key: cord-0742588-m2mwxntx
authors: Jiang, Congshan; Li, Xiaowei; Ge, Changrong; Ding, Yuanyuan; Zhang, Tao; Cao, Shuai; Meng, Liesu; Lu, Shemin
title: Molecular detection of SARS-CoV-2 being challenged by virus variation and asymptomatic infection
date: 2021-03-26
journal: J Pharm Anal
DOI: 10.1016/j.jpha.2021.03.006
sha: bae2e12bfd44bba4bd259d2ba8010d50b61b3e6a
doc_id: 742588
cord_uid: m2mwxntx

Coronavirus disease 2019 (COVID-19) has been a pandemic for more than a year. With the expanding second wave of pandemic in winter, the continuous evolution of SARS-CoV-2 has brought new issues, including the significance of virus mutations in infection and the detection of asymptomatic infection. In this review, we firstly introduced several major SARS-CoV-2 mutations since the COVID-19 outbreak and then mentioned the widely used molecular detection techniques to diagnose COVID-19, primarily focusing on their strengths and limitations. We further discussed the effects of viral genetic variation and asymptomatic infection on the molecular detection of SARS-CoV-2 infection. The review finally summarized useful insights into the molecular diagnosis of COVID-19 under the special situation challenging by virus mutation and asymptomatic infection.

. At least three mutations have potential biological significance [17] . The mutation N501Y occurs at the critical contact residues between RBD and ACE2, and it may enhance the binding affinity of SARS-CoV-2 to human ACE2 [21] . The P681H mutation locates near the S protein's furin cleavage site, which is a significant region of known infection and spread, but the specific effect of this mutation on J o u r n a l P r e -p r o o f SARS-CoV-2 is unclear [17, 22] . Besides, the deletion of the two amino acids at [21] . K417 of the S protein in SARS-CoV-2 is a unique residue that interacts with D30 of human ACE2 to form a salt bridge in the central contact area [24] . This area is the most significant difference in the RBD-ACE2

complex between SARS-CoV-2 and SARS-CoV, which will enhance the binding affinity of SARS-CoV-2 and human ACE2 [24, 25] . Deep mutational scanning showed that K417N mutation has little effect on human ACE2 binding affinity [21] . At present, The SARS-CoV-2 S protein sequence was obtained on the website (https://cov3d.ibbr.umd.edu) and analyzed by PyMol software.

Up technologies, high-throughput sequencing, hybridization technologies and so on [26] .

An analysis of 112 detection assays for SARS-CoV-2 RNA found that 90% of these detection assays use PCR or RT-PCR technology, 6% use isothermal amplification technology, 2% use CRISPR technology, and 2% use hybridization technology [26] .

Rapid and accurate molecular tools detecting the SARS-CoV-2 viral nucleic acid are in great need for efficient public health responses to the viral threat and are considered the first step to combat COVID-19.

RT-qPCR uses specific primers to amplify and identify trace amounts of viral genetic material in samples, which is considered as the gold standard for SARS-CoV-2 virus identification due to its shorter assay time, high sensitivity, and specificity. Based on the SARS-CoV-2 genome information, various RT-qPCR assays were designed to detect certain specific viral gene regions with the RNA extracted from clinical samples [27] , and some of them were validated and recommended by authorities such as the Centers for Disease Control and Prevention (CDCP) in China.

Its subordinate National Institute for Viral Disease Control and Prevention released its international teams worked at speed to distribute reliable RT-qPCR kits for better diagnostics with different techniques, specifications, and turnaround time all along [28] .

In the daily clinical practice of SARS-CoV-2 screening in multiple countries,

RT-qPCR plays a vital role as a game-changer and has been so effectively, extensively, and massively employed (by vast orders of magnitude) that even for many medical staffs it is referred to as "nucleic acid detection" itself. However, on some occasions, the RT-qPCR could be troubled with its false-negative effect. It was reported that some individuals with the signature CT change of ground-glass opacity was not initially identified as SARS-CoV-2 positive until the second repeat of RT-qPCR detection with their swab samples [29] . According to the information from 81, 554

reported confirmed SARS-CoV-2 cases of COVID-19 by March 31 st , 2020 in mainland China, a single round of RT-qPCR could only help diagnose 61.8% of the total cases. The high false-negative rate of nearly 40% mainly comes from the entire sampling process to the end of PCR testing within the diagnosis, but not the false-negative rate of PCR test kits themselves [30] . Hence the inpatients with high clinical suspicion of COVID-19 should participate in the second round of test, even when they get a negative result on the initial RT-qPCR test. Such protocol is presently widely applied. For example, in China, the medical staffs follow such rule of 2 RT-qPCR swabs and 2 chest radiographs (CXRs) with more than 48 hours apart for inpatients and 2 RT-qPCR swabs (at the beginning and the end of quarantine) for the J o u r n a l P r e -p r o o f population during the 14-day quarantine period at home or the assembly site [31] .

Suitable samples for RT-qPCR could come from diverse origins, including nasopharyngeal swabs, oropharyngeal swabs, sputum, feces [30] , blood, pleural fluid [32] , bronchoalveolar lavage (BAL) [33] , and breastmilk [34] Many scientists are working on RT-qPCR optimizing, and there are some good news. For example, microneedle-based oropharyngeal swabs are introduced to improve the quality and quantity of virus collection for COVID-19 testing [37] . For high-scale screening in regions at low prevalence, pooled tests could be applied for financial consideration [38] . Similar techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) have been developed to overcome certain RT-qPCR shortcomings. This assay could detect as less as 50 copies per μL in viral transport medium within 30 min [39] . In a RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swabs after COVID-19 testing, an excellent performance with a sensitivity of 97.5% and a specificity of 99.7% was validated [40] . The reverse transcription-recombinase polymerase amplification (RT-RPA) method also exhibits its excellent potential. The inexpensive enhanced RT-RPA could detect as less as 5 copies within 45 minutes, does not require RNA purification, and support the high-scale detection [41] . It was optimized to one-copy sensitivity, field-deployable, and could simultaneously detect N and S genes from SARS-CoV-2 [42] . Droplet digital PCR is also established in a small sample volume of 50 nasopharyngeal swabs, and the results showed that this direct quantitation could reach a sensitivity of 93.33% and specificity of 100% [43] . Such methods might be promising if validated with a larger number of specimens.

High-throughput sequencing of the genome is a powerful technique to obtain the full-length genome sequences of SARS-CoV-2 isolated from patients, which offers critical clues for potential transmission and virus evolution [1, 44] . Although the equipment dependency and high-cost limit its application, its application is irreplaceable as a potent detection tool. For example, SARS-CoV-2 viral genome might stay persistently detectable in swabs from the upper respiratory tract via J o u r n a l P r e -p r o o f RT-qPCR even after several weeks since the patients were fully cured [45] . Such RT-qPCR re-positive results are confusing since it is difficult to tell whether these individuals are still actively infectious or already cured. This confusing phenomenon could be interpreted by the nanopore sequencing result of the SARS-CoV-2 genome from individuals [35] , which showed the degraded viral genome could be responsible for a relatively lower transmission risk for those re-positive outpatients [46] .

There have been several CRISPR/Cas-based methods established for SARS-CoV-2 detection. Generally speaking, such CRISPR-Cas-based strategy used Cas proteins' nuclease activity after the gRNA was specifically bond to viral genes such as ORF1ab, and the produced fluorescent signals released from the cleaved single-stranded (ss) RNA reporter probe help reflect the existing SARS-CoV-2 [47] .

Multiplex CRISPR/Cas13a-based diagnosis methods have been developed for SARS-CoV-2, and some of them could reach a nearly 100% (single-copy) sensitivity [48] . Its fulfill period of 40 minutes is very time-saving. No thermo-cycle help reduce the potential cross-sample aerosol contamination within the equipment and detection facilities. In Cas13-based detection system optimized by Arizti-Sanz et al. [49] , it was validated with 90% sensitivity and 100% specificity with a turnaround time of 50 minutes. It is more exciting that CRISPR-Cas12a assay could bring about an ultrasensitive, instrument-free, and visual detection (for few copies) within as soon as CRISPR-Cas12a and RT-RPA techniques have been developed for potential portable use [51] .

Besides the RNA detection system, some DNA-based platform using DNA nanoswitch with very reasonable cost and detection speed has been designed in labs [52] . So far, multiple platforms and strategies with a high diversity of assay systems were developed for nucleic acid detection of SARS-CoV-2, including in a tube, a Chip, a box, a cartridge, and even on a drone. J o u r n a l P r e -p r o o f

Immunochemical testing is a powerful technique for detecting viral infections.

Although RT-qPCR is the most commonly used detection method for SARS-CoV-2 virus identification [54] , it also leads to potential misdiagnosis. So other effective complementary detection methods are needed considering the false negative and false positive results of RT-qPCR.

For medical surveillance, the need for serologic tests on SARS-CoV-2 antigen and antibody emerged. A multiplex diagnostic pipeline named ReScan was developed to perform proteome-wide profiling of SARS-CoV-2 antigens enriched by pan-98 patients' sera, and 2 essential antigens, including spike and nucleocapsid proteins, were found out, which are widely used in SARS-CoV-2 serologic assays [55] . Besides, some wireless electrochemical platforms were established to detect SARS-CoV-2 antigen using two-dimensional monoelemental materials (Xenes), named as SARS-CoV-2 Rapid-Plex [56] . Such electrochemical antigen detection method brings us a quick, low-cost way for SARS-CoV-2 detection.

Infection of SARS-CoV-2, just like many other viruses, could stimulate the human immune system for defense. Antibodies such as IgG and IgM are produced during the infection process with a well-recognized pattern and specific binding to N and S viral proteins [57] , which could become the molecular basis of the present SARS-CoV-2 antibody detection. The ELISA assay of 2019-SARS-CoV-2 antibodies J o u r n a l P r e -p r o o f such as IgG and IgM was one of the earliest validated methods to explore individuals' infection state [1] . The COVID-2019 patients displayed significantly evaluated IgG and IgM, with an outstanding cut-off value compared with healthy control in ELISA assay. SARS-CoV-2-specific IgM and IgG antibodies could be detectable in serum between the first to second weeks after the onset of symptoms, and the IgG specific to SARS-CoV-2 spike protein stays detectable in serum up to 2 months after symptom onset [58] . Seropositivity for IgG and IgM was detected in 32% of patients with mild symptoms after 2 weeks of symptom onset and 3% of healthy blood donors [59] . A study on 175 once-infected outpatients and asymptomatic individuals found that their SARS-CoV-2 antibodies, including IgG, are quite long-lasting until they progressively decrease after five months post-infection [60] . IgM antibodies reduced sharply, while serum and saliva IgG antibodies stably remained in most COVID-19 patients for at least 3 months post symptom onset [61] . As early as in February, 2020, a rapid recommended to apply to the entire general population just as we did with RT-qPCR tests due to its high false-positive ratio against real positive results [68] . SARS-CoV-2 serology is very complicated, for example, the cross-reactivity with seasonal (non-severe acute respiratory syndrome) coronaviruses [69] as well as autoantibodies in autoimmune diseases [70] could contribute to the false-positive result, while immunodeficiency can lead to false-negative reaction [71] .

Besides, there is still much uncertainty about the situation when the result of SARS-CoV-2 nucleic acid is negative, anti-SARS-CoV-2 IgM is negative, and anti-SARS-CoV-2 IgG is positive. He/she might be once-infected while recovering.

Any false-negative for nucleic acid may leave out an infected individual experiencing an infection with few or mild symptoms. The individual could obtain the preexisting immunity (anti-SARS-CoV-2 IgG) through vaccine inoculation [72] , and a few individuals might even produce preexisting neutralizing antibodies without J o u r n a l P r e -p r o o f SARS-CoV-2 pathological infection after naturally exposed to deactivated zoonotic SARS-CoV-2. Hence, it is vital to distinguish the situation of preexisting and de novo immunity before and/or after the serological antibody tests [73] . Indeed, such exceptional immunological condition of healthy individuals makes the management of pandemic surveillance more complicated than we could ever imagine. The test results of healthy and safe individuals with antibody protection could be very similar to those of most worrying asymptotic infected ones. Therefore, we suggest that the molecular diagnosis strategy should always combine the RT-qPCR based nucleic acid detection and the serological antibody tests whenever financial conditions permit.

Although the people from all walks of life have made huge efforts and great advances against the pandemic, COVID-19 has been becoming rampant to threaten human society. The reasons are complicated from both sides, human and virus. But two things probably contribute much more to the situation, virus mutations and existence of asymptomatic cases. also be used to detect mutations of viruses [25, 74] . However, some traditional detection methods are often complicated and time-consuming. Another significant effect of SARS-CoV-2 mutation is that it might decrease the detection sensitivity.

Artesi et al. [75] reported that the failed detection of SARS-CoV-2 E protein gene in 8 patients is associated with the C-to-U transition at position 26340 of the SARS-CoV-2 genome. A study uploaded to bioRxiv in January 2021 conducted an in silico survey on SARS-CoV-2 sequence variability within the binding regions of primer/probe and performed RT-qPCR detection using synthetic RNA containing these mutations [76] .

It highlights the necessity of genomic monitoring for SARS-CoV-2 and selection for RT-qPCR primers. Moreover, there is an urgent need for more efficient, easy-to-operate, and straightforward detection methods to monitor SARS-CoV-2 mutations.

As far as we concern, the presently reported mutations in virus variants are mainly located in the S gene region. As we mentioned before, for many of the individuals for any mutations.

In addition to SARS-CoV-2 variations, asymptomatic infection is another challenge for virus detection. According to the information from 81, 554 reported confirmed SARS-CoV-2 cases of COVID-19 by March 31 st 2020 in mainland China, 1.2% of cases are so-called asymptomatic infected [30] . In many other countries, the ratio is as high as 10-30% [78] . For some of the asymptomatic infected individuals, their virus copies within the upper respiratory tract might still be accumulating below the limit of nucleic acid detection with the specific anti-SARS-CoV-2 IgG and IgM also undetectable. This is why the "false negative" results are very likely found in asymptomatic infected individuals. However, it was also believed that most transmission incidences could come from the pre-symptomatic stage and the asymptomatic infections. Hence it is even more vital to determine asymptomatic infections with high specificity and sensitivity.

Hence, establishing efficient high-throughput tests for the detection of asymptomatic carriers is more urgent. Moreover, effective screening depends mostly on the testing frequency and reporting speed, secondarily on high test sensitivity. Thus, extensive, frequent, and large-scale screening with short sample-to-answer time should be above all [79] . Accordingly, we consider that RT-qPCR with even insufficient sensitivity could still play its colossal role. A further recommendation is choosing a combination of appropriately timed and multiple rounds of RT-qPCR (e.g., once per week on Day 0, 7, and 14 within the quarantine period at home or the assembly site) and serological antibody testing for anti-SARS-CoV-2 IgM and IgG (e.g., on Day 0 and Day 14 within the quarantine period at home or the assembly site).

In addition, for those who just passed the 14-day quarantine period at home or the assembly site, a self-disciplined isolated lifestyle with daily health checking for any abnormal subclinical signs, mask-wearing, and minimum public social contact is benefit to public health. Any of their voluntary tests for RT-qPCR and serological antibody testing should always be highly recommended (encouraged at moral and financial levels).

Based on the strengths mentioned above and limitations of various methods to diagnose SARS-CoV-2, a comparison chart of their turnaround time cost, dependency on sophisticated equipment, requirements for the capacity of reagent supplies, and the diagnosis value are analyzed and displayed in Table 1 .

Based on the recent advances, we reviewed the widely used molecular diagnostic techniques for SARS-CoV-2, primarily focusing on their strengths and limitations in discussing several concerning issues such as genetic variation of virus and asymptomatic infection during our global battle against COVID-19. Some more sensitive, efficient, easy-to-operate, and straightforward detection methods are still in urgent demand for COVID-19 due to the virus variation and asymptomatic infection.

J o u r n a l P r e -p r o o f

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Molecular immune pathogenesis and diagnosis of COVID-19

Emerging genetic diversity among clinical isolates of SARS-CoV-2: Lessons for today

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus

A new coronavirus associated with human respiratory disease in China

AXL is a candidate receptor for SARS-CoV-2 that promotes J o u r n a l P r e -p r o o f infection of pulmonary and bronchial epithelial cells

HDL-scavenger receptor B type 1 facilitates SARS-CoV-2 entry

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Structures of the SARS-CoV-2 nucleocapsid and their perspectives for drug design

Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity in COVID-19 Convalescent Individuals

Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) membrane (M) protein inhibits type I and III interferon production by targeting RIG-I/MDA-5 signaling

Genetic Recombination, and Pathogenesis of Coronaviruses

Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA

Mutations Strengthened SARS-CoV-2 Infectivity

Emergence of a SARS-CoV-2 variant of concern with mutations in spike glycoprotein

Estimated transmissibility and severity of novel SARS-CoV-2 Variant of Concern 202012/01 in England

Spike mutation D614G alters SARS-CoV-2 fitness

D614G Spike Mutation Increases SARS CoV-2 Susceptibility to Neutralization

Making Sense of Mutation: What D614G Means for the COVID-19 Pandemic Remains Unclear

Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

Recurrent emergence and transmission of a SARS-CoV-2 Spike deletion ΔH69/V70

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactions

Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies

Assay Techniques and Test Development for COVID-19 Diagnosis

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Coronavirus and the race to distribute reliable diagnostics

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First Detection of SARS-CoV-2 by Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay in Pleural Fluid

Delayed diagnosis of COVID-19 in a 34-year-old man with atypical presentation

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Rapid isothermal amplification and portable detection system for SARS-CoV-2

A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples

An enhanced isothermal amplification assay for viral detection

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A smartphone-read ultrasensitive and quantitative saliva test for COVID-19

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Base-Paired Double Helixes

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Test sensitivity is secondary to frequence and turnaround time for COVID-19 screening

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This work was supported by the National Natural Science Foundation of China 

The authors declare that there are no conflicts of interest.