key: cord-0958036-b0cwjvjq
authors: Kumar, Vinod; Mishra, Suman; Sharma, Rajni; Agarwal, Jyotsna; Ghoshal, Ujjala; Khanna, Tripti; Sharma, Lokendra K.; Verma, Santosh Kumar; Mishra, Prabhakar; Tiwari, Swasti
title: Development of RNA-based assay for rapid detection of SARS-CoV-2 in clinical samples
date: 2020-09-18
journal: bioRxiv
DOI: 10.1101/2020.06.30.172833
sha: d8eb3b0bd3a4499bb20e22b8607a264cc40b3e29
doc_id: 958036
cord_uid: b0cwjvjq

The ongoing spread of pandemic coronavirus disease (COVID-19) is caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). In the lack of specific drugs or vaccines for SARS-CoV-2, demands rapid diagnosis and management are crucial for controlling the outbreak in the community. Here we report the development of the first rapid-colorimetric assay capable of detecting SARS-CoV-2 in the human nasopharyngeal RNA sample in less than 30 minutes. We utilized a nanomaterial-based optical sensing platform to detect RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2, where the formation of oligo probe-target hybrid led to salt-induced aggregation and changes in gold-colloid color from pink to blue in visible range. Accordingly, we found a change in colloid color from pink to blue in assay containing nasopharyngeal RNA sample from the subject with clinically diagnosed COVID-19. The colloid retained pink color when the test includes samples from COVID-19 negative subjects or human papillomavirus (HPV) infected women. The results were validated using nasopharangeal RNA samples from suspected COVID-19 subjects (n=136). Using RT-PCR as gold standard, the assay was found to have 85.29% sensitivity and 94.12% specificity. The optimized method has detection limit as little as 0.5 ng of SARS-CoV-2 RNA. Overall, the developed assay rapidly detects SARS-CoV-2 RNA in clinical samples in a cost-effective manner and would be useful in pandemic management by facilitating mass screening.

Coronavirus disease is rapidly spreading across the world and raising severe global health 50 concerns. In December 2019, China reported the first disease case in its Hubei Province. Based 51 on the phylogenetic analysis, the identified novel coronavirus is named as Severe Acute 52

Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and the disease spread by SARS-CoV-2 53 is known as "COVID-19", declared as a pandemic by World Health Organization 54 (WHO)(https://www.who.int/emergencies/diseases/novel-coronavirus-55 2019?gclid=EAIaIQobChMI-u-Pz9vW6QIVUyUrCh0kjAlOEAAYASAAEgJ0pvD_BwERemuzzi). 56

Despite global massive efforts to control the outbreak of COVID-19, this pandemic is still on the 57 rise. To date, lack of approved medicine or vaccine impede escalated the management of the 58 positive patients or IVT RNA. The RdRp oligo probe (5'-GTGATATGGTCATGTGTGGCGG-3') 126 was used to specifically detect the presence of SARS-CoV-2 RNA in the assay. In parallel, to 127 measure the specificity of the assay, input genomes from different source such nasopharyngeal 128 RNA from COVID negative subjects and HPV DNA from cervical cancer positive samples were 129 used as negative controls. Similarly, non-template control (NTC) was also included to measure 130 the background reactivity. In addition, to confirm the working principle of the assay, a different 131 RNA template (isolated from pancreas) and pancreas specific REG-3 (Regenerating islet-132 derived protein 3) gene oligo probe (5'-GTGCCTATGGCTCCTATTGCT-3') were used 133 separately. 134

The final reaction mixture with above combinations was then denatured at 95°C for 30 135 seconds, annealed at 60°C for 60 seconds and then cooled to room temperature for 10 minutes. 136

Subsequently, 10 nM colloidal AuNPs (~10 nm) were added to the assay mixture and allowed to 137 develop color for 1-2 minute. 138

Spectral studies and measurement of sensitivity: Absorption spectrum of the assay mixture 139 was recorded in the range of 300 -700 nm. The peak shift from 520 nm (known as red-shift) and 140 peak broadening after 520 nm were measured as a characteristic feature of the salt induced 141 aggregation. Using various combinations of positive and negative controls (as discussed in the 142 previous section) the specificity of reaction and aggregation were compared. Assay sensitivity 143 was determined by serially diluting the input SARS-CoV-2 RNA from both the IVT synthesized 144 and, synthetic SARS-CoV-2 control (nCov19 control kit by Applied Biosystems) ranging from 5-145 0.1ng and 1-0.1ng concentrations respectively. 146

Statistical Analysis: Diagnostic accuracy of the new colour test was calculated by assuming 147 RT-PCR as gold standard method. Sensitivity (True Positive rate), Specificity (True Negative 148 rate) and overall accuracy (True positive and true negative rate) were calculated with 95% 149 confidence interval. Likelihood ratio positive (sensitivity / false positive rate), Likelihood ratio 150 negative (false negative rate / specificity), Positive predictive value (True positive value / Total positive results predicted by colour test), Negative predictive value (True negative value / Total 152 negativeresults predicted by colour test) were also calculated. Measured accuracy was 153 considered statistically significant (p<0.05) when 50% did not falling within the confidence limit 154 for values given in % whereas LR are considered significant when 1 was falling within 155 confidence limit. Statistical analyses were performed using software MedCalc for 156

Windows(MedCalc Software, Ostend, Belgium). 157

In this study, we report for the first time the development of a rapid and affordable RNA-based 159 assay for the visual detection of the SARS-CoV-2 genome in human samples. Using RT-PCR 160 as gold standard, the developed assay was found to have a sensitivity of 85.29% and 161 sepcififcity of 94.12%. For the assay, we use surface plasmon resonance property of gold 162 nanoparticles/colloids (AuNP) and targeted the RdRp specific gene sequence of SARS-CoV-163 2. RdRp is essential for viral replication and has higher analytical power than E (envelope 164 protein) and N (nucleocapsid protein) genes of SARS-CoV-2 (Corman et al. 2020). Our current 165 established assay, salt-induced aggregation and color change of the gold colloids occurs 166

after RdRp oligo probe hybridizes with its specific target RNA of SARS-CoV-2. With the current 167 escalated demand of cost effective, easy and sensitive diagnostic for COVID-19, the test was 168 developed using commercially available nCoV19 synthetic DNA and validated further using 169 clinical samples from COVID-19 subjects (as confirmed using Taqman based RT-PCR method, 170 Table S1 ). In our study, we demonstrated a visual change in gold colloid color from pink to blue 171 when RNA samples from subjects with clinically diagnosed COVID-19 infection hybridize 172

with RdRp oligo probe. Simultaneously, the color remained pink in SARS-CoV-2 negative 173 samples due to the absence of hybridization. 174

In the study, out of 136 samples, 50% samples (n=68) were true positive (COVID-19 Positive) 175 and 50% samples (n=68) true negative (Free from COVID-19 disease) confirmed by gold 176 standard diagnostic test RT-PCR. These true positive and negative samples were again tested by New Colour test to assess the diagnostic accuracy of our new test ( In this assay, SARS-CoV-2 RNA from human patients or IVT synthesized RNA was added into 209 hybridization buffer (containing oligo probe), followed by denaturation and annealing at 95°C 210 (30s) and 60°C (60s), respectively. After cooling at room temperature, the gold colloid was 211 added into the above reaction mixture. The colloid color changes in visible range from pink to 212 blue, indicate the formation of hybridized product (Figure 2b -c, and Figure 3 ). Broadening of the 213 peak with red-shift (~30 nm) was observed in the spectrum of aggregated colloids than non-214 aggregated, confirms the success of developed assay for detection of an unamplified target with 215 unmodified colloids in a quick and facile way. The principle of binding oligo probe to its specific 216 target leading to change in color of the solution was independently verified using a different 217 template RNA (isolated from pancreas tissue) and pancreas specific gene REG3 oligo probe in 218 a separate assay. This assay also resulted in a similar change in color and absorption spectra 219 as optimized earlier for SARS-CoV-2 RNA and RdRp oligo probe. It established the working 220 principle and specificity of the test ( Figure S1 ). 221

We determined the cross-reactivity using a cervical-DNA sample from women diagnosed with 222 HPV infection (non-specific target control). No color change of gold colloids was observed with 223 HPV DNA, indicating no hybridization, and specificity of the developed assay (Figure 3 b, right 224 vial). Contrary to HPV DNA-negative control and NTC, a positive control sample shows 225 development of blue color (Figure 3 b, middle vial). Absorption spectrum (of colloids) with HPV 226 DNA-negative control exhibited characteristics similar to that of NTC, and no red-shift or peak 227 broadening as found with positive samples (Figure 3a ). Cross-reactivity of the developed assay 228 with other respiratory viruses is warranted. However, we do not anticipate the same as the test utilizes the detection of the RdRp gene of the SARS-CoV-2 virus. The oligo probe sequence 230 used in our assay is not complementary to any human mRNAs and other members of the SARS 231 family, as verified by BLAST using the NCBI database. 232

IVT synthesized SARS-CoV-2 RNA was used to test the sensitivity of the developed assay. 233 RNA ranging from 0.1 to 5ng resulted in a gradual change in colloid color from light pink to blue 234 

We have successfully developed an affordable gold nanoparticles-based colorimetric test for the 250 rapid detection of SARS-CoV-2 RNA in humans. The assay can detect up to 0.5 ng of SARS-251

CoV-2 RNA. The turnaround time of our assay is less than 30 minutes. Moreover, the 252 developed test will be helpful for mass screening, as it does not require sophisticated 253 equipment. However, while analyzing the clinical samples we observed the assay works best 254 with the freshly isolated RNA samples. In addition, pH and salt concentration in the elution 255 buffer, used for RNA isolation, may affect the result and hence further optimization may be 256 needed when using a different kit for RNA isolation. 257

The study was supported by overhead funds from the extramural grants to ST from DBT, ICMR 

Schematic illustrates the assay flow to detect RdRp (RNA dependent RNA polymerase) gene sequence of SARS-CoV-2 in the nasopharyngeal RNA sample from subject clinically diagnosed with nCOVID infection (positive control). Hybridization buffer with RdRp oligo probe (forward) was mixed with RNA sample. The reaction mixture was denatured at 95°C for 30 seconds, followed by annealing at 60° C for 60 seconds. After annealing, the tube was kept at room temperature for 10 minutes before colloidal AuNPs were added. A pure colloid is pink in color. It turns blue in the vial containing RNA sample from positive control due to salt-induced aggregation upon successful hybridization between oligo probe and target RNA. The color remained pink in the absence of target RNA, or the presence of a non-specific target. (a) Comparative absorption spectra of unmodified AuNPs (Green curve) and oligo probe stabilized AuNPs i.e., NTC (Grey curve). Both spectra are exhibiting characteristics absorption peak at λmax 520 nm, however, the reduced peak intensity in NTC is due to dilution of colloid solution. In NTC, no red-shift in peak position, approve the stabilizing property of single-stranded oligo probe, Optical images of gold colloids (left vial) and NTC (right vial) are shown in the inset. (b) Comparative absorption spectra of NTC (Grey curve) and positive control i.e., nasopharyngeal RNA sample from subject clinically diagnosed with nCOVID infection (Brown curve). Broadening of the peak as well as red-shift in peak position confirm the salt-induced aggregation of AuNP due to successful hybrid formation in control. Optical images shown in inset demonstrate the evident change in the color of the solution from pink to blue in the control vial (right) while no change in color of NTC vial (left). (c) representative absorption spectra, and in the inset shows optical images comparing assay performed with NTC (Grey curves, left vial), RNA from clinically diagnosed nCOVID infected subjects (Brown curve, middle vial), and RNA from subjects without nCOVID infection (Yellow curve, right vial). Samples from a total of eighteen infected and eighteen uninfected individuals were analyzed (optical images were attached as supplimentry figure S2) . Above estimation are based on equal number of disease-and disease-free cases in study samples detected by Gold standard method. 366 Table S1 397 398 Table S1 . The clinical diagnosis of the subjects based on Taqman RT-PCR analysis. RNA from the nasopharyngeal samples were analyzed by the new test (optical images shown in Figure S2 ). RNA samples received from clinical laboratories were re-elluted in RNAase free water before testing with new method. Figure S3 ). Nasopharyngeal samples Samples were collected and RNA was isolated at RMLIMS (Lucknow). 

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