A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection

Abstract

A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13 Assisted Saliva-based & Smartphone Integrated Testing), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.

Keywords: COVID-19; CRISPR Diagnostics; Crispr-Cas13a; SARS-CoV-2; saliva.

Figure 1

Validation of saliva-based detection of SARS-CoV-2 in clinical samples (A) Standardization of SARS-CoV-2 specific primer pairs for S and N gene. Primers are labeled as S-P1 to S-P-4 for S gene and N-P1 to N-P-3 for N gene, N1 represents the CDC approved primer for N gene. Dotted line indicates lower level of detection; ND indicates not detected (B) Determination of limit of detection using RNA of S spiked into RNase free water with serial dilutions. (C) RNA extracted from four normal saliva samples was used at various copy numbers to find any interference for detection of spiked-in SARS-CoV-2 S gene RNA. (D) Heat map of 105 saliva samples showing RT-qPCR results represented as Ct values for E, N, RdRp and S genes respectively. The test results of these samples were validated by the hospital using swab samples, and represented as Swab. IC is the internal control. Green boxes represent the samples with Ct values not detected and were labeled as SARS-CoV-2 negative, and yellow boxes indicate swab samples positive for SARS-CoV-2. (E1-E3) Shows the Ct value comparison between S gene with E, RdRp, and N gene respectively. (F1-F3) Shows the spearman correlation of Ct values between S gene with E, RdRp, and N gene respectively.

Figure 2

RNA extraction-free detection of SARS-CoV-2 in saliva samples (A) Heat map of Ct values obtained with 10⁵ copies of S gene standard RNA spiked into normal saliva and subjected to various heat inactivation and chemical treatments. PK: Proteinase K; NAC: N-acetyl cysteine and TX100: Triton X-100. Saliva samples with water was used instead of spiked-in RNA as negative control, which showed no detectable Ct value; blue boxes. (B) Standard curve showing various dilutions of S gene RNA spiked into normal saliva to obtain the LoD. (C1, C2) Comparison of Ct detected when S gene RNA was spiked into the saliva samples of eight SARS-CoV-2 negative volunteers vs the same spiked into water as control. (D) Optimization of various RNA storage agents like RNAlater, guanidinium hydrochloride (GuHCL) and without RNA storage agent for detection of SARS-CoV-2 in eight clinical saliva samples. (E) Comparison of N and S gene amplification in saliva samples after undergoing heat and chemical denaturation. (F) Heat map of Ct values obtained for N gene for 76 samples with RNA extraction (N-RNA_Ex) and RNA extraction-free (N-RNA_ExF) method. Human RNaseP (RP) was used as the experimental control to find the RNA integrity of the samples used. Green boxes represent the samples with not detected Ct values. (G) Shows the individual Ct values of N-RNA-Ex and N-RNA_ExF along with the RNaseP with dotted line indicating lower level of detection. (H) Correlation of Ct values between N-RNA_Ex and N-RNA_ExF method (I) Median of Ct values of two methods as indicated by solid lines. The dotted line represents lower Ct value below which samples were labeled as not detected (ND). (J) Comparison of Ct values obtained from saliva samples when stored at room temperature (RT) for 6 h with same samples processed without storage (direct).

Figure 3

Optimization of SHERLOCK-based detection on extraction free saliva samples (A) Schematic representation of various steps involved in SHERLOCK-based detection when the starting genetic material is RNA. Cas13a enzyme is used for the target recognition and reporter cleavage. For visual detection using LFA, RNA reporter molecule conjugated with 6-Carboxyfluorescein (FAM) and biotin is used. (B) Images of paper-strips after lateral-flow assay obtained from spiked-in saliva samples using S gene standard RNA with a range from 0 to 4, 000 copies of RNA/reaction. A consistent detection of test lane signal was obtained in all three samples with 200 copies of RNA, which was considered as LoD for visual readout. (C) Similarly, Orf1ab standard RNA was subjected to LFA and paper-strip images were obtained. The LoD for Orf1ab was found to be higher than S gene at 400 copies/reaction.

Figure 4

Validation of SHERLOCK on RNA extraction-free saliva samples (A) RNA extraction-free samples were used for the detection with SHERLOCK-based method using visual lateral-flow. Samples were divided into five groups, based on the Ct values with Group-I Ct below 25; Group-II Ct between 26-30, Group-III Ct between 31-35, Group-IV Ct above 36, and Group-V Ct not detected (ND). The LFA images of samples with respective Ct values is shown along with the patient ID corresponding to samples in (Figure 2). (B) Representative images of the seven paperstrips with 200, 100, 50, and 0 RNA copies spiked-in to the saliva samples and subjected to SHERLOCK. (C) To obtain semi-quantitative analysis of the data, 10 images of paper-strips corresponding to 200, 100, 50, and 0 RNA copies as shown in Figure 3B and Figure 4B, were subjected to image quantitation. The threshold value (T/C ratio) was obtained based on the signal in the control (C) and test (T) lane. The T/C ratio was considered positive above the background value of 0.15. Based on T/C ratio, the detection sensitivity was found to be 80% with100 copies of RNA and 100% above 200 copies of RNA per reaction. (D) Similarly, visual results of paper-strips shown in Figure 4A, were subjected to signal quantitation and T/C ratio was calculated. One sample (blue arrow head, ID: 39), which was difficult to characterize by visual detection, was correctly characterized as positive using T/C ratio (blue arrow head).

Figure 5

Schematic representation of the CASSPIT workflow (A) Self-collection of saliva samples by the patient in a sample collection tube which contains the RNA release chemical agents. (B) The samples will be subjected to heat inactivation to inactivate the virus and simultaneously release the viral RNA. (C) Released RNA will be transferred into one-pot or two-pot SHERLOCK master mix tube to amplify the signal with RT-RPA and detect the target by Cas13a. After target detection, activated Cas13 will cleave the reporter. (D) Paper-strips will be immersed in the SHERLOCK reaction mix and subjected to LFA to obtain the visual results. (E) Using mobile phone camera, images of the paper-strip will be captured and subjected to processed using the app. Test results will be provided based on the signal detection in Test and Control lanes of the paper-strip. (F) Further the app will have a provision to store the images, test results and help with online assistance if needed. The overall workflow with saliva as test samples is named as Cas13 Assisted Saliva-based & Smartphone Integrated Testing (CASSPIT).