key: cord-0723043-1j4anmho
authors: Haque, Muhammad Farhan Ul; Bukhari, Syeda Sadia; Ejaz, Rabia; Zaman, Faheem Uz; Sreejith, Kamalalayam Rajan; Rashid, Naeem; Umer, Muhammad; Shahzad, Naveed
title: A novel RdRp-based colorimetric RT-LAMP assay for rapid and sensitive detection of SARS-CoV-2 in clinical and sewage samples from Pakistan
date: 2021-06-17
journal: Virus Res
DOI: 10.1016/j.virusres.2021.198484
sha: 6bbcd38b10063baf048f2e2654006f4a39086669
doc_id: 723043
cord_uid: 1j4anmho

Novel corona virus SARS-CoV-2, causing coronavirus disease 2019 (COVID-19), has become a global health challenge particularly for developing countries like Pakistan where overcrowded cities, inadequate sanitation, little health awareness and poor socioeconomic conditions exist. The SARS-CoV-2 has been known to spread primarily through direct contact and respiratory droplets. However, detection of SARS-CoV-2 in stool and sewage, have raised the possibility of fecal-oral mode of transmission. Currently, quantitative reverse-transcriptase PCR (qRT-PCR) is the only method being used for SARS-CoV-2 detection, which requires expensive instrumentation, dedicated laboratory setup, highly skilled staff, and several hours to report results. Considering the high transmissibility and rapid spread, a robust, sensitive, specific and cheaper assay for rapid SARS-CoV-2 detection is highly needed. Herein, we report a novel colorimetric RT-LAMP assay for naked-eye detection of SARS-COV-2 in clinical as well as sewage samples. Our SARS-CoV-2 RdRp-based LAMP assay could successfully detect the virus RNA in 26/28 (93%) of RT-PCR positive COVID-19 clinical samples with 100% specificity (n = 7) within 20 min. We also tested the effect of various additives on the performance of LAMP assay and found that addition of 1 mg/ml bovine serum albumin (BSA) could increase the sensitivity of assay up to 10(1) copies of target sequence. Moreover, we also successfully applied this assay to detect SARS-CoV-2 in sewage waters collected from those areas of Lahore, a city of Punjab province of Pakistan, declared as virus hotspots by local government. Our optimized LAMP assay could provide a sensitive first tier strategy for SARS-CoV-2 screening and can potentially help diagnostic laboratories in better handling of high sample turnout during pandemic situation. By providing rapid naked-eye SARS-CoV-2 detection in sewage samples, this assay may support pandemic readiness and emergency response to any possible virus outbreaks in future.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the associated disease referred to as coronavirus disease 2019 (COVID-19) were first described during an unusual outbreak of pneumonia in Wuhan city of China in December 2019 and has since become a global health challenge. According to the latest tally from WHO, there are more than 120 million confirmed cases of COVID-19 and over 2.6 million fatalities globally (WHO, 2021) . SARS-CoV-2 is a member of the enveloped single-stranded, positive-sense RNA virus family Coronaviridae which are known to infect birds, amphibians, and mammals (Pal et al., 2020) .

Genetic analysis showed that SARS-CoV-2 shares 96.2% homology with bat-derived SARS-like coronaviruses and 80% identity with SARS-CoV of 2003 outbreak . The >29 kb long genome of SARS-CoV-2 has a 5′ methylated cap and 3′ polyadenylated tail, and is structurally divided into 14 open reading frames (ORFs) that encode for a variety of structural, non-structural and accessory proteins responsible for virus structure, replication, survival in host, and pathogenesis (Astuti, & Yasrafil, 2020; Romano et al., 2020) .

SARS-CoV-2 infection was initially thought to cause mild to severe respiratory problems only.

However, numerous studies subsequently described the induction of gastrointestinal symptoms including diarrhea, nausea and vomiting Gao et al., 2020; Wang et al., 2020) .

A meta-analysis, based on 29 published studies, concluded that approximately 12% of COVID-19 patients manifest gastrointestinal symptoms (Parasa et al., 2020) . The secretion of both viral genome and live virus have recently been reported in the feces/anal or rectal swabs and urine of mild to severely affected as well as asymptomatic COVID-19 patients Guan et al., 2020; Sun et al., 2020; Song et al., 2020; Zhang et al., 2020) . Continuous fecal shedding of SARS-CoV-2 was reported even after clearance of the respiratory tract with negative throat swab results (Ling et al., 2020 . Similarly, viral load was reported to be higher in feces when compared with respiratory samples . These evidences not only suggest the possibility of fecal-oral or fecal-respiratory SARS-CoV-2 transmission but also emphasize the need and importance of SARS-CoV-2 monitoring in wastewater.

The surveillance of sewage water has become an extremely important tool to trace the circulation of viruses in a population, to describe their prevalence and genetic diversity and to study molecular epidemiology and geographical distribution (Sinclair et al., 2008; La Rosa & Muscillo, 2013) . Sewage water monitoring may be more useful for viruses that do not manifest clinical symptoms or present at a very low level (Asghar et al., 2014) . Earlier studies during previous coronavirus outbreaks reported the detection of SARS-CoV RNA in sewage samples (Wang et al., 2015) . Likewise, during the current outbreak, molecular detection of SARS-CoV-2 in wastewater has been reported in several studies conducted in Australia, Netherlands, USA, France, and China. These studies successfully detected SARS-CoV-2 RNA in untreated wastewater with high viral loads Lodder et al., 2020; Medema et al., 2020 , La Rosa et al., 2020 Nemudryi et al., 2020; & Wurtzer et al., 2020 . Findings of these studies have opened new horizons in the ongoing battle against this deadly pandemic.

Sewage and wastewater surveillance may help in development of early warning systems for a feared second wave, rapid implementation of data driven non-pharmaceutical interventions for curtailing the virus spread, as well as real time monitoring of the efficacy of implemented interventions [Nemudryi et al., 2020) . Numerous such studies are underway in different parts of the world. However, in Pakistan, not much has been done in this regard. To the best of our knowledge, currently only one non-peer reviewed preprint has reported sewage water testing for SARS-CoV-2. The said study has a major limitation that it relies on conventional Real-time reverse transcriptase (rRT-PCR) method. Moreover, the authors have tested sewage water from only a single source (Yaqub et al., 2020) .

Real-time reverse transcriptase PCR (rRT-PCR) is currently the gold standard method for detection of SARS-CoV-2 in clinical as well as in environmental samples. However, rRT-PCR is expensive, cumbersome, time consuming, and requires sophisticated instrumentation, dedicated laboratory setup and highly skilled staff (Corman et al., 2020) . Rapid, robust, cost effective and simple diagnostic methods, which can be performed without the need of advanced instruments and highly trained technicians, may be a better diagnostic and surveillance approach during the times of emergency like COVID-19 outbreak. Loop-mediated isothermal amplification (LAMP) was originally developed as a rapid and reliable method to amplify small amounts of target sequences at a single reaction temperature, preventing the need of thermocycler (Notomi et al., 2000) . Having the advantage of simplicity to perform and isothermal amplification, this method gained immense popularity for the effective diagnosis of pathogens. Herein, we report a novel colorimetric LAMP assay targeting RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2 genome for sensitive and specific detection of the virus in clinical and sewage water samples from Pakistan.

A set of six sequence specific LAMP primers targeting a 225 nt long sequence spanning 

For optimization of LAMP assay, a target DNA fragment (225 nt) corresponding to SARS-CoV-2 RdRp gene region cloned into pTOP Blunt V2 vector was obtained from Clonetech. The said fragment was amplified by PCR using outer primers (Cor-RdRp-F3 and Cor-RdRp-B3). The resulting PCR product was purified using GeneJet purification column and stored at -20 o C until further use. For optimization of the LAMP assay, we used this purified DNA fragment as a template and LAMP assay was performed using previously described reaction conditions . Since, addition of certain additives in LAMP reaction has been described to influence the efficiency of LAMP assay (Xu et al., 2016; Siyafiraf et al., 2018) , we tested three of the additives: MgSO 4 (8 mM), DMSO (5 %) and/or BSA (1 mg/ml) in the reaction mix. We performed the LAMP assay over a wide range of temperature (50 to 65 o C) and color change was observed after every 10 min till 60 min. We also performed the LAMP assay reactions in 10 and 20 µl volumes and compared efficiencies. For a 10 µl total reaction volume, components were after 20 min at 65 o C was considered as negative.

Mughal Diagnostics and Research Lab (MDRL) (Johar Town, Lahore, Pakistan) is among the approved Laboratories by Government of Pakistan for the detection of SARS-CoV-2 in clinical samples. Nasopharyngeal swabs were collected from suspected individuals at the designated laboratory and placed in vials containing viral transport media (VTM). Briefly, samples were inactivated at 56 °C for 30 min in BSL-3 laboratory as described previously .

These samples were processed for total RNA isolation at above mentioned laboratory using GF1 viral DNA/RNA extraction kit (Cat # GF-RD-300, Vivantis), following manufacturers protocol.

All these processes were carried out by wearing proper personal protective equipments (PPEs) and protocols recommended by WHO. 1 The qRT-PCR was performed at MDRL by using COVID-19 detection kit (Cat # Z-Path-COVID-19-CE, Genesig). In total, 35 RNA samples (28 positive and 7 negative) were retrieved from MDRL along with patients' history and Ct values.

These RNA samples were transported to School of Biological Sciences, University of the Punjab Lahore, and stored at -80 o C until further processing. The present study was approved by Ethical

Review Board of School of Biological Sciences, University of the Punjab Lahore, Pakistan. A written consent was obtained from each participant to use their samples for the validation of LAMP assay.

Raw sewage (1 L) samples were collected from different drain localities in Lahore in sterile collection jars. Adequate PPEs were used by personnel collecting sewage samples. Collected sample was mixed properly before aliquoting and stored at -20 o C. The viruses were concentrated by PEG method described previously (Hejelmsø et al., 2017) . Briefly, glycine buffer (glycine 0.05 M, beef extract 3%, pH 9.6) was added to sewage water to extricate virions from organic materials. The samples were centrifuged at 7000 rpm for 30 min at 4 o C.

Supernatants were collected and filtered through 0.45 µm polyethersulfone membrane (PES) to eliminate eukaryotic and bacterial cells. pH of filtrate was adjusted to 3.7 and filtrate was incubated with PEG-8000 (80 g/L) and NaCl (17.5 g/L) in shaker (100 rpm) overnight at 4 o C.

The incubated filtrate was centrifuged at 7000 rpm for 2 h to precipitate viruses in a pellet. The resulting pellet was then dissolved in 1X phosphate buffered saline (PBS) and stored at -80 o C until further processing.

RNA was isolated from concentrated sewage water samples by Trizol method. Briefly, 400 µl of each sample concentrate was mixed with 1 ml Trizol reagent following addition of 300 µl of chloroform in sterile micro centrifuge tubes. Mixture was vortexed for 10 sec, incubated at room temperature for 3 min and subjected to centrifugation at 12000 rpm for 10 min at 4 o C. After centrifugation, upper layer was transferred to a new micro centrifuge tube, mixed with equal volume of chilled 100% isopropanol and incubated at room temperature for 10 min following centrifugation. Obtained pellet was washed with 70% ethanol, air dried and dissolved in 50 µl DEPC treated water. Isolated RNA was quantified using Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). To synthesize cDNA, the reverse transcription was performed.

Eleven microliters of extracted RNA were mixed with 1µl of RdRp reverse outer primer Cor-RdRp-B3 (10 µmol/ml) and incubated at 65 o C for 5 min. After incubation, 8 µl of second mixture containing 4 µl of 5X reaction buffer, 1 µl of ribolock RNase inhibitor, 2 µl of 10 mM dNTPs and 1 µl of RevertAid reverse transcriptase was added and incubated at 25 o C for 5 min followed by an incubation of 60 min at 42 o C and final incubation at 70 o C for 5 min.

After cDNA synthesis, RT-PCR was performed using RdRp outer primer set. In total, 10 µl reaction mixture was prepared using 5 µl of PCR MasterMix (LUNA), 0.25 µM of each forward and reverse outer primer and 2 µl of template cDNA and volume was made up to 10 µl.

Amplification was performed using initial denaturation at 95 o C for 5 min followed 40 cycles of denaturation of 30 sec at 95 o C, annealing for 30 sec at 58 o C and extension for 30 sec at 72 o C with final extension at 72 o C for 5 min. Each PCR contained RdRp synthetic fragment as positive control and nuclease free sterile water as negative control. PCR products were analyzed on 1.5% agarose gel.

The LAMP assay was performed on the clinical samples (n =35) as well as the sewage water samples in 10 µl reaction volume using optimized reaction conditions described above. Each sample was assayed three times. The assay results for clinical samples were compared with the Ct values retrieved from the data bank of MDRL. For sewage samples, assay results were compared with the results of in-house RT-PCR.

Sequences of the 4 primers (F3, B3, LF, LB) as well as F1c, F2, B1c, and B2 regions of primers Primers were aligned with the subject sequences using NCBI program BLASTN 2.11.0 (Altschul et al., 1997) using following parameters: Word size 7, Expect value 1000, Match/Mismatch scores 1,-3, Gapcosts 5,2. Specificity of each primer was determined by analyzing the E-values obtained.

We used synthetic RdRp gene fragment as template for the optimization of our newly developed LAMP assay. Genomic location of the target region within the SARS-CoV-2 genome and the map of plasmid having cloned target fragment is shown as Figure 1 and Supplementary Figure   1 , respectively. Initially, no amplification was obtained after 30 min of incubation when LAMP assay was performed according to the previously described procedure .

However, LAMP assay produced yellow color when we further incubated the reaction for 60 min at 65 o C (Supplementary Figure 2) . With the aim to improve the sensitivity of the assay as well as to reduce the reaction time, we optimized several parameters. We first tested the effect of We further tested the effect of incubation time and temperature on efficiency of the assay. Our findings showed that the time to obtain amplification signal in LAMP assay reduced with the increase in the incubation temperature. There was a complete colour change from pink to yellow at 65 o C in 20 min. On the other hand, incubation time also seemed to be having an impact on the assay. For instance, assay produced color even at as low as low temperature of 55 o C when incubated for 60 min ( Figure 2B ). Based on these observations, we slightly modified the reaction conditions for further testing. In contrast to the procedure reported by Zhang et al. (2020) and also the protocol recommended by the manufacturer, where a 30 min incubation at 65 o C is suggested, we used only 20 min incubation for a reaction volume of 10 µl, in the presence of BSA at a final concentration of 1 mg/ml.

For the determination of sensitivity and detection limit of the assay, LAMP assay was performed with 10-fold serial dilutions of synthetic DNA fragment. We tested a range of starting copies (10 6 to 10 0 ) of the target RdRp synthetic DNA fragment. Our results showed that using optimized parameters the assay was able to detect as low as 10 1 copies of the target region ( Figure 3A) . Moreover, the sensitivity range of our LAMP assay spanned over several orders of magnitude for serial dilutions of RNA, obtained from a qRT-PCR confirmed clinical sample of COVID-19 ( Figure 3B ).

In order to test the capability of our optimized assay in detecting clinically relevant SARS-CoV- 

One of the primary objectives of this study was to apply the LAMP assay for the detection of SARS-CoV-2 in the environmental samples, particularly the sewage water that receives feces and urine from COVID-19 affected communities. For this purpose, samples were collected from 6 major drains of Lahore (Supplementary Figure 3) . The sample collection sites were chosen to cover especially the areas declared as infection hotspots by local government. These samples were concentrated and processed for RNA isolation. SARS-CoV-2 RNA was detected using our optimized LAMP assay in all of the sewage water samples. Tap water and water collected from Lahore canal were used as controls. The control samples were subjected to the same RNA isolation procedure and LAMP assay. No amplification (change in color) was observed in any of the controls ( Figure 5A) . We further verified the results of our LAMP assay by carrying out standard qRT-PCR analysis of sewage and control samples. qRT-PCR results substantiated the findings of our LAMP assay ( Figure 5B and Supplementary Table 2 ).

In Despite slight similarity of certain primer sequences with unrelated coronavirus sequences, it is highly unlikely that this similarity will result in any significant non-specific amplifications.

Firstly, as F2, and B2 regions do not have any significant similarity with any of the closely related coronaviruses initial binding of FIP and BIP primers with the template is not possible.

Additionally, F3 and B3 sequences are also not identical to any other coronaviruses except SARS-CoV-2 therefore strand displacement also cannot ensue. Despite slight similarity of F1c region with Tor2 genome or being completely identical to HKU1 genome, it will not lead to any loop formation and amplification from this loop seems highly unlikely due to noncomplementarity at the F2 region of FIP. Similarly, high similarity of LB primer with Tor2 genome alone in the absence of any significant similarity at B1c and B2 regions will not affect the overall specificity of the assay.

On the other hand, all the eight regions targeted by LAMP primers showed 100% similarity with various SARS-CoV-2 sequences. In addition to the sequence of Pakistani isolate, which was used to design the LAMP assay, the primers were also 100% identical to the reference sequence (Wuhan-1 isolate) as well as various newly emerging SARS-CoV-2 variants of concern. These results indicate that our novel LAMP assay is capable of highly specific detection of SARS-

CoV-2 as no amplification of any of the related coronavirus sequences is possible. Moreover, it can possibly detect most of the common SARS-CoV-2 variants as the assay targets one of the most conserved viral regions i.e. RdRp.

On 31 Although Loop-mediated isothermal amplification (LAMP) assay for SARS-CoV-2 has been reported targeting ORF and N-genes , to the best of our knowledge none of the studies published so far have reported on the use of RNA dependent RNA polymerase (RdRp) gene for virus detection using LAMP assay. RdRp gene is an excellent choice for being highly conserved (Chana, Yip, & To, 2020; Nawattanapaiboon et al., 2020) . Remarkably, RT-LAMP is reported to be ~ 100-fold more sensitive than conventional RT-PCR methods (Khan et al., 2018) . Moreover, RT-LAMP assay is faster relative to qRT-PCR and therefore it could be used as a reliable method in laboratories to keep up with the increasing demand of screening the potential patients during the pandemic situations.

Primarily, our assay was meant to be qualitative. As limit of detection and reproducibility are the key performance matrices for a diagnostic assay, we used synthetic target fragment to demonstrate the performance of our assay. Our assay showed optimal reproducibility not only for synthetic fragment but also for clinical and environmental samples. Our assay demonstrated a reliable limit of detection of 10 copies per reaction in less than 20 min, which is better as compared to the previously reported RT-LAMP assays. For example, Lamb et al., (2020) were able to detect nearly 300 viral copies and Bharda et al (2020) reported the detection of 100 copies of SARS-CoV-2 genomic RNA. We successfully evaluated the performance of our assay in clinical settings, recruiting 35 RNA samples including 28 confirmed COVID-19 cases. The results of our assay for these clinical samples were largely consistent with those of qRT-PCR.

Only two samples tested positive by qRT-PCR were negative by our RT-LAMP and while none of the samples tested negative by qRT-PCR were tested positive by our assay. The observed difference in positive predictive value of RT-LAMP and qRT-PCR may be caused by less/more RNA inputs (Jiang et al., 2020) . In addition to assay performance, cost per test is also an important consideration, particularly in rapidly spreading pandemic situations. Cheaper, yet reliable, tests may help reducing the financial burden of testing and the resources can be diverted to other healthcare facilities. We optimized our assay using 10 µl reaction volume compared to the volume recommended by the manufacturer (25 µl), thus reducing the cost per test 2.5 times.

In recent months, the emergence of novel SARS-CoV-2 variants has led to unexpected rise in number of cases of COVID-19 in various countries. UK, South Africa, and Brazil are among the countries which appear to be most severely hit by emergence of novel variants. Each of these countries has been suspected to be the place where a particular variant emerged. For example, the B.1.1.7 clade emerged in UK, while B.1.351 is known to have emerged in South Africa. Both of these variants harbor a mutation (N501Y) in the receptor binding domain of spike protein which confers high transmissibility to these variants. It has been estimated that these variants have 40-70% higher transmission rates compared to wild type. B.1.351 has at least two known additional mutations (E484K and K417N) in spike protein which can potentially make the variant less susceptible to neutralization by antibodies. The third variant P.1 which is thought to have emerged in Brazil harbors the N501Y mutation as well as two additional mutation E484K and K417T. While emergence of these variants is a major concern for vaccination efforts, performance of diagnostic assays may also be affected. Although based on the available data the effect of genome mutations in the newly emerging variants on performance of most commonly used diagnostic assays seems to be minimal, a cautious approach may be needed as several mutations have been reported around the regions targeted by available diagnostic assays. We carried out in-silico analysis of the specificity of primers used in our assay and demonstrated that all of them show 100% similarity to various SARS-CoV-2 isolates and more importantly three of the newly emerged variants of concern (Fontanet et al., 2021) .

Recent discoveries about detection of SARS-CoV-2 RNA as well as viable virus in stools and sewage samples has suggested the possibility of fecal-oral transmission that warrants farreaching consequences for public health and for pandemic control strategies (Heller et al., 2020) .

Furthermore, a few studies have reported the secretion of both viral genome and viable viruses in the feces of mild to severely affected as well as asymptomatic COVID-19 patients Holshue et al., 2020) and this fecal shedding of SARS-CoV-2 were reported to be continued even after clearance of respiratory tract and negative throat swab results (Kitajima et al 2020) . Considering the implications of these reports, we reasoned that detection of SARS-CoV-2 in sewage water may offer a good strategy to measure the true scale of coronavirus outbreak (Mallapaty, 2020) . We selected 6 different drains located in various urban areas of the city of Lahore that received the sewage water from areas marked as hotspots by the local government. Recently, SARS-CoV-2 RNA has been detected in wastewater samples from various countries like Netherlands (Medema et al., 2020) , Australia and

USA . However, all these testes were based on qRT-PCR. Here, we successfully report detection of SARS-CoV-2 in the sewage water samples based on our optimized RT-LAMP assay, and the results were further validated by conducting RT-PCR. To the best of our knowledge, this is the first study that reports the detection of SARS-CoV-2 in sewage samples using a highly sensitive LAMP assay. Although in this study we have demonstrated the applicability of LAMP assay on sewage sample isolated RNA, further work may be needed to develop a standardized assay. Currently one of the major issues being faced in this regard is that none of the reported studies have provided information on the percent recovery of SARS-CoV-2 from sewage samples due to the risk associated with handling SARS-CoV-2 and the requirements for a BSL-3 facility. To the best of our knowledge, only one study has to date reported the percent recovery of SARS-CoV-2 from sewage samples which was estimated to be only 1% using an electropositive membrane . Since the characteristics of SARS-CoV-2 are different from other enteric viruses, more research may be needed to streamline protocols for effective recovery and downstream LAMP based detection of SARS-CoV-2 from sewage and/or other environmental samples.

One of the biggest challenges, in using sewage water SARS-CoV-2 detection to assess the scale of virus spread in corresponding community, would be to establish correlation between quantities detected in sewage water and the actual number of cases in the community. Medema et al., (2020) (Medema et al., 2020) reported RT-qPCR data, while another recent study reported ~250 copies/ml SARS-CoV-2 in sewage water in Massachusetts, USA . The authors acknowledged that the estimated concentration was much greater (5% of all fecal samples in the catchment) than the confirmed cases (0.026%). The authors listed several factors and assumptions for this discrepancy and considered their results conservative. Another challenge in using sewage water for SARS-CoV-2 detection is the possible contamination of sewage water with related coronaviruses. These related coronaviruses show high overall sequence similarity with the SARS-CoV-2 thus there is a possibility of false positive results due to the contamination of sewage water with related coronaviruses if the molecular assay being used is not highly specific. We carried out in silico analysis of specificity of our primers. Our analysis showed that most of the primer sequences were highly specific for SARS-CoV-2 with no significant similarity to any of the six related coronaviruses included in the analysis. Although

F1c and LB regions showed similarity to Tor2 and HUK1, and Tor2 viruses, respectively, as discussed in section 3.5 these similarities cannot possibly lead to non-specific amplifications.

Thus, due to inherent specificity of LAMP assay (as 6 primers are used which target 8 distinct regions in target), as well as careful design of primers, we demonstrate that our LAMP assay can potentially differentiate SARS-CoV-2 from various related coronaviruses, and at the same time can detect most common SARS-CoV-2 variants with equal efficiency.

For application of sewage-based epidemiology to assess SARS-CoV-2 spread, further systematic research is needed covering aspects from effective sampling and preservation through to data interpretation. Development of effective enveloped virus concentration methods and understanding the decay of SARS-CoV-2 in sewage are some of the major research avenues needing attention. Information on the composition of sewage and environmental factors such as stormflow and its impact on sewage may also be useful. Since application of such a rapid isothermal viral RNA detection platform may be particularly useful in point-of-need and offlaboratory settings, integration of LAMP based SARS-CoV-2 detection in novel handheld and miniaturized microfluidic devices is also one of the most important research area for development of rapid, simple and low-cost viral detection methods. These requirements are expected to be achievable and it is anticipated that LAMP based SARS-CoV-2 detection will not only support routine viral diagnostics but may also enable better approaches for rapid assessment of the disease burden in the community.

In this study, we report a novel RdRp based sensitive RT-LAMP assay for SARS-CoV-2 detection in clinical as well as sewage water samples. Rapid and low-cost detection ability may potentially help in controlling the outbreak and spread of the disease by enabling implementation of data-driven smart control interventions.

We are highly thankful to School of Biological Sciences, University of the Punjab, Lahore, Pakistan, to provide research facilities to conduct this study.

There is no conflict of interest among all contributing authors.

No funding was received for this study.

Author statement: All the authors have read the manuscript and are agreed with the current version.

The authors declare no conflict of interest performed by using serial dilutions (copy number 10 6 to 10 0 ) of synthetic fragment of RdRpgene of SARS-CoV-2 as template. NTC: no template control. B) Images are results of LAMP assay performed by using dilutions (10 -1 to 10 -9 ) of RNA from a COVID-19 clinical sample.

Validation of RdRp-gene LAMP assay for COVID-19 clinical samples. Images are of LAMP assay performed on representative clinical samples obtained from COVID-19 patients (two +ve and one -ve). Synthetic RdRP gene was used as positive control while nuclease free water as negative control. Assay was repeated three times for each sample and the representative image is presented here. 

First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

Environmental surveillance for polioviruses in the Global Polio Eradication Initiative. The Journal of infectious diseases

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response

High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes. bioRxiv

Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific 2 COVID-19-RdRp/Hel real time reverse transcription-polymerase chain reaction assay validated 3 in vitro and with clinical specimens

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

SARS-CoV-2 variants and ending the COVID-19 pandemic

novel coronavirus infection and gastrointestinal tract

COVID-19: gastrointestinal manifestations and potential fecal-oraltransmission

Clinical characteristics of coronavirus disease 2019 in China

COVID-19 faecal-oral transmission: Are we asking the right questions? Science of The Total Environment

Evaluation of Methods for the Concentration and Extraction of Viruses from Sewage in the Context of Metagenomic Sequencing

First case of 2019 novel coronavirus in the United States

Development and validation of a rapid single-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) system potentially to be used for reliable and highthroughput screening of COVID-19. medRxiv

Comparative evaluation of the LAMP assay and PCR-based assays for the rapid detection of Alternaria solani

SARS-CoV-2 in wastewater: State of the knowledge and research needs

Molecular detection of viruses in water and sewage

SARS-CoV-2 has been circulating in northern Italy since December 2019: evidence from environmental monitoring

Rapid detection of novel coronavirus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification

Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients

SARS-CoV-2 in wastewater: potential health risk, but also data source

Genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

How sewage could reveal true scale of coronavirus outbreak

Samphaongern, C. Colorimetric reverse transcription loopmediated isothermal amplification (RT-LAMP) as a visual diagnostic platform for the detection of the emerging coronavirus SARS-CoV-2

Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater

Loop-mediated isothermal amplification of DNA

Severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2): An update

Prevalence of Gastrointestinal Symptoms and Fecal Viral Shedding in Patients With Coronavirus Disease 2019: A Systematic Review and Meta-analysis

A Structural View of SARS-CoV-2 RNA Replication Machinery: RNA Synthesis, Proofreading and Final Capping

Pathogen surveillance through monitoring of sewer systems

SARS-CoV-2 induced diarrhoea as onset symptom in patient with COVID-19

Isolation of infectious SARS-CoV-2 from urine of a COVID-19 patient. Emerging microbes & infections

An ambient temperature stable and ready-to-use loop-mediated isothermal amplification assay for detection of toxigenic Vibrio cholerae in outbreak settings

Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification

Detection of SARS-CoV-2 in different types of clinical specimens

WHO Coronavirus (COVID-2019) dashboard. Available from

SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases

SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases

Prolonged presence of SARS-CoV-2 viral RNA in faecal samples

Time course quantitative detection of SARS-CoV-2 in Parisian wastewaters correlates with COVID-19 confirmed cases

Infectious SARS-CoV-2 in feces of patient with severe COVID-19

Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection

A longitudinal survey for genome-based identification of SARS-CoV-2 in sewage water in selected lockdown areas of Lahore city, Pakistan; a potential approach for future smart lockdown strategy

Isolation of 2019-nCoV from a stool specimen of a laboratory-confirmed case of the coronavirus disease 2019 (COVID-19)

Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP

A novel coronavirus from patients with pneumonia in China

A set of specific LAMP primers targeting a 225 bp long sequence spanning nucleotide positions 15319 to 15543 of Pakistani SARS-CoV-2 isolate (MT240479.1) within the RdRp gene region (RNA dependent RNA polymerase) of viral genome, were designed