key: cord-0707393-6splke53
authors: Chen, Ying; Huang, Shengxiong; Zhou, Liuyan; Wang, Xin; Yang, Huan; Li, Wenqing
title: Coronavirus Disease 2019 (COVID‐19): Emerging detection technologies and auxiliary analysis
date: 2021-12-10
journal: J Clin Lab Anal
DOI: 10.1002/jcla.24152
sha: c768aa81decfad0cb7b8de0d7b0150076691d254
doc_id: 707393
cord_uid: 6splke53

The ongoing COVID‐19 pandemic constitutes a new challenge for public health. Prevention and control of infection have become urgent and serious issues. To meet the clinical demand for higher accuracy of COVID‐19 detection, the development of fast and efficient methods represents an important step. The most common methods of COVID‐19 diagnosis, relying on real‐time fluorescent quantitative PCR(RT‐qPCR), computed tomography, and new‐generation sequencing technologies, have a series of advantages, especially for early diagnosis and screening. In addition, joint efforts of researchers all over the world have led to the development of other rapid detection methods with high sensitivity, ease of use, cost‐effectiveness, or allowing multiplex analysis based on technologies such as dPCR, ELISA, fluorescence immunochromatography assay, and the microfluidic detection chip method. The main goal of this review was to provide a critical discussion on the development and application of these different analytical methods, which based on etiology, serology, and molecular biology, as well as to compare their respective advantages and disadvantages. In addition to these methods, hematology and biochemistry, as well as auxiliary analysis based on pathological anatomy, ultrasonography, and cytokine detection, will help understand COVID‐19 pathogenesis. Together, these technologies may promote and open new windows to unravel issues surrounding symptomatic and asymptomatic COVID‐19 infections and improve clinical strategies toward reducing mortality.

infected by direct contact of mucous membranes with hands that previously touched a surface contaminated with SARS-CoV-2. 4 It should be noted that SARS-CoV-2 can be transmitted through the oral-fecal route. 5 In addition, the existence of SARS-CoV-2infected asymptomatic persons adds complexity, uncertainty, difficulties, and challenges to epidemic prevention and control because of their invisibility and lack of clinical symptoms. The monitoring, tracking, isolation, and treatment of asymptomatic infected persons are crucial. 6 Dry cough, fever, shortness of breath, and respiratory distress 7 are the main manifestations of SARS-CoV-2 infection. In severe cases, patients progress to acute respiratory distress syndrome (ARDS). 8 A previous study 9 has shown that elderly people or individuals with underlying diseases are likely to progress to severe and critically severe pneumonia once infected with SARS-CoV-2.

Without timely treatment, they can easily develop acute respiratory distress syndrome, which causes respiratory failure. Therefore, early Rapid detection of COVID-19 is of great significance for epidemic control and clinical diagnosis. In this review, we provide a brief description of the rapid detection and analytical methods for COVID-19 and evaluate their advantages and disadvantages with regards to sensitivity, specificity, and ease of operation (Table 1) . Furthermore, we briefly summarize some of the auxiliary analytical methods that are of great significance for the study of this disease.

For safety purposes, personnel performing sampling must use eye protection goggles, gloves, a full-sleeved gown, an N95 respirator, higher-level respirator, or a face shield. 10, 11 The quality and time of sample collection substantially affect the test results. Therefore, professional training should be provided to the sampling personnel in order to reduce the number of false-negative results. Two types of samples are usually collected, depending on the nature of the probed molecules. 12 Ryan et al. used exhaled breath condensate (EBC) as a non-invasive lower respiratory tract sampling method and showed that the false-negative rate with this test was lower than that with nasopharyngeal swabs. 13 For antigen detection, samples are eluted in viral transport medium (VTM) or suspended in phosphate-buffered saline (PBS). 14 In nucleic acid-based detection, the nucleic acid needs to be prepared automatically when the RNA-containing samples are collected. The procedure of opening the lid and adding normal saline can be avoided by using a pharyngeal swab or nasopharyngeal swab with virus preservation solution. 15 The viral RNA is extracted after sample collection. 

choose simple and efficient nucleic acid extraction methods, such as a rapid method using proteinase K digestion and magnetic bead separation. 16 

Immunological technology for detection of anti-SARS-CoV-2 antibodies is better applied to serum samples collected in both the acute and recovery phases. The first serum sample should be collected as early as possible (preferably within seven days of disease onset), and the second should be collected 3-4 weeks after disease onset. The sample volume is 5 ml, and it is recommended to use a vacuum blood collection vessel without anticoagulant. 17

Currently, the analytical methods of COVID-19 mainly include methods based on PCR, methods based on immunological test, the single nucleotide detection methods (sequencing), imaging test, and other methods such as nucleic acid microfluidic detection chip.

The basic idea of traditional PCR is similar to the natural replication process of DNA, including denaturation, annealing, and elongation.

In the amplification process, single-stranded DNA is used as the 

Theoretically, compared with traditional PCR, multiplex PCR is more efficient, but the sensitivity is reduced. Therefore, the parameters such as primer concentration, annealing temperature, annealing time, and DNA polymerase content of multiple PCR reaction system need to optimize. By replacing primers, the result of the coverage at both regions targeted by the products can successfully be improved, which expected to be used for the detection of lower viral load SARS-CoV-2 samples. 34

As an unbiased technique that does not need pathogen culture, mNGS, based on next-generation sequencing, is a preferential method of pathogen detection. After high-throughput sequencing of DNA or RNA directly extracted from clinical samples, the sequences are submitted to databases for comparison and biological information analysis. 35, 36 A variety of pathogens such as bacteria, fungi, viruses, and parasites can be tested at once. Currently, pathogenic gene sequencing is the most commonly used method in clinical practice.

The mNGS approach could rapidly identify the novel coronavirus, which was the sole pathogen detected in the sample. 37 Ren et al. 38 

By translocating nucleotides through nano-scale pores, NTS can quickly discriminate single nucleotides of target DNA strands due

to the ion-current blockades 39 ( Figure 3 ). Solid-state nanopore and protein-pore channels have been studied extensively for NTS applications. 40 Recently, NTS was used for simultaneous detection of respiratory viruses, including SARS-CoV-2, 41 within 6-10 h. Sixtyone nucleic acid samples were tested with qPCR kits and NTS. The NTS method has been confirmed to have the capacity of higher sensitivity and accuracy, as well as monitoring mutated nucleic acid sequences. 42 Phylogenetic analyses demonstrated that SARS-CoV-2

is the closest relative of the bat SARS-related coronaviruses found in Chinese horseshoe bats.

3.3.1 | Colloidal gold immunochromatography assay, GICA

In recent years, GICA has become a fast-developing solid-phase marker immunoassay technique (Figure 4b ). 

Assay, ELISA ELISA relies on specific antigen/antibody interaction and enzymelinked amplification of the reaction signal ( Figure 4d ). The enzyme conjugated with the antibody or antigen retains its activity, while the immunological reactivity of either the antibody or the antigen is preserved. Thus, the enzyme-labeled antigen or antibody can combine with its respective cognate antigen or antibody. Upon addition of a colorless enzyme substrate, catalysis produces chemical reactions such as hydrolysis, oxidation, or reduction that form colored products that can be qualitatively estimated with the naked eyes or

quantitatively measured with a spectrometer. 45 This colored signal is proportional to the level of antibodies or antigens in the sample. 46 ELISAs are critical tools to define previous exposure and determine seroprevalence in a population. These assays, using plasma/serum, have proven to be specific and sensitive for the screening of individuals who have undergone seroconversion upon SARS-CoV-2 exposure as early as three days after disease onset. 47 With this method, the total Igs, IgGs, and IgMs against SARS-CoV-2 in plasma samples can be detected, constituting a significant sensitivity improvement for the diagnosis of COVID-19 patients. 48 ELISA has the advantages of rapid, sensitive, simple, and easy to standardize. However, difference exists between different in-house and commercial ELISAs. It is demonstrated that in-house ELISAs show higher specificity. 49 In addition, this method relies heavily on antibodies. Moreover, the primary antibody in the test has to be labeled with enzymes, but not every antibody is suitable for labeling, which may limit its application.

FICA is a new detection technique performed on a membrane support and based on specific antibody/antigen recognition. This new immunoassay not only retains the advantage of allowing rapid detection, such as the common colloidal gold strips usable on the spot, but also adds the high sensitivity of the fluorescent detection technology to improve the detection performance of immunochromatography. 50 This method has been tested for the detection of the nucleocapsid protein of SARS-CoV-2 within 10 min. To evaluate this approach, nasopharyngeal swab samples and urine from 239 participants were tested in parallel with a nucleic acid-based test as a reference standard. 51 The results showed that this method provides a rapid, simple, and accurate assay for the diagnosis of COVID-19 ( Figure 4a ). diagnosis. 52 CMIA was used to evaluate the immune response in COVID-19 patients co-infected with HIV-1 or HCV by testing plasma IgM and total Igs specific for SARS-CoV-2. 53 The results

showed that HIV-1-induced immune dysfunction can influence early SARS-CoV-2 clearance (Figure 4c ). The serology of total Igs, IgG, and

IgM after SARS-CoV-2 infection was studied with GICA, FICA, and ELISA methods. 54 The antibody level increased rapidly from 6 days postonset, which correlated with the decrease in the viral load. The antibodies showed the highest sensitivity for patients at the early stage of illness.

Chest CT scans and X-rays are important imaging methods for the preliminary diagnosis of chest diseases. 55 The results showed that COVID-19 pneumonia was characterized by chest CT abnormalities. In asymptomatic patients, rapid evolution from local unilateral to diffuse bilateral ground-glass opacity could even be observed, and complications developed within 1-3 weeks.

An automated COVID- 19 

The Compared with RT-PCR, this method amplifies DNA with high specificity, efficiency, and rapidity, without the need for expensive instruments, under isothermal conditions. These characteristics are a huge advantage for POCT.

The fastest way to test for the presence of a coronavirus may be by using the CRISPR genome editor, better known for adding or deleting DNA in cells. 67 Researchers have recently adapted accurate CRISPR-Cas12-based lateral flow assay for SARS-CoV-2 detection. 68 This method uses RT combined with LAMP assays, followed MS-based methods for viral antigen detection may deliver higher throughput and could complement RT-qPCR as a diagnostic tool. 75 

Biosensors are inexpensive, sensitive, rapid, miniaturized, and portable platforms. In recent years, biosensors developed rapidly, and today, they provide more approaches for the detection of SARS-CoV-2. 76 An article reported the development of a field-effect transistor 

Research shows that patients with COVID-19 have lower lymphocyte counts, higher leukocyte counts, and neutrophil-lymphocyte X-rays and chest CT scans, especially high-resolution CT scans, are better used for the detection of early changes in lungs. [100] [101] [102] Combined with medical history and other comprehensive analytic tools, clinicians can make early diagnoses and implement treatment.

However, due to infection control issues related to patient transport to CT suites, the inefficacy of CT room decontamination, and lack of CT availability in parts of the world, portable chest radiography will likely be the most commonly utilized mean of identification and follow-up of lung abnormalities. 103 This emergence brings greater challenges for the prevention and control of the pandemic. Detection methods to distinguish between these mutants need to be thoroughly studied, and whether the original detection targets are mutated also needs to be carefully examined. Lastly, multisectoral partnerships can minimize social and economic impact. 113 All scientists should work together and endeavor to overcome the pandemic, protect lives and livelihoods, and dedicate more research and development funding for vaccines, medicines, and diagnosis methods, as well as strengthen international scientific cooperation and leverage digital technologies.

We thank Accdon (www.accdon.com) for its linguistic assistance during the preparation of this manuscript.

All authors have completed the ICMJE uniform disclosure form. The authors have no conflicts of interest to declare.

YC conceived of and designed the study. YC, SXH, LYZ, and XW wrote the article. HY and WQL generated the table and figures.

All authors reviewed the article and approved its submission for publication.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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