key: cord-0956623-zcmzpat5
authors: Jalandra, Rekha; Yadav, Amit K.; Verma, Damini; Dalal, Nishu; Sharma, Minakshi; Singh, Rajeev; Kumar, Anil; Solanki, Pratima R.
title: STRATEGIES AND PERSPECTIVES TO DEVELOP SARS-CoV-2 DETECTION METHODS AND DIAGNOSTICS
date: 2020-06-19
journal: Biomed Pharmacother
DOI: 10.1016/j.biopha.2020.110446
sha: fec7931bbbc35a5f4ecaa2ee76f4d5e4a694228f
doc_id: 956623
cord_uid: zcmzpat5

To develop diagnostics and detection methods, current research is focussed on targeting the detection of coronavirus based on its RNA. Besides the RNA target, research reports are coming to develop diagnostics by targeting structure and other parts of coronavirus. PCR based detection system is widely used and various improvements in the PCR based detection system can be seen in the recent research reports. This review will discuss multiple detection methods for coronavirus for developing appropriate, reliable, and fast alternative detection methods. Considering the current scenario of COVID-19 diagnostics around the world and an urgent need for the development of reliable and cheap diagnostic, various techniques based on CRISPR technology, antibody, MIP, LAMP, microarray, etc. should be discussed and tried.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected almost every country with 7410510 total cases and 418294 deaths globally as of 12 June 2020 [1] . Since this virus is new and lack of any approved drug or vaccine, there is urgent need for a highly specific and sensitive diagnostic measures to identify infected people and isolate them to avoid the further spread of the virus [2, 3] . Testing is paramount in the fight to slow down an minimize the spread of the virus. Effective diagnostic approaches need to be discussed and tried to detect the virus itself or the response of the host body to the virus. Currently, numerous diagnostic technologies are available for SARS-CoV-2 detection, and many are under development [4] . Recently, the World Health Organization (WHO) has enumerated the first two diagnostic tests for emergency use to detect COVID-19 to increase the accurate assessment of disease. The tests are genesis Real-Time polymerase chain reaction (RT-PCR). Corona virus and Cobas SARS-CoV-2, a qualitative assay for use on the Cobas® 6800/8800 Systems, and are for in-vitro diagnosis of COVID-19 [5] . Currently, the most popular diagnostic tool that identifies the viral RNA through amplification is RT-PCR. It is a sensitive technique that requires a small amount of input RNA but takes hours to get results [6] .

Other tools that target virus includes microarray relying on the binding of the viral genomespecific probe and CRISPR technology that recruits Cas12/13 enzyme specific for viral genes for detection of SARS-CoV-2 [7, 8] . As SARS-COV-2 infected individuals produce antibodies against the virus, these antibodies can be used for the identification of infected individuals.

Antibody-based diagnostics give a positive result if they find binding antibodies that might have generated earlier during the infection and would serve to provide protection against reinfection thereby, limits their use in the detection of active diseases [9] . The sensors are another approach that presents low-cost promising diagnostic tools with high sensitivity to detect COVID-19 [10] .

Most of the diagnostic techniques used now-a-days are time-consuming and require skilled J o u r n a l P r e -p r o o f crisis, where a collective pool of knowledge is a prerequisite, here, we recapitulate available updates on diagnostic methods such as PCR, microarray, molecularly imprinted polymer-based sensor, CRISPR, etc. for COVID-19 detection.

As current and future COVID-19 detection methods are based on genomics and structure of SARS-CoV-2, it is pertinent to review the recent progress on these aspects. SARS-CoV-2, a novel coronavirus species, placed under the betacoronavirus genus based on genomic similarity and phylogenetic relationship with SARS-CoV (Fig. 1 ). Genome sequence of SARS-CoV-2 has 88% similarity with SARS-like bat derived coronaviruses, SL-CoVZC45, and SL-CoVZXC21.

Among different coronaviruses, RNA-dependent RNA polymerase (RdRp) gene sequence is an extremely conserved sequence. According to the International Committee on Taxonomy of Viruses criteria, if a species shows less than 90% similarity for conserved RdRp sequence, it would be considered as novel species. RdRp sequence of isolated strain in Wuhan, China exhibits 86% similarity with existing SL-CoVZC45 coronavirus; therefore, CoVs were declared as a new species (SARS-CoV-2). SARS-CoV-2 has a single-stranded positive helix RNA genome of 30 kb with a GC content of 38% [12] . Whole-genome sequencing showed that the virus genome from different parts of the world exhibited sequence homology of more than 99.9% with SARS-CoV-2 isolated from Wuhan, China [13] . Homology modeling showed that the receptor-binding domain of SARS-CoV-2 and SARS-CoV differs only in a few amino acid residues [14] . The genome of COVID-2 consists of an open reading frame (ORF) 1a/b-coding region and four protein-coding regions flanking with the non-coding region on both sides.

Starting from 5' end protein-coding region, an s-region coding for spike protein and e-region coding for envelope protein, m-region coding for a membrane protein, and n-region coding for nucleocapsid protein are present [15] . Structural and accessory proteins (S, M, E, N-proteins) are translated from sgRNAs (single guide RNAs). The most abundant structural protein in coronavirus is membrane glycoprotein (~25-30kDa), spans the lipid membrane thrice with the N-J o u r n a l P r e -p r o o f terminal domain on the outside and C-terminal domain inside the virion. S-protein (~150kDa) recognizes and binds to the receptor present on the host cell, thereby responsible for viral infectivity. Scanning electron micrograph of the virus revealed that it is oval or spherical with stalk-like projections ending in round structure (spike) like other viruses of coronaviridae family.

Spikes are essential for viral infectivity and host specificity. While invading host cell, furin-like proteases cleave S-protein into two parts: a receptor binding unit (S1) and a membrane-anchored fusion unit (S2). Envelope protein (8-12kDa) determines the formation and composition of the viral membrane. Nucleocapsid protein protects and enfolds the viral RNA [16] (Fig. 2) . SARS-CoV-2 binds to receptors on the cell surface via Receptor-Binding Domain (RBD) present in their S1 subunit. RBD of SARS-CoV-2 is an almost identical 3-D structure with that of SARS-CoV using computer modeling and 76.47% amino acid sequence similarity, which uses spike proteins to bind with Angiotensin-Converting Enzyme 2 (ACE2) on host cell [17] . Thereby, it is believed that SARS-CoV-2 also enters cells by binding spike proteins to ACE2. SARS-CoV-2 contains ORF3 and whole ORF8 gene regions, which are characteristic features of bat-origin coronaviruses [12] . Scanning electron micrograph revealed that virus particle size ranges from 70-90 nm and invades various intracellular organelles, especially vesicles [13] .

Immunofluorescent assays of the culture of Vero cells showing cytopathic effect with the convalescent serum from patients showed green signals in the cytoplasm; in contrast, no signal was detected in control serum. Though viruses recruit error-prone RNA polymerase for replication, so it is more likely to get mutated [18] . The emergence of new strains is a consequence of mutation. Mutation in SARS-CoV-2 enabled it to spread more efficiently from animal to humans and then from human to human. Mutation in ORF8 region at 28144 and ORF1b region at 8872 occurred in the early stages of the outbreak of SARS-CoV-2 (SARS-CoV was also mutated in the ORF region during 2002-03 outbreak). Mutation in the ORF8 gene at 28144 positions is stable and increases population frequency from 0-29% as the epidemic progressed (Fig. 3) . These mutations became fixed during the outbreak and increase population frequency gradually. The phylogenetic analysis shows SARS-CoV-2 is the mutant product of SARS-CoV. The phylogeny indicates that the initial emergence of novel strain in Wuhan, China gained transmission between humans and was followed by worldwide outbreak transmission ( Fig. 4) . All the research reports on genomics and structure of SARS-CoV-2 are essential, which J o u r n a l P r e -p r o o f can become the premise of the development of more accurate, sensitive, and effective detection methods.

Due to the increasing incidence of SARS-CoV-2, PCR based detection is routinely used for the diagnosis of COVID-19.RT-PCR or qPCR is a molecular biology technique used to study gene expression analysis at the transcript level. It involves the following steps: RNA isolation from samples and cDNA synthesis using reverse transcription kit. PCR was set by making a working mix of the buffer, dNTPs, primers of the target gene, Taq polymerase, cDNA template and, SYBR green dye. PCR mix incubated in a PCR machine and fluorescence generated by PCR amplification is measured by machine to give Cycle threshold (Ct) values. Ct values of control and experimental samples are compared, and relative expression is estimated (Fig. 5 ).

On 14 January 2020, the protocol of RT-PCR for detection of 2019-nCoV was published on the WHO website for its use of COVID-19 detection [19] . Other than regularly used RT-PCR for diagnosis of COVID-19, some other studies used other diagnostic techniques like RT-insulated isothermal PCR (RT-iiPCR) [20] , reverse transcription loop-mediated isothermal amplification (RT-LAMP) [21] and a one-step rRT-PCR assay [22] in which unique TaqMan probes is used.

RT-LAMP has a high specificity for detection of MERS-CoV, and its sensitivity is comparable to RT-PCR. RT-iiPCR and a one-step rRT-PCR assay also show similar sensitivity and specificity, but RT-PCR is used mainly for the diagnosis of MERS-CoV. The selection of specimen type and time of taking that sample for RT-PCR is also a crucial point for the diagnosis of SARS-CoV-2.

The PCR-based first test for diagnosis of SARS-CoV-2 was constructed in Germany by Corman et al., at first, before the release of sequence, RT-PCR assay was designed following SARS or SARS-like coronavirus because it was supposed that 2019-nCoV is SARS-related.

After getting the sequence information, the RT-PCR assay was designed according to sequence similarity with SARS-CoV-2 after sequence alignment. Corman et al., developed a detection method that can differentiate between SARS-CoV and SARS-CoV-2. At first, three assays were done: E gene assay, N gene assay, and RdRp gene assay. But due to less sensitivity of N gene assay, only two assays were selected where E gene assay work as a first-line screening tool and The protocol and thermal conditions of each assay were adjusted according to the manufacturer, but the concentration of probes was taken the same as published before by Corman et al., in 2020. These assays show unspecific E gene signals but using SuperScript III these unspecific signals reduce to 5%, so SuperScript III RT-PCR is better as given by Corman et al. This research also recommended RealStar SARS-CoV-2 RT-PCR kit 1.0 (Altona) as it does not give any unspecific E gene signals [25] . In a study, a combined approach was done by using RT-PCR, CRISPER-based assay, and metagenomic next-generation sequencing (mNGS) for diagnosis of pneumonia patient, which later finds positive according to RT-PCR and CRISPERbased assay [26] . So, by combining these approaches, equitable diagnostic results can be found.

An article published in a magazine named Semiconductor Digest stated that Cepheid, a California company, made a new point of care detection test: Xpert® Xpress SARS-CoV-2. This test can give results only in 45 minutes, and it requires less than one min for sample preparation, so even healthcare workers can use it easily [27] . As point-of-care (POC) devices are easy to use and less time taking so these can be used primarily for diagnostic purposes in the next few months.

viruses, which involves using antibodies to the disease as a drug. Because of exquisitely affinity, target specificity, designed and engineered molecules nature, they have played a significant role in the number of diagnostic approaches [29] . Currently, a variety of COVID-19 tests are already available that determine the viral genome from mucosal membrane swabs till now, eleven pointof-care diagnostics for COVID-19 are reported, which is mainly based on molecular and antibody-based tests. Various research groups and companies are trying hard to develop antibody tests which include lateral flow immunoassays (BioMedomics rapid test and Surescreen rapid test cassette), time-resolved fluorescence immunoassay (Goldsite diagnostics kit), colloidal gold immunoassay (Assay Genie POC kit and VivaDiag COVID-19 IgG-IgM test), ELISA, etc [30] .

While a variety of antibody tests are also available in the market, there is a lot of confusion regarding the efficacy of such tests given the high percentage of the asymptomatic population and the fact that detectable antibodies typically develop later in the disease. Therefore, diagnostic approach proposals will have to describe their novelty in terms of process, sensitivity, specificity, the strength of risk stratification, and scalability.

We believe that the electrochemical immunosensor technique could be a potential approach for the POC device application in the detection of SARS-CoV-2, as shown in (Fig. 6A ). There are several recent research studies about a variety of virus detection using immunosensors [31] [32] [33] [34] [35] [36] [37] [38] . For example, the electrochemical based influenza A virus H5N1 biosensors are used for the detection of influenza A virus H5N1 to prevent massive death and to control the transmission from one country to the other due to their sensitivity, selectivity and economically affordable than the conventional detection method [39] . In another study, reduced graphene oxide-based electrochemical immunosensor was developed using EDC-NHS coupling chemistry between the COOH group of graphene attached on the gold surface of the working electrode and NH2 of antibody specific for H1 of H1N1 influenza and the reported value of the limit of detection is 0.5 PFU ml −1 [40] .

Thus, several studies are still going on based on immunosensor, which aims to achieve good sensitivity and specificity via different electrodes surface modifications. In the overall view, the best advantage of these hypothesized assays is easy handling, no requirement of any sophisticated instruments, rapid detection efficacy, and cost-effectiveness than other molecular approaches. Identification of major antigenic and protective epitopes of target virus particles, is J o u r n a l P r e -p r o o f crucial in understanding the antibody response while developing these detection strategies, as antigenic variations make it difficult to generate similar antibodies, thus affecting the development of these diagnostic assays.

Deoxyribonucleic acids (DNA) are perhaps the most essential of all biomolecules and has been the foundation for genetic study over the past few years. However, the use of aptamers, which are small-sized, single-stranded artificial nucleotides (RNA or DNA), having 10-100 nucleotides, are now gaining enormous importance due to its specific binding just like in case of antibodies. The selection technique of aptamers is done via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process that binds to a broad range of target analytes with high affinity and specificity. Aptasensors can measure analytes in tiny quantities where such small amounts cannot be identified with most of the other methods being used [41, 42] . Furthermore, the low cost of the aptasensor design compared with other virus diagnostic methods should be regarded as one of the economic benefits [43, 44] . Many vital benefits of aptasensors compared to other available diagnostic techniques include low detection time and a quick detection method [45, 46] . Though aptamers have massive potential as a viable tool in therapeutics and virus detection, so aptamer-based biosensors also known as aptasensors, have reported the various methods for the immobilization of the DNA oligonucleotides sequences and have used different biomaterials for enhancing the stability and sensitivity of the electrode [47] .

Several electrochemical DNA biosensors have been developed for the detection of various viruses [48] [49] [50] [51] [52] [53] and, based on that, a hypothetical schematic illustration of aptasensor based direct detection of SARS-CoV-2 using the different platform has been shown in (Fig. 6B) . In a study, DNA influenza virus A H5N1 aptasensor has been developed, which is a reliable and suitable analytical device with newly emerged designed for the detection of AIV H5N1 [10] . The active biological element on the DNA biosensor has used the specific oligonucleotide sequences single-stranded DNA (ssDNA) for identifying the viral genome of complementary ssDNA during the hybridization process.

The standard diagnostic methods implemented for SARS-CoV-2 are often invasive and costly; these methods often cannot detect the presence of SARS-CoV-2 virus in tiny amounts early. For the identification of the many viruses with high affinity, aptamers as molecular structures were used in the aptasensor system. The use of advanced nanostructures and the J o u r n a l P r e -p r o o f functionalized aptamer with certain organic materials will improve the aptasensor's diagnostic sensitivity and specificity towards SARS-CoV-2. As the aptasensor specificity and sensitivity are higher, make them suitable commercial diagnostic devices for early diagnosis of SARS-CoV-2.

The work is hoped to reduce the effects of interfering factors in SARS-CoV-2 aptasensors; the more reliable and compact SARS-CoV-2 diagnostic tools such as commercial aptasensors will also be developed by incorporating other technologies. 30 min from the sample to result in the detection limit of 70-300 copies/µl [54] .

In the queue of detection of SARS-CoV-2 using CRISPR, Lucia et al., employed Cas12 for targeting RdRp, ORF1b, and ORF1ab genes, and used WH-human1 sequence as control. They employed synthetic RNA fragments and amplified isothermally using the RPA kit. Amplified fragments were mixed with Cas12, single guide RNA (sgRNA), and reporters. Detection of SARS-CoV-2 was performed using plate reader-based assay and paper strips. ssDNA reporters were labeled with fluorescein amidites (FAM)and biotin for fluorescence and paper assay, respectively. The limit of detection for both assays was ten copies/µl. The study was carried out using synthetic fragments because of the non-availability of real samples in the nearby regions.

The study represents the usefulness of CRISPR-Cas12 technology for the detection of the virus [55] .

Besides detection, CRISPR/Cas13 approach can also be used for the treatment of COVID19. 

While a variety of bioassays and biosensors have been developed, low-cost, disposable, or reusable biosensors are still needed that can quickly detect and accurately identify the SARS-CoV-2. The use of molecular imprinting technology as elements of biorecognition for the design of molecular impressed polymers (MIPs) offers a real alternative to antibodies due to their robustness and reproducibility [58] . The molecular imprinting method allows the host components to selectively, sensitively, and rapidly identify and detect the numerous molecules.

In the previous years, MIPs based sensors have formed an exciting horizon for surface modification methods by creating specific recognition cavities in the template molecules. They have to turn out to be fascinating, having sensitivity to small structural changes in the biomolecule structure. There are several research reports which are aimed at MIP based approach for medical diagnostics for the detection of various types of viruses such as Influenza virus, Dengue virus, Japanese encephalitis virus, human immunodeficiency virus, Hepatitis virus A and B, Adenovirus, Picornavirus, etc. [59] [60] [61] [62] [63] [64] [65] [66] .

For example, a MIPs based nanosensor was developed to detect human papillomavirus derived E7 protein by Cai and co-workers [67] . Their EIS data analysis showed the detection of E7 protein could be as low as sub-pg L -1 levels. Ma et al., have fabricated an electrochemical biosensor based on multi-walled carbon nanotubes modified MIPs for detecting HIV-p24 in human serum samples whose linear range was in the range of 1.0×10 -4 -2.0 ng cm -3 and limit of detection (LOD) was found to be 0.083 pg cm -3 [68] . Recently, Tancharoen et al., have proposed an electrochemical sensor based on graphene oxide polymers imprinted for the detection of Zika virus. The detection limit of this sensor was then compared to the commercial method and found the observed value to be similar to the reverse transcription [69] .

One of the key advantages of MIPs is their robustness, high selectivity, long-term stability, and cost-efficiency, which cannot be accomplished by using fragile biomolecules. MIPs were often used for selective and sensitive viral detection and the distinction of viral subtypes. Thus, it is anticipated that the many advantages of molecular imprinting technology should find its home in the frontlines of possible SARS-CoV-2 detection (Fig. 7) . There was no signal in negative and blank controls, and results showed that microarray could be used for the detection of SARS coronavirus [70] . As studies find out that there are many point accurately with 100% accuracy [74] . So, the microarray is a better technique for the detection of the virus with a high mutation rate. Microarray-based diagnostics can be a better approach for detection of SARS-CoV-2 because PCR based methods can detect only a few numbers of genes per experiment, but microarray can detect a higher number of DNA fragments synchronously. technique that amplifies DNA quickly with high specificity and simplicity at a constant temperature [75] . Now, after two decades of its development, LAMP seems a most promising approach to tackle the burden of COVID-19 testing as it is easy to use and does not require specialized machines such as PCR and costly reagents kits. The LAMP test can be done in a single tube at a constant temperature, hence very economical. DNA amplification can be detected by colorimetric, turbidity, or fluorescent-based methods [76] . There are increasing research reports which strongly hint at the possibility of employing the LAMP test for COVID-19 detection. Naama Geva-Zatorsky group from Israel developed a test for COVID-19 detection using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP) technique.

From the nose and throat swabs, SARS-CoV-2 can be detected by the developed test without RNA purification step [77] . The test was applied to 180 suspected patients, and results were found comparable to standard detection methods for COVID-19.

Another fascinating research report can also be seen on the preprint server (bioRxiv) in which a group from 'Weill Cornell Medicine' designed a 30-minute colorimetric LAMP test for COVID-19 detection and also developed a shotgun metatranscriptomic profiling platform technology for nose swabs [78] . Simplicity and low cost of the LAMP technique will increase its chance to be chosen as a favored diagnostic tool for COVID-19 detection, and this can be used as a POC device at workplaces, clinics, and clinics and entry points.

Testing is a valuable tool to control the spread of COVID-19. In our fight with the COVID- samples seem more appropriate for upcoming technologies. For sensitivity, reproducibility, and reliability, all COVID-19 testing technologies need to be studied and compared. The next few months will witness the comparison, advantage, and disadvantage of these technologies and how suitable they are for mass screening of healthy and suspected patients. Techniques such as LAMP, which requires minimum resources and reagents may be favored and increase the testing capabilities of hospitals and clinics. In the coming months, we can witness more comprehensive technologies that will provide more insights for viral and host biology besides just detection.

MIP, rolling circle amplification [79] real-time NASBA [80] AI, and smart phone are some examples of future technologies that can be pursued and optimized for the detection of COVID-19. Besides, development of therapeutics and vaccines for the control of COVID-19, the development of new testing technologies is equally important to fight against COVID and lessen the socio-economic harm, we all are facing in current time due to this pandemic.

The authors declare that there are no conflicts of interest. blue. Image has been taken from GISAID database (https://www.gisaid.org/).

Diagram showing the single stranded RNA genome and proteins present in coronavirus.

The trend of the mutation sites over time is displayed in the form of heatmap. Population frequency of site 28,144 located in gene ORF8 slowly raised from 0 to 35.98%, and then dropped to 16.96% as the epidemic progressed (see the area that gradually changes from blue to red). Image has been taken from http://bigd.big.ac.cn/ncov/variation/heatmap.

Phylogenetic tree shows the 4645 genomes sampled from December, 2019 to April, 2020 in different geographical regions. Each color represent specific country where genome has been sequenced. Image has been taken from GISAID database (https://www.gisaid.org/epifluapplications/next-hcov-19-app/). Electrode is fabricated and SARS-CoV-2 could be used as a template to create specific

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