts of the extraction reagents on extraction performance. The ratio of Buffer AVL, absolute ethyl alcohol, Buffer AW1, and Buffer AW2 strictly follows the kit's instructions. Heat‐inactivated SARS‐CoV‐2 was provided by BEI Resources. Three replicates (Assays 1–3) were set up for each test with the heat‐inactivated SARS‐CoV‐2.

Figure 5

Performance of the pH‐EVD method using various amounts of heat‐inactivated SARS‐CoV‐2. A) Procedures of the pH‐EVD method and the parallel assays including direct pH‐paper‐based visual detection, extraction‐free colorimetric RT‐DAMP using cresol red, and extraction‐free TaqMan probe‐based RT‐qPCR. B) Comparison of visual detection results. C) Percent positives for various assays. All of the visual detections were incubated for 40 min. GE, genome equivalents. NTC, solutions without any heat‐inactivated SARS‐CoV‐2. Three independent experiments (Assays 1–3) were set up for each test group.

Figure 6

Performance of the pH‐EVD method on detecting various amounts of heat‐inactivated SARS‐CoV‐2 spiked in human saliva. A) Procedures of the pH‐EVD method with lysate and heat‐treatment of saliva, and the parallel assays with heat‐treated saliva for direct pH‐paper‐based visual detection, extraction‐free colorimetric RT‐DAMP using cresol red, and extraction‐free TaqMan probe‐based RT‐qPCR. B) Comparison of visual detection results. C) Percent positives for various assays. All the visual detections were incubated for 40 min. GE, genome equivalents. NTC, solutions without any heat‐inactivated SARS‐CoV‐2. Three independent experiments (Assays 1–3) were set up for each test group.

Figure 7

Clinical validation of rapid instrument‐free SARS‐CoV‐2 detection by the pH‐EVD method. A) Procedures of the instrument‐free detection method and the routine RT‐qPCR assay for testing clinical NP samples and contrived saliva samples. B,C) C _q values of the 33 saliva samples and 30 clinical NP samples by RT‐qPCR following spin column‐based extraction, respectively. D,E) Visual detection results of the instrument‐free pH‐EVD method for the saliva and NP samples, respectively. F) Confusion matrix describing the overall performances of the two assays between positive and negative samples. The cutoff of C _q was 40. The spin column‐based extraction and RT‐qPCR are considered as the standards. The samples marked with red were tested to be negative by the pH‐EVD method, while positive by RT‐qPCR. Each experiment was repeated three times.