Detection Techniques of SARS-CoV-2

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1		Yulin Deng	--	3950	2022-06-27 14:07:33	\|
2	format correct	Catherine Yang	Meta information modification	3950	2022-06-29 02:55:20	\| \|
3	format correct	Catherine Yang	-2 word(s)	3948	2022-06-30 09:02:08	\|

This entry is adapted from the peer-reviewed paper 10.3390/bios12060410

The SARS-CoV-2 coronavirus, also known as the disease-causing agent for COVID-19, is a virulent pathogen that may infect people and certain animals. The global spread of COVID-19 and its emerging variation necessitates the development of rapid, reliable, simple, and low-cost diagnostic tools. Many methodologies and devices have been developed for the highly sensitive, selective, cost-effective, and rapid diagnosis of COVID-19. The diagnosis platforms into four groups: imaging, molecular-based detection, serological testing, and biosensors was organized. Each platform’s principle, advancement, utilization, and challenges for monitoring SARS-CoV-2 are discussed in detail. In addition, an overview of the impact of variants on detection, commercially available kits, and readout signal analysis has been presented. This would expand our understanding of developing advanced diagnostic approaches to evolve into susceptible, precise, and reproducible technologies to combat any future outbreak.

SARS-CoV-2 coronavirus variants diagnosis immunoassays

1. High Throughput Sequencing

Next-generation sequencing (NGS)-based strategies are used to trace the evolutionary history and to investigate the chain of transmission of disease during the outbreak. The complete genomic sequence of SARS-CoV-2 was released in January 2020 ^[1]. The sequence has over 82% similarity to those in SARS-CoV and bat SARS (SL-CoV) ^[2]. Nasopharyngeal swabs from the respiratory tract were used to analyze the viral load ^[3]. The NGS proved significant for the diagnosis of severe infections or the patient that carries pathogens of unknown origin ^[4]. Recently, variants of SARS-CoV-2 have also been traced by SNP genotyping ^[5]. This ultrasensitive high-throughput sequencing method is expensive, time-consuming, and dependent on stringent laboratory equipment, limiting its use in the clinical diagnosis of COVID-19. Therefore, a cost-effective and fast testing procedure is needed to develop for further investigations.

2. Imaging

Initially, imaging techniques were used to diagnose and observe the severity of COVID-19 infection. The Computed Tomography (CT) scan images of 63 patients were taken from a hospital possessing some variant results of affected lungs images. The symptoms observed included affected lobes, patchy consolidation and fibrous stripes, and some complex irregular solid nodules while enlarged, which varied differently in different patients. This imaging was a supportive method for the diagnosis before the submission of the genomic sequence of the virus ^[6]. However, this high-resolution CT scan is an indicative but non-confirmative method due to the lack of difference between viral and non-viral infection of respiratory tracts, which limits its diagnostic applications ^[7]^[8]. So, there is a need to diagnose the disease at the molecular level with more specification and authenticity at the individual level before further treatments.

Advanced imaging optical coherence tomography (OCT) has been applied for the diagnosis of acute respiratory failure. This real-time 3D imaging technique is used to have a visual demonstration of alveolar compartments and air pathways. The potential application of OCT for COVID-19 patients can be devised to observe the lungs of the patients ^[9]. Alternatively, the application of photoacoustic imaging has potential for the analysis of the inflammatory markers in the lungs for the clinical diagnostics ^[10].

3. Microarrays

Nucleotide-assisted microarray-based detection strategies have been adopted for the detection of newly invaded coronavirus into the SARS family in the past years. The 60 mer oligonucleotides were designed for the detection of coronavirus from the SARS family. Thirty designed oligonucleotides of 60 mer (TOR2) were able to detect the whole genome of the submitted new coronavirus strain ^[11]. In another study, a microarray was designed for the detection of a mutated spike gene (27 single nucleotide polymorphism), which was correlated to the pathogenicity and epidemiology of the disease by SARS-CoV. The designed SNP DNA microarray served as the detection tool with ≈100% accuracy. A non-fluorescent-based low-density oligonucleotide assay was utilized for the point-of-care testing of SARS-CoV-2. This approach has a comparable sensitivity with RT-PCR with the limit of detection of 15.7 copies per reaction for the other HCoVs ^[12]. Similarly, a more sensitive platform was used for the diagnosis of respiratory infection and MERS that may also be useful for the detection of SARS-CoV-2 ^[13].

4. Molecular Assay-Based Diagnosis

Nucleotide-based detection methods are considered as most reliable for individual-level testing. The developing kits have been designed in many ways of targeted sites, sets of primers, the principle of the test, and final signal readout in a way to be more specific.

4.1. PCR-Based Methods

The polymerase chain reaction (PCR) methods applied for nucleic acid amplification testing include a list of delicate steps such as sample collection and its transport, viral extraction, amplification, and signal readouts. The primary step of sample collection is performed by the swabs, or forks containing CDC recommended materials nylon or polyester fibers on a plastic stick over wooden shafts or, even better, the calcium alginate sticks to avoid contaminations. At the beginning of the pandemic, a short supply of swabs created a bottleneck. To overcome this challenge, 3D-printed nasopharyngeal swabs were designed ^[14]. A clinically validated medium, phosphate buffer, was used as stable transportation of the viral medium. This can keep samples stable for up to 18 h for qPCR testing without compromising the detection of N, S, and Orf1ab targets ^[15].

After pre-analytical steps, analytical accuracy depends on the nucleic acid extraction before amplification and final signal readouts. False-negative testing also increases if the contaminated or low viral load is further processed. Conventionally, organic phenol-chloroform extracts nucleic acid by simple degradation of protein and non-nucleic acid parts by the action of SDS and proteolytic enzyme K ^[16]. In contrast, a more efficient acid-pH method is utilized for the nucleic acid extraction of SARS-CoV-2 ^[17]. In this method, the sample is directly incubated with proteinase K and heated at 98 °C for 5 min ^[18]. Subsequently, the PEARL (precipitation enhanced analyte retrieval) is performed to break down the non-nucleic acid components by a lysis solution which yields a precipitate of alcohol-based nucleic acid ^[19]. Due to the use of various sample reagents and centrifugation steps that elute bonded nucleic acid to the column supports, silica-functionalized magnetic microbeads were applied on a testing LionX system platform to devoid the elution step.

The extracted genome is then added to the target gene primer, probe, and a master mixture. Amidst other amplification, the qRT-PCR is the most reliable clinical testing for detecting infectious pathogens alternative to Northern blotting-based assays ^[17]. The previously reported coronaviruses were detected by the same method ^[19]^[20]^[21]^[22]. In the prevailing period, RT-PCR is a gold standard for several facts such as the specificity of the particular target strain of the SARS-CoV-2 without cross-reactivity with preceding human CoVs. The key to the specificity is primer-probe binding to the target gene; a comparative evaluation of the sensitivity of different primer-probes sets was checked according to the target gene (N, N2, and N3). RNA isolation were performed by using Vero cell culture, QIAamp Viral RNA Mini Kit (#1020953, Qiagen, Hilden, Germany) and RT-ddPCR was performed using primers and probe published by the Chines CDC. The target genes Orf1ab (Beijing, China), NIID_2019-nCoV_N (Tokyo, Japan), and 2019-nCoV-N2 (US-CDC) were found more sensitive to apply for the clinical analysis; later, excluding the target N3 did not affect the sensitivity of the assay ^[23]^[24]. The multiple target detection makes it more accurate and high throughput to apply on multiple parallel assays (384-wells plate).

Being most frequently available, it is a highly significant and sensitive, direct, and rapid procedure in routine practice ^[25]. The amplification of cDNA through PCR proceeding to quantification and detection was performed on conventionally accepted agarose gel or DNA sequencing procedures ^[26]^[27]. Despite some clinical limitations such as time consumption and dependency on the instrument or trained workers, in the past studies, a single-tube RT PCR method was applied for the identification of respiratory pathogens such as HCoV-OC43 and HCoV-229E targeting the gene Orf1b ^[28]^[29]^[30]. In addition, RT-qPCR has other disadvantages of potential biological safety risks, nuclear extraction, and sophisticated laboratory equipment such as biosafety cabinets, which are often available in a few central laboratories, and sample transportation and processing to the laboratories ^[29].

According to FIND diagnostics, a total of 435 SARS-CoV-2 RT-PCR-based kits have been designed, and among these, 235 kits have been approved by the Food and Drug Administration (FDA) for the commercial applications. Most of these have multiplex targets to attain more sensitive results. More targets serve as the templates to transcribe into complementary DNA, which further act as templates for the extension. During extension, the Taq polymerase released the reporter dye from the 3′quenching dye after cleaving annealed probes, which increases the fluorescence relevant to the amplified part. The companies and researchers are modifying the techniques by improving various steps of the molecular assay. Unique molecular testing is introduced in less than 13 min ID Now ^TM by Abbott, based on the rapid isothermal amplification of the target pathogen to generate a short segment of the target pathogen, which is later recognized by its fluorescent probes. Moreover, various modifications such as the automated extraction of nucleic acids, amplification procedures, and better signal readouts can improve the test run time, minimize the cost, process a large sample, reusability, and multiplex detection.

Isothermal Amplification

Isothermal amplification is an excellent alternative to PCR amplification to avoid highly expensive thermal cyclers, and it is a rapid and efficient amplification process to amplify nucleic acid sequences at a constant temperature. Amplicons produced by this procedure are far better at producing nucleic acid base nanomaterials to utilize in biomedicines, biosensing, and bio imaging. For biosensing DNA, RNA, cells, peptides, some molecular, and sub-molecular species are the selected targets.

RT-Loop-Mediated Isothermal Amplification (LAMP)

LAMP assays have been used in many studies for the detection of SARS-CoV and also for other human coronaviruses, particularly HCoV-NL63 ^[31]^[32]. Quantitative RT-LAMP tests were designed for the early analysis of SARS-CoV ^[33]. For instance, a rapid, reliable, reusable, and robust point-of-care RT-LAMP was introduced for the detection of SARS-CoV-2, which is 12 times more sensitive and 10 times less expensive than the conventional RT-PCR ^[33]. The RT-LAMP assay has several advantages over RT-PCR, including direct detection without the laborious step of RNA extraction ^[33]^[34], lack of cross-reactivity with other respiratory pathogens ^[35], and colorimetric and fluorescent-based signal detection within 20–30 min at a temperature of 63–65 °C ^[36]^[37]. The improved and more specific method was used for the detection of MERS-CoV in past studies to overcome the problem of turbidity due to the production of pyrophosphates (white precipitates) during the polymerization reaction. The fluorescent dyes intercalate in double-stranded amplicons, which cause non-specificity to produce a signal from primer dimerization or non-primer involvement ^[38]. Therefore, a temperature-specific DNA amplification LAMP method and quenching probes were introduced to track the true signal in the reaction for the specific diagnosis of MERS-CoV ^[39]. A similar method has been utilized for the detection of SARS-CoV-2 ^[40]^[41]. Yet, this method required a fluorophore and a quencher for labeling, because the toehold is located at the end position of the hairpin stem. So, it may cause an improper quenching due to high background signals. In addition, these molecular techniques have limitations for full-length genome analysis ^[36]^[37]. In recent advancements, LAMP has been improved by introducing artificial intelligence-based results interpretation. A smart palm top diagnostic device was designed to produce automatic image and algorithmic data processing through artificial intelligence. Such devices have improved the run time of tests and pH-dependent colorimetric detection. The specificity, sensitivity, and reliability of the test procedure were performed on 200 suspected patients and were provided by NHS to validate against the target RdRp ^[42].

5. CRISPR Based Detection

A nucleic acid-based detection by clustered regularly interspaced short palindromic repeat (CRISPR (Cas-9, Cas-12a, Cas-12b, and Cas-13)) is a gene-editing technique that aids researchers to add, delete, or modify the genome at a required specific domain on the gene map ^[43]^[44]^[45]^[46]. A previous study implanted to delete the RNA-based viruses by using Cas-13 from mammals ^[47]. The same procedure was used for the detection of the dengue, Zika virus ssRNA genome. The SHERLOCK protocol is a specific and highly sensitive enzymatic reporter unlocking for a portable and multiple nucleic acid base detection from clinical samples. The whole essay includes a series of reactions of pre-amplification of DNA or RNA and subsequent enzymes followed by Cas-12 or Cas-13 mediated detection through colorimetric and fluorescent signals. The observed run time of the assay is less than 15 min, and total signal readouts are provided in less than 60 min ^[48]. Moreover, Cas12a, Cas12b, and Cas 13a nucleases cleavage activity are to develop point-of-care testing of SARS-CoV-2 in different studies’ workflows schemes after modifying according to required setups. A more sensitive RT-PCR assay detection was performed (10 copies/reaction in 40 min) by RPA-mediated DNA amplification and signal amplification by CRISPR-Cas-12a ^[49]. Another CREST, Cas-13-based detection was performed on low-cost thermocyclers and accessible enzymes such as Taq polymerase based on fluorescence signal amplification to detect ten copies/μL ^[48]. All-in-one CRISPR-Cas 12a (AIOD-CRISPR) assay was modified without pre-amplification steps of RNA directly; all incubated components in a single reaction with 4.6 copies of SARS-CoV-2 within 40 min ^[46]. Regardless of the advantages, the procedure has some limitations, such as expertise dependent on the preparation of reaction components and reaction steps such as protein purification and RNA extraction. Moreover, multistep amplification and digital quantification may affect precise testing. Another rapid (≈30 min), inexpensive, and easy to handle diagnostic technique introduced the CRISPR-Cas-12 platform as DETECTR (DNA endonuclease-targeted CRISPR Trans reporter for the detection of viral infections) ^[45]^[50]. DETECTR is based on the lateral flow assay alternative to PCR testing, with 95% positive predictive agreement and 100% negative predictive agreement for the viral detection in <40 min ^[49].

6. Limitations of Molecular Diagnosis

It is hard to validate all molecular diagnostic techniques in a specific way. There are several pre-analytical and analytical factors responsible for false-negative testing such as low viral loads, viral shedding time, the sample collection site (nasal swabs, upper respiratory tracts, or lower respiratory tract), and the time of sample collection day after on-set of symptoms (0–7, 8–14 or ≥15 days) ^[51]. For example, samples from lower respiratory tracts and the sputum have the highest positivity rates of 93% and 72%, respectively, while samples taken from nasal swabs, upper pharyngeal, feces, and blood have corresponding low positive rates of 63%, 32%, 29%, and 1%. A systematic study reported up to 54% of initial false-negative RT-PCR testing rates ^[52]. In another study conducted in New York, the clinical performance of SARS-CoV-2 was evaluated by retesting the negatively tested patients on the same day. An increase in positive rate from the negative rate was observed due to inadequate sample, incorrect sample collection, and stochastic bias from the low viral load ^[53]. The detection sensitivity of RT PCR assay is varied from different target regions because highly conserved region targets and multiple targets could be applied to reduce invalid false-negative results. Mutations in a primer region also affect primer-probe binding ^[54]. A one-step quantitative assay has 10 times greater sensitivity for the N-gene over open reading frame 1 ab. At the same time, CDC proposed that the set of primer probes for the N-gene (N1, N2, and N3) and E-gene have better sensitivity for N1, N2, and the E-gene over N3 and RdRp. The impartial application of metagenomic assay and unauthentic information on possible coinfections would urge the researchers to develop improved treatment. Moreover, CRISPR-based strategies continue to grow, but their extensive clinical application for the rapid detection of pathogens can be restricted in limited resources settings. While the sensitive PCR tools are not available or lack suitable sample collections, a significant viral load for a small genetic expression of the virus hampers their applications. Moreover, the turnaround time of sequencing is far greater than the widely accepted techniques. Metagenomics, RT-PCR and CRISPR hold great promise as a distinct diagnosis, especially in patients carrying novel respiratory pathogens. All these three methods involve a 1 h RNA extraction step. The remaining RT-PCR and CRISPR workflows are also designed to prepare the library, while the mNGS flow has several parts with different periods of time: 3 h for library preparation, 18 h for mNGS sequence and 3 h for data analysis. Therefore, the turnaround time (TAT) for RT-PCR, CRISPR and mNGS is about 3, 2, and 24 h, respectively. Moreover, RNA extraction is a must by depending upon biohazard safety labs to handle sensitive testings. Despite efficient diagnostics, 1-step detection devices or biosensors for point-of-care testing is the need of the hour ^[4]^[55]. Recently, portable CRISPR-based COVID testing miSHERLOCK was introduced by combining two unrelated CRISPR nucleases (Cas 13 and Csm 9) in a tandem assay on a portable microfluidic chip ^[56]. A minimum dependency on the instrument, alternative to PCR testing, and portable chip with sensitivity (100 copies/µL) within 20 min can be advancement toward molecular-based testing.

7. Signal Readout Methods

Signal readouts are the quantification steps of the reaction yield for an efficient diagnosis. These methods include optical, electronic, colorimetric, and electrochemical, following their different strategies. The means used to read signals varied according to the design. The possible means of optical signaling are naked-eye, color change, fluorescence emission, and optical reflections, which are usually carried on the platforms such as microfluidic devices, microplate readers, spectrophotometers, cartridges, strips, and cupids. Florescence and colorimetric-based optical signaling are the most applied strategies during the development of SARS-CoV-2 detecting kits. The conjugated fluorescent dyes nonspecifically bind to the double-stranded amplicons and non-specifically to the single-stranded target probe to quantify the amplification product visually. These signal readers are integrated electronically with smart gadgets or plate readers to measure fluorescence. The improvement in signal detection methods improves the sensitivity of the procedure ^[57]. The conventional signal readout method is highly delicate to interpret the final results. Conventionally, detected SARS-CoV gives one copy per reaction, while SARS-CoV-2 detected by fluorescent signal readout gives 100 copies/mL. A portable detection platform was developed by incorporating PCR with a smartphone ^[58]. Further CRISPR methods also used the modified DNA fragments with fluorescent probe quencher combos.

8. Serological Testing

Antibodies are the proteins that are produced in the body within 1–3 weeks of the infection. Combat to the disease depends upon the antigenicity (the ability of an antigen to induce an immune response) of the invaded pathogen and the host’s immunogenicity (responses from the host to produce antibodies). The challenges such as unavailability of RT-PCR, denaturation of viral RNA during sample collection and extraction steps, shortage of primers, and mutations in viral genomes urge us to use an alternative, cheaper testing method. Serological testing procedures are helpful to trace the contact and the extent of body response toward infection as well as conduct an epidemiological survey and identify past and post-infection responses of the body. An accessible sampling collection and transportation of blood samples taken from finger sticks or veins compared to the PCR testing sample collection of mucus from the nasopharyngeal tracts is more sensitive to carry in ultra-care test performing procedures. Both testing procedures have their sampling, testing formats, and targets within the limitations to use. A comparative study reveals RT-PCR 36.6% results, and 17.3% serological assay tested positive while 6.8% showed positive serological testing and negative RT-PCR results ^[59]. So, it is not a confirmed shot to hit. Nevertheless, uncertainty regarding serological assays is much higher than in other methods. Serological testing includes antigen-based and antibody-based detection.

8.1. Antigen-Based Detection

The SARS-CoV-2 contains the spike protein (S-protein), nucleoprotein (N-protein), envelope protein (E-protein), and membrane protein (M-protein) ^[60]. The S-protein exhibits a heavily intact glycosylated bond ^[61] and is a maximum mutation region, which may affect the effectiveness of the immunoassays. The N-protein is abundant to contribute to the identification of virus particles and RNA packages ^[62]. The antibodies IgA, IgG, and IgM against the N-antigen have an optimal expression in corona patient serum to run Immunoblast assays. Previously, N-antigen is taken as an impeccable detection marker for the prognosis of SARS-CoV and MERS ^[63], while these have a more genomic expression of N-antigen than SARS-CoV-2. The SARS-CoV-2 protein detection by the mass spectrometer method identifies samples collected from the gargles of the patients containing the viral load of 105 to 106 genome equivalents/μL, which is much smaller than the RT-PCR viral load requirement, suggesting its significance as an efficient diagnostic tool ^[64]. Comparing and selecting the more sensitive and specific recombinant antigens is essential before designing the serological assays. The S-antigen is involved in viral neutralization due to the domain (S1, RBD, and S2); as a result, it generates protective neutralizing antibodies, which can be commonly immobilized to develop assays ^[65]. The S-antigen-based assays’ sensitivity and specificity are higher than those of the N-antigen and have a lower cross-reactivity ^[66]. In a study, a quantum dot-based lateral flow immunoassay was found to be more sensitive for the detection of N-antigen than a particular conventional ELISA with a relevant recorded LOD of 100 ng/mL and 10 ng/mL ^[67]. Furthermore, an immunochromatographic fluorescence assay was designed to detect N-antigen from the patients’ nasopharyngeal swabs and urine samples in 10 min. The urine samples of corona patients were detected with 73.6% (14/19) N-antigen; overall, a 100% positive results accuracy was shown even after the negative nucleic acid test results of the same samples ^[68], while the urine sample of SARS recorded 8% ^[69]. The possibility of low viral protein expression in body fluids of SARS-CoV-2 patients verifies the data on a more significant number of samples ^[70]. It is more convenient to test the N-antigen of SARS-CoV-2 than other antigens because it is abundantly expressed in serum, although both antigens have greater immunogenicity to the viral proteins.

8.2. Antibody-Based Detection

A lateral flow immunoassay was designed to target the serum antibodies of (IgG/IgM) receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. This sensor enabled the simultaneous screening of IgM and IgG from the serum of the patient with a precision of 90.63% and sensitivity of 88.66%. Samples from a fingerstick, plasma of venous blood, and serum gave consistently positive results in less than 30 min ^[68]. An IgM ELISA against N-protein was designed for the carrier’s humoral response to the infection from the onset of the symptoms until the patient recovered. The recorded positive detection rate between the sets of 0–7 days, 8–14 days, and 14–21 days from 208 plasma samples was 188/208 (90.4%) for IgM and 194/208 (93.3%) for IgA against the target. Comparatively, the IgG detection rate was 162/208 (77.9%) within seven days. Gradually, the rates stopped increasing within 14 days for both (markers of acute infection), while IgG kept increasing after 21 days. Considerably, the detection of IgM and IgA identifies the current infection and IgA for the post-infection response to the body. IgM’s positive rate reached 93.1% (54/58) within the 5th day after the onset of the symptoms. Therefore, it is considered a competent testing method compared to the qPCR testing with an increased positive rate of qPCR ^[51]^[69]. Similarly, the response found for SARS-CoV in 2003 for IgM developed in 3–6 days, and IgG was detectable after eight days of onset of symptoms ^[71]^[72]. However, IgM gives the first line of defense against infection than IgG ^[73].

9. Challenges in Immunoassay-Based Diagnostics

Immunoassay is an efficient detection method yet has some uncertainties. Serological testing helps us to interpret the disease severity, ascertain the immunization level, identify updates against the past infection, and measure the vaccine efficacy. Each person before and after the onset of symptoms until the recovery has their immunity responses. A cascade of antibodies generation according to specific antigens is produced at the different periods within 0–7, 7–14, and 14–21 days after infection. The seroconversion-based detection depends upon the antigenicity of antigens. A similar trend of antibody generation was observed in the past infections such as SARS-CoV and MERS. Due to specific antibodies, IgM against RBD is much stronger compared to the IgM-N-protein depending upon the antigenicity. Because it usually takes up to 1–2 weeks for the human B-cells to produce and secrete antibodies in the blood serum after a viral infection and during the early stages of the COVID-19 pandemic, an optimal level of antibodies was not known ^[74]. The antigenicity of both antigens is highly noticeable, as SARS-CoV patients had higher anti-N than anti-S opposite to SARS-CoV-2, which has higher anti-S [nAbs of RBD] because of the highly conserved spike region. IgG rates against both antigens are higher than the IgM, while total seropositivity (IgG/IgM) against anti-S is higher than anti-N. In conclusion, S-antigen has higher antigenicity than N-antigen; while entering into the host cell, the viral (S1, RBD, and S2) domains become fused with the ACE 2, resulting in a quicker immunological response to develop protective neutralizing antibodies.

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