A smartphone-read ultrasensitive and quantitative saliva test for COVID-19

Optimization of CRISPR-FDS for extraction-free detection of SARS-COV-2 RNA in saliva

A saliva-based point-of-care COVID-19 assay should, ideally, function as a single-step assay that can be read without any additional equipment or infrastructure, while retaining high sensitivity (fig. S1). Because CRISPR-FDS has high sensitivity using a simple workflow that requires minimal equipment, we used its assay protocol as a starting approach and optimized aspects of its lysis procedure (sample-to-buffer ratio, and incubation temperature and time) and RT-PCR CRISPR reaction [Cas12/guide RNA(gRNA)–to–probe ratio, and reaction temperature and time] to improve workflow and performance (Fig. 1A).

Most high-sensitivity NAA assays analyze purified RNA samples isolated in multistep procedures that require additional laboratory equipment. This additional step must be eliminated to allow the development of an integrated assay. We thus optimized the viral lysis procedure to determine the conditions that would allow viral lysis samples to be directly analyzed by CRISPR-FDS without a separate isolation step, using a cell lysis procedure compatible with PCR as the base condition. CRISPR-FDS exhibited robust performance when healthy donor saliva samples spiked with SARS-CoV-2 were mixed with increasing lysis buffer volumes (1:1 to 1:10, saliva to buffer) before standard heat denaturation (65°C for 5 min) and direct analysis by CRISPR-FDS (Fig. 1B). SARS-CoV-2 RNA signal increased with lysis buffer concentration up to a 1:5 ratio. However, signals detected at the 1:1 and 1:10 ratios were similar and ~40% less than the signal detected at the 1:5 ratio, indicating that CRISPR-FDS results were not markedly affected by the sample-to-buffer ratio. Given this robust response, we elected to use a 1:1 sample-to-buffer ratio for all further studies.

Because temperature control can be a notable factor in point-of-care assays, we next analyzed the effect of lysis temperature on CRISPR-FDS signal. No signal was detected when lysis reactions were performed at room temperature (~22°C), similar CRISPR-FDS signal was detected at 37° and 65°C, the manufacturer-recommended lysis temperature, with the most signal detected at 95°C (Fig. 1C). We next examined the effect of lysis time on CRISPR-FDS signal when using a 1:1 saliva-to-buffer ratio at 65°C and found that signal increased with incubation time out to the final 15-min time point (Fig. 1D). Given that there was no difference between 65° and 37°C lysis, we elected to use 37°C for the lysis temperature, with the assumption that this minimum temperature would be the simplest to achieve, maintain, and measure in most point-of-care settings, although temperature deviations above this minimum would not negatively influence the lysis reaction. We also chose to use a 5-min lysis time to minimize overall assay performance time.

We next evaluated CRISPR-FDS saliva assay performance at 42°C, the maximum recommended temperature for Cas12a reactions, varying reaction time and Cas12a/gRNA-to-probe ratio to determine the optimal conditions. Increasing probe concentration enhanced CRISPR-FDS signal at 37°C but increased reaction completion time, with 1:20 and 1:25 ratios requiring 20 and 40% longer, respectively, than 1:15 assays, while producing 35 and 70% more signal (Fig. 1E). Reducing the CRISPR-FDS reaction temperature increased assay completion times but did not alter the final signal, with CRISPR-FDS assays performed at 42°, 37°, and 22°C reaching completion after 11, 15, and 27 min of incubation, respectively (Fig. 1F). CRISPR-FDS signals detected after 10 min at 42°, 37°, and 22°C reached 97, 84, and 50% of the signal detected at reaction completion, respectively, allowing robust detection over a broad temperature range at this time point. We next evaluated the specificity of our RT-RPA-CRISPR assay using 39 negative control samples that each contained >10⁶ genome copies/ml of different bacterial/viral/fungal species that can cause respiratory infections, including influenza A and B and respiratory syncytial virus (RSV). No positive results were detected for any of these negative control samples in triplicate assays (table S3). Individual CRISPR-FDS assays could be generated for each of these respiratory infections by replacing the gRNA used in the current assay with gRNAs that recognize gene targets specific for each of these pathogens. Multiplex detection within a signal assay reaction is not feasible, however, because CRISPR-FDS uses the nonspecific single-strand DNA cleavage activity of the bound Cas12a/gRNA-DNA complex to derepress the fluorescent signal from a quenched oligonucleotide probe.

On the basis of these results, we next examined the diagnostic potential of a rapid saliva-based CRISPR-FDS assay in which lysis (5 min; 1:1 sample to buffer) and CRISPR (10 min) incubation steps were performed at 37°C, using a 1:25 CRISPR/gRNA-to-probe ratio. CRISPR-FDS assay results obtained upon saliva from a patient with confirmed COVID-19 and from a healthy individual indicated that this assay can readily distinguish between these individuals (Fig. 1G).

Evaluation of a saliva-based CRISPR-FDS assay for quantitative COVID-19 diagnosis

Next, we analyzed the linear range and other properties of the optimized saliva-based CRISPR-FDS COVID-19 assay using a standard curve generated by spiking a known amount of heat-inactivated virus into healthy donor saliva and performing serial dilutions in healthy donor saliva. Results from this analysis demonstrated good linearity (R² = 0.90) across a broad range of virus concentrations (1 to 10⁷ copies/μl; Fig. 2A), with an LOD (24) of 0.05 copies/μl. Subsequent CRISPR-FDS analysis of 20 replicate samples containing 0.05, 0.1, and 0.25 copies/μl detected positive signal in all samples, suggesting that the actual LOD is <0.05 copies/μl (Fig. 2B). CRISPR-FDS demonstrated complete concordance with RT-qPCR when analyzing saliva aliquots spiked with or without virus, at concentrations matching 1× to 2× the LOD (0.05 and 0.1 copies/μl, respectively) of the CRISP-FDS assay (Fig. 2C). PPA and negative PA (NPA) estimates remained high after adjusting for sample size [100%; 95% confidence interval (CI), 85.8 to 100]. Comparison of CRISPR-FDS results for paired saliva and nasal swab samples obtained from a cohort of 31 individuals screened for COVID-19 (Fig. 2D) indicated that viral loads were similar in these samples and demonstrated reasonable correlation (r = 0.8029; P < 0.0001), and SARS-CoV-2 RNA levels remained stable in saliva stored at 4°C for up to 7 days after collection (fig. S2).

Saliva virus levels are elevated throughout infection in a rhesus macaque model of COVID-19

Saliva from patients with COVID-19 contains SARS-CoV-2 RNA (25–27), and saliva and nasal samples exhibit high PPA when analyzed within 7 days of COVID-19 symptom onset, which declines thereafter, with an indication that saliva samples may remain positive longer (28). Given the uncertainty involved in human exposure events, it is not possible to evaluate the relative diagnostic use of saliva and nasopharyngeal swab samples early in infection. To address this question, we analyzed paired nasal and oropharyngeal (saliva analog) swabs obtained from seven nonhuman primates before and after SARS-CoV-2 infection. CRISPR-FDS determined that mean SARS-CoV-2 RNA levels were 3.6-fold to 124-fold higher, and more stable, in oropharyngeal versus nasal swab samples at all time points after infection (Fig. 3A). These data, and limited human data (28), suggest that saliva may represent a more robust diagnostic sample than nasal swabs both early and later in infection.

Anecdotal evidence also suggests that saliva CRISPR-FDS results may have greater diagnostic sensitivity than nasopharyngeal swab RT-qPCR results, as illustrated by a COVID-19 patient whose nasal and nasopharyngeal swabs tested negative by RT-PCR but whose saliva tested positive by CRISPR-FDS (29). In this case, a 53-year-old woman with Philadelphia-negative B cell acute lymphoblastic leukemia presented with fever, shortness of breath, cough, and tachycardia. A chest x-ray revealed bilateral lung infiltrates that were confirmed by a chest computed tomography scan that revealed ground glass opacities suggestive of COVID-19 (Fig. 3B). However, the nasal and nasopharyngeal RT-PCR swab tests (Abbott and Roche) conducted as her respiratory status deteriorated to require 4 liter/min oxygen via nasal cannula were negative for SARS-CoV-2. Additional infectious causes were ruled out using the BioFire respiratory panel, serum fungal markers, and serum tests for cytomegalovirus, Epstein-Barr virus, adenovirus, Legionella, and Cryptococcus. Continued suspicion of COVID-19 led to investigational RT-PCR CRISPR-FDS testing of her saliva, which was determined to be SARS-CoV-2 positive (Fig. 3C). This patient was transferred to the COVID-19 ward for isolation, where she received one unit (200 ml) of COVID-19 convalescent plasma. Within 24 hours, she reported improvements in shortness of breath and cough, exhibited fever resolution, and reduction in her oxygen requirements leading to her eventual discharge without supplemental oxygen. This report, albeit anecdotal, supports the ability of a saliva CRISPR-FDS assay to detect COVID-19 cases missed by RT-PCR assay. In this case, the CRISPR-FDS saliva assay results were instrumental in identifying the false-negative RT-PCR results, which allowed rapid initiation of SARS-CoV-2–directed therapy with convalescent plasma to reverse COVID-19 pathology.

Subsequent CRISPR-FDS analysis of 103 paired saliva and nasal swab samples obtained from individuals screened for COVID-19 detected SARS-CoV-2 RNA in more saliva than nasal swab samples (44 versus 28, Fig. 3D). Viral load was strongly correlated in the 24 individuals with two positive samples (Fig. 3E).

Design and diagnostic performance of the COVID-19 CRISP-FDS saliva assay read on a chip

These results support the hypothesis that saliva is a valid specimen for COVID-19 diagnosis and can yield results comparable or superior to those obtained with nasopharyngeal or nasal swab samples to reduce the materials, expertise, and infrastructure required to for sample collection. However, this assay still requires a benchtop fluorescence reader for assay evaluation and quantification, limiting its use to sites equipped with such devices. Smartphone-based devices have become an appealing handheld platform to read multiple different assay types (30–34), and we hypothesized that a smartphone device could similarly allow sensitive and quantitative readout of saliva CRISPR-FDS assays at sites lacking basic laboratory equipment or infrastructure.

Such an approach would require that the CRISPR-FDS assay be reformatted to be easily read by a smartphone camera. To address this issue, we designed and fabricated a proof-of-concept compact assay chip (25 × 35 × 4 mm) suitable for an on-chip CRISPR-FDS saliva assay inserted into a smartphone device functioning as a fluorescence microscope for capture by the field of view (FOV) of its camera. This prototype chip consisted of a layer of polydimethylsiloxane (PDMS) mounted on a glass microscope slide. PDMS was selected for this application because it is a chemically inert and optically clear silicone elastomer that can spontaneously adhere to glass surfaces after plasmonic oxidation, because it allows assay wells to be excited by low-angle laser illumination, and because this inexpensive PDMS/glass format (∼$0.7 per chip) can be readily modified to improve assay functionality.

Our chip design contained five reaction wells [internal diameter (I.D.) = 3.5 mm, maximum volume ≈ 28 μl] to allow the analysis of five assays in parallel (e.g., three test wells, one PC well, and one NC well), where wells were designed to contain sufficient volume for sensitive detection. Reaction wells were arranged in a pentagonal array illuminated by a laser diffused to cover the ~20 × 20 mm FOV of the smartphone camera (Fig. 4, A to C). A pentagonal array was chosen to minimize illumination differences in our proof-of-concept experiments; more compact arrays containing more wells could be used to simultaneously analyze samples from multiple individuals and/or to accommodate a standard curve to quantify viral load. Alternate designs could also use microfluidic channels to load multiple wells from a single inlet port and use film to seal the chip after sample loading to prevent environmental contamination by assay amplicons. To verify the use of this chip design, we used an on-chip CRISPR-FDS assay to analyze saliva from 12 patients with COVID-19 and 6 healthy controls. Images from this proof-of-concept assay approach that were captured and analyzed by a fluorescence microplate reader (Fig. 4, D and E) distinguished saliva from patients with positive and negative nasal RT-qPCR results, supporting the feasibility of this on-chip approach for COVID-19 diagnosis.

Analytical and diagnostic performance of on-chip assays read by a smartphone device

We next developed a smartphone fluorescence microscope device to read the on-chip CRISPR-FDS assay. This device incorporated a smartphone socket, external lens filter, laser diode powered by AAA batteries, a power switch, chip slot, and an emission filter for the smartphone camera (Fig. 5A). This integrated system was designed to use a straightforward workflow, in which a typical saliva volume of 0.5 to 3 ml (35) is collected in a tube prefilled with 3 ml of lysis buffer, which is then capped and heated at >37°C for ≥5 min, after which ~5 μl of the lysed sample is added to each sample well of an assay chip containing 10 μl per well of premixed RPA and CRISPR solution. This chip is then incubated for ≥10 min at room temperature and then inserted into the smartphone reader, the laser diode is turned on, and assay chip images are captured by the smartphone camera (Fig. 5B).

The FOV of this device was increased relative to previous smartphone microscope designs (36) by adding an external lens with a 50-mm focal length. This yielded an FOV compatible with the diameter of the reaction well array on our assay chip without producing notable aberration. This device also used a 100-mW laser diode with a high-incidence angle to allow sensitive detection of reaction products while minimizing background noise (Fig. 5C and fig. S3).

We examined the analytical performance of this device by analyzing on-chip assays of a SARS-CoV-2 RNA concentration curve generated serially diluting heat-inactivated SARS-CoV-2 virus in healthy donor saliva. This standard curve demonstrated good linearity (R² = 0.91) over a broad viral concentration range (1 to 10⁵ copies/μl) and a calculated LOD of 0.38 copy/μl when read on the smartphone device (Fig. 5D) (24). We then used both CRISPR-FDS and RT-qPCR to blindly analyze 103 saliva samples from individuals screened for COVID-19 (Fig. 5E) and found that the CRISPR-FDS plate reader and smartphone assays and the standard RT-qPCR assay detected similar numbers of SARS-CoV-2–positive saliva samples (43 versus 44) (table S1). In an analysis using RT-qPCR as the reference standard (table S1), CRISPR smartphone results exhibited a 1.3% false-positive rate with saliva but complete concordance with RT-qPCR results for swab samples, while CRISPR plate reader results perfectly matched RT-qPCR saliva results, but exhibited a 2.3% false-negative rate with nasal swab samples. Viral load was strongly correlated in the 43 saliva samples that tested positive by both the on-chip smartphone assay and conventional RT-PCR analysis (Fig. 5F) and exhibited similar mean values (3803 versus 1797 copies/μl).