key: cord-0985214-qr2m698q
authors: Mukhopadhyay, Chitrangada Das; Sharma, Pramita; Sinha, Koel; Rajarshi, Keshav
title: Recent trends in analytical and digital techniques for the detection of the SARS-Cov-2
date: 2020-12-28
journal: Biophys Chem
DOI: 10.1016/j.bpc.2020.106538
sha: 1364b35d914b141f5d1a017fbf00f0196d4b076f
doc_id: 985214
cord_uid: qr2m698q

The current global outbreak of COVID-19 due to SARS-CoV-2 is an unprecedented humanitarian crisis. Considering the gravity of its impact there is an immediate need to develop a detection technique that is sensitive, specific, fast, and affordable for the clinical diagnosis of the disease. Real time Polymerase Chain Reaction (RT-PCR)-based detection platforms are contemplated to be the gold standard to detect viral RNA. However, that may be susceptible to errors, and there is a risk of obtaining false results, which ultimately compromises the strategy of efficient disease management. Several modern techniques exhibiting assured results with enhanced sensitivity and specificity against the SARS-CoV-2 associated viral components or immune response against it have been developed and may be implemented. The review deals with the conventional RT-PCR detection techniques and compares them to other detection platforms viz., biosensor based detection of antigens, fluorescent or colorimetric detection systems including CRISPR-Cas 13 based SHERLOCK kit, CRISPR Cas-9 based FELUDA test kit, CRISPR DETECTR kit, Next Generation Sequencing or microarray-based kits. These modern techniques are great as a point of care detection methods but should be followed by RT PCR based detection for the confirmation of COVID-19 status.

instrument. After collection, there is a chance of contamination of the sample which should be considered with the utmost care.

Then viral RNA is extracted by solid-phase extraction method where stationary phase consists of a silica gel membrane on which the RNA binds and later eluted. If the sample is either contaminated or contain viral RNA inadequately, the PCR may give false results. It is difficult to extract RNA if the viral load is very low in the sample and carrying the sample to the laboratory may cause denaturation of the RNA [19] . So, skilled personnel is required for the collection, handling, and RNA extraction.

At least two important enzymes with high efficacy are required for the protocol of RT-PCR viz., reverse transcriptase and DNA polymerase. Several manufacturers offer improved quality of enzymes, the right choice of the enzyme is very crucial for nucleotide amplification.

During early infection, a swab may contain very little amount of viral RNA. The minor mistake in the collection and extraction of the sample can create a major difference in the result of test analysis since DNA is amplified exponentially in each step of PCR. SARS-CoV-2 can mutate rapidly [20] . Therefore, the mutations in the probe and the primer targeted regions in the genome of SARS-CoV-2 may lead to false-negative results. results and thus affecting the performance of the assay. This problem could be avoided by targeting multiple genes. To meet up the huge demand for detection kits, initially, several real-time RT-PCR kits for detection of SARS-CoV-2 have been rapidly approved, but their quality varies. Some of the RT-PCR based detection kits targeting genes are highlighted in Table 2 .

The RT-PCR technique, however, has some flaws and constraints. Viral preservation solutions vary by different manufacturers so the sensitivity and quality of the detection kits, all may lead to the risk of obtaining false-negative results and thus compromising the accuracy of detection [16] . More importantly, the viral load may be undetectable in patients with persistent and frequent antiviral medications like anti-HIV drugs [21] . Therefore, some novel virus detection techniques with more precision than real-time RT-PCR are needed to be developed and implemented for accurate detections.

Loop-mediated isothermal amplification or LAMP is a new isothermal nucleic acid amplification technique having a high efficacy. The LAMP-based detection technique shows enhanced sensitivity and specificity as it possesses the feature to amplify exponentially and six target sequences, which are detected by the four different primers concomitantly, and where the ORF1b region was selected to detect the virus SARS-CoV-2 which was then amplified by LAMP technique using 6 primers, and the result so obtained after amplification has undergone gel electrophoresis [23] . It was found that the sensitivity and the rate of detection were the same as the conventional PCR-based detection technique. In another study, the LAMP technique was successfully implemented by executing agarose gel electrophoresis for diagnosing HCoV-NL63, with desired specificity and sensitivity in clinical specimens and cell cultures. The detection limit came around a copy of the RNA template per reaction [24] . The amplification is usually noticed by the fluorescence dye or magnesium pyrophosphate which allowed real-time analysis of the fluorescence or turbidity of pyrophosphate which has efficiently surpassed the endpoint detection limitations [25] .

The RT-lamp technique was refined with the help of a quenching probe (QProbe), which was used to detect signs and have similar efficiency to real-time RT-PCR [26] . To make the LAMP-detection technique more reliable researchers replaced the intercalating dye and were implemented for sequence-specific validation of LAMP amplicons on a real-time basis [27] .

However, the optimum temperature for LAMP-assay's efficient performance is around 65°C, thus limiting its utilization. Another type of LAMP-based technique involving the use of phosphorothioated primers, known as PS-LAMP, was developed which allowed the more effective formation of the hairpin and growing concatamers' termini extension, thus enabling the technique to function at relatively lower temperatures, and specific and sensitive detection

The rolling circle amplification (RCA) can amplify the signal of each circle up to 10 9 -fold within 90 minutes, and therefore, RCA has gathered remarkable attention in the determination of nucleic acid. To detect SARS-CoV using RCA, an effective assay was set up in solid as well as liquid phases, and on a fewer number of respiratory specimens, the initial results were put up. The RCA can be carried out under isothermal conditions without obtaining any false-positive results and requires minimum reagents, thus making it a better alternative for detection techniques based on PCR [10] . However, this technique is not deployed for the detection of the SARS-CoV-2 virus and should be worked on for its further clinical trial.

Due to the affordable instrumentation, easy operation, fast analysis proficiency, and minimum or no sample pre-treatment, the biosensors can serve to be a potential method to rationalize diagnostic protocols for viruses [12] . Therefore, biosensing techniques have been developed into the detection tools for specific, rapid detection of pathogens.

The virus biosensors based on affinity interactions are categorized into four groups [29] based on the viral targets and affinity reagents. They are immune/ antibody-based biosensors, DNA-based biosensors, cell-based biosensors, and antigen-based biosensors. Immunosensor is a kind of biosensor which mainly depends on the antigen-antibody specific interactions resulting in generations of responses that can be quantified and assessed. Aptamers also can recognize the viral antigens of interest with high affinity and precision. These aptamers either include peptide molecules comprised of approximately 40 to 60 bases or single-stranded DNA/RNA or peptides. The Cell-SELEX (Systematic Evolution of Ligands by exponential enrichment) method is used to select aptamers from the mixture of random oligonucleic acid [30] . The peptide aptamers are manipulated proteins comprised of specific target binding J o u r n a l P r e -p r o o f Journal Pre-proof sites situated at peptide loops or surfaces, and thus, these peptide aptamers mimic the function of antibodies [31] . By acquiring a three-dimensionally preferred orientation that distinguishes targets based on fine structural variations, the virus targets are identified by the DNA aptamers [32] . RNA aptamer-based detection of C-terminal region of N protein of SARS virus was developed by Ahn et al, which could detect the antigen up to 2pg/ml concentration and with a dissociation constant of 1.65nm [33] .

The diagnostic applications of antigenic probe-based biosensors mostly rely on the detection of surface antigens such as nucleocapsid and envelope proteins or the whole virus particles using antigen-specific antibodies originated from patient sera [34, 35] . The reliability of these antigen probe-based biosensors is constrained by the number of antibodies produced during various stages of infection, similar to the conventional enzyme-linked assays and serological evaluations.

Nucleic acid hybridization is the principle on which the DNA-based biosensors function.

Short single-stranded DNAs consisting of around 20-40 base pairs are immobilized on the surface of the sensor retaining their reactivity, accessibility, and stability towards the target strands of DNA. Later peptide nucleic acids (PNAs) and their use were emphasized in biosensing techniques. PNAs are remarkably stable and can hybridize rapidly, strongly, and specifically, which makes them an essential and propitious tool in the detection of DNA [36] . 

Pathogens like viruses and bacteria and the components associated with them, which are airborne and cannot be seen with naked eyes; pose a significant threat to health even when present in small amounts. To quantify the total bioaerosol and accurately detect the presence of SARS-CoV-2, plasmonic biosensors have been developed [39] . A localized surface plasmon resonance biosensor (LSPR) relying on succinimidyl-ester-functionalized gold nanoislands (SEF-AuNIs) was developed to quickly analyze the total concentration of bioaerosol [40] .

The combination of LSPR sensing transduction and plasmonic photothermal (PPT) effect along with the plasmonic biosensor, possessing a dual functionality, gives an assured solution to the issue of SARS-CoV-2 detection. The AuNIs can carry out the detection of selected sequences from SARS-CoV-2 sensitively via the nucleic acid hybridization when functionalized with cDNA receptors. The AuNIs chip generates thermo-plasmonic heat when they are illuminated at their plasmonic resonance frequency, to enhance the sensing performance. The escalation in the in situ hybridization temperature is regulated by the localized PPT heat, thus facilitating the precise differentiation of two gene sequences that are alike. High sensitivity with a lower detection limit as low as 0.22 pM is exhibited by the LSPR biosensor, thus enabling accurate detection of the specified target in a mixture of multiple genes. As this virus sensor can provide fast detection and continuous monitoring of

A novel biosensor for the detection of the S1-spike protein of SARS-CoV-2 was developed for the steady monitoring of affected patients, especially the asymptomatic ones. The changes in the bioelectric responses of membrane-engineered mammalian Vero cells consisting of the human chimeric spike S1 antibody is measured by the biosensor according to the bioelectric recognition assay principle [42] and the membrane engineering-based molecular identification technology [43] . It was demonstrated that a substantial and marked change in the cellular bioelectric properties occurred due to the adherence of spike protein to the membrane-bound antibodies. Additionally, any cross-reactivity against the SARS-CoV-2 nucleocapsid protein was not reported. The detection with high specificity (pg/ng level) and selectivity of viral antigen as possible within 3 minutes using this biosensor, and did not require any prior processing of sample [44] . The reduced cell viability is a great concern for cell-based biosensors.

Graphene is a chemically stable two-dimensional material exhibiting extremely well electronic properties. The Gr-FET (graphene field-effect transistor), which has an unmatched sensitivity, was combined with highly specific antigen/antibody interaction for the development of immunosensors, which can rapidly detect SARS-CoV-2 with ease. GrFET involves the use of FET structure with graphene acting as the channel material and the body sequencing detection kit, which can diagnose and detect the novel coronavirus as well as other coronaviruses and respiratory disorders, and thus can rapid detection of viral sequences can be attained. The NMPA (National Medical Products Administration) of China sanctioned the testing kits called NGS COVID-19 kit, designed by the company BGI Biotechnology

[14].

"Clusters of regularly interspaced short palindromic repeats" abbreviated as "CRISPR" have the repeated sequences of nucleotides and small bits of spacer sequences and CAS stands for CRISPR-associated proteins which function as nuclease enzyme. These are found as bacterial defense systems protecting them foreign invaders and are now widely employed for RNA editing, gene therapy, and even in the detection of viral genome detection. In recent years, CRISPR is being widely employed in the in vitro diagnostic field because of its allele specificity, which is critical for its successful application in the development of high-precision treatment and diagnosis. CRISPR-based DETECTR assay targets the E and the N2 gene of SARS-CoV-2 based on the CRISPR-Cas 12 system [57] . A lateral flow strip was developed where the probes were tagged with the streptavidin-biotin complex. This can detect as low as 10 copies per microlitre of viral RNA at about 40 minutes. CRISPR-Cas13 is another such system used for rapid nucleic acid-sensing [58] . It was demonstrated in a recent study that Cas13 could affect and knockdown the genomes of many single-stranded RNA viruses that reside in mammals. The enzyme can be programmed to about an hour. Beginning from nucleic acid extraction, which is commonly employed for qRT-PCR tests, it is supposed to be completed in 1 h. Very similar to SHERLOCK diagnostic TEST another All-in-one dual CRISPR-Cas 12a (AIOD-CRISPR) assay was developed which is a very rapid and ultrasensitive visual detection method. The limit of detection is up 

A hypothesis is being made regarding detection of COVID-19 asymptomatic subjects, using forced-cough cell phone recording harvesting Artificial Intelligence [63] . A survey was done Table 3 . Nowadays multiplexed RT-PCR kits manufactured by several Healthcare units can detect two target sequences simultaneously, reverse transcription and PCR amplification are done in a single step with no cross-reactivity with other human coronaviruses, and thus required time also is reduced.

It is essential to choose the detection methods which are appropriate, accurate, sensitive, rapid, and specific. The collection of samples and various detection techniques are represented in Figure 2 . Techniques like LAMP and Microarray-based detection have limitations in their implementation, e.g., LAMP requires a high temperature for its functioning, whereas microarray-based platforms are quite expensive. Thus, the development of more practical techniques with enhanced efficiency is the need of the hour. Significant attempts have been and are still being made to improve the diagnosis of COVID-19. The RT-PCR based methods are still the most reliable and can be a gold standard, and at least at present none of the new methods based on the sophisticated technology can be 'alternative' to real-time RT-PCR based methods. Modern sophisticated techniques definitely can be additional and supportive tools for diagnosis of COVID-19 and they can be the best for certain purposes, but would not be 'alternative' to real-time RT-PCR based methods for the first line of diagnosis of COVID-19 (detection of SARS-CoV-2). Some of the methods may be potentially applicable to asymptomatic or mild COVID-19 cases clinically, but may not be considered as the gold standard, or alternative to real-time RT-PCR. The RT may give a negative report when the test is performed at an early stage of infection due to lack of viral particle in the swab, secondly, improper collection of the swab. The antibody kits are based on predominantly IgG, or both for IgM and IgG. But when we are looking at picking up 

and its viruses-a statement of the Coronavirus Study Group

Drug treatment options for the 2019-new coronavirus (2019-nCoV)

An Introduction to COVID-19

Springer Briefs in Applied Sciences and Technology

Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction, Cold Spring Harbor symposia on quant. boil

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding, The Lancet

Electrochemical SARS-CoV-2 Sensing at Point-of-Care and Artificial Intelligence for Intelligent COVID-19 Management

Loop-mediated isothermal amplification of DNA, Nucleic acids research

Detection of severe acute respiratory syndrome coronavirus RNA in plasma during the course of infection

An ultrafast one-step assay for the visual detection of RNA virus

Recent trends in in-vitro nanodiagnostics for detection of pathogens

Comprehensive detection and identification of seven animal coronaviruses and human respiratory coronavirus 229E

Assay Techniques and Test Development for COVID-19 Diagnosis

RT-qPCR Testing of SARS-CoV-2: A Primer

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays, Jl of molecular endocrinology

Frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction

Molecular Diagnosis of COVID-19: Challenges and Research Needs

Challenges in Laboratory Diagnosis of the Novel Coronavirus SARS-CoV-2

Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis, Jl of clinical microbiology

Rapid detection of the severe acute respiratory syndrome (SARS) coronavirus by a loop-mediated isothermal amplification assay

Development of loop-mediated isothermal amplification assay for detection of human coronavirus-NL63

Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation

Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification

Phosphorothioated Primers Lead to Loop-Mediated Isothermal Amplification at Low Temperatures, Analytical chemistry

Review-Chemical and Biological Sensors for Viral Detection

In vitro selection of RNA molecules that bind specific ligands

Development of the PANI/MWCNT Nanocomposite-Based Fluorescent Sensor for Selective Detection of Aqueous Ammonia

RNA aptamer-based sensitive detection of SARS coronavirus nucleocapsid protein

Gold nanoparticle-based electrochemical magnetoimmunosensor for rapid detection of anti-hepatitis B virus antibodies in human serum

Label-free optical diagnosis of hepatitis B virus with genetically engineered fusion proteins

Direct detection and discrimination of double-stranded oligonucleotide corresponding to hepatitis C virus genotype 3a using an electrochemical DNA biosensor based on peptide nucleic acid and double-stranded DNA hybridization

Monitoring viral-induced

Functionalized Plasmonic Biosensor To Reveal Different Characteristics at Three Locations in Switzerland

Dual-Functional Plasmonic Photothermal Biosensors for Highly Accurate Severe Acute Respiratory Syndrome Coronavirus 2 Detection

Nanotechnology-Based Surface Plasmon Resonance Affinity Biosensors for In Vitro Diagnostics

Bioelectric recognition assay (BERA)

Visualization of the membrane engineering concept: evidence for the specific orientation of electro inserted antibodies and selective binding of target analytes

High mobility graphene ion-sensitive field-effect transistors by noncovalent functionalization

Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirusspecific human monoclonal antibody, Emerging microbes & infections

Electrical probing of COVID-19 spike protein receptor binding domain via a graphene field-effect transistor

Design and application of 60mer oligonucleotide microarray in SARS coronavirus detection, Chinese science bulletin Kexue tongbao

Generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-PCR and nonfluorescent low-density microarray

Initial performance evaluation of a spotted array Mobile Analysis Platform (MAP) detection of influenza A/B, RSV, and MERS coronavirus, Diagnostic microbiology and infectious disease

In vitro biochemical and thermodynamic characterization of nucleocapsid protein of SARS

Host-membrane interacting interface of the SARS coronavirus envelope protein: Immense functional potential of C-terminal domain

Orientation of immobilized antigens on common surfaces by a simple computational model: Exposition of SARS-CoV-2 Spike protein RBD epitopes

Evaluation of Nucleocapsid and Spike Protein-Based Enzyme-Linked Immunosorbent Assays for Detecting Antibodies against SARS-CoV-2

Rapid Microbiology. Next-Generation Sequencing Delivers 30x Faster COVID-19

CRISPR-Cas12-based detection of SARS-CoV-2

Nucleic acid detection with CRISPR-Cas13a/C2c2

Programmable Inhibition and Detection of RNA Viruses Using Cas13

CRISPR-Cas system for biomedical diagnostic platforms

All-in-One Dual CRISPR-Cas12a

Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

Rapid, field-deployable nucleobase detection and identification using FnCas9, bioRxiv

COVID-19 Artificial Intelligence Diagnosis using only Cough Recordings

A Framework for Biomarkers

Based on Coordination of Speech-Production Subsystems

Signs of COVID-19 may be hidden in speech signals

Testing for SARS-CoV-2 (COVID-19): a systematic review and clinical guide to molecular and serological in-vitro diagnostic assays

Comparison of seven commercial RT-PCR diagnostic kits for COVID-19