key: cord-312278-rin733w4 authors: Wang, Yung‐Chih; Lee, Yi‐Tzu; Yang, Ting; Sun, Jun‐Ren; Shen, Ching‐Fen; Cheng, Chao‐Min title: Current diagnostic tools for coronaviruses–From laboratory diagnosis to POC diagnosis for COVID‐19 date: 2020-08-13 journal: Bioeng Transl Med DOI: 10.1002/btm2.10177 sha: doc_id: 312278 cord_uid: rin733w4 The Coronavirus‐2019 (COVID‐19) pandemic has put tremendous strain on healthcare systems worldwide. It is challenging for clinicians to differentiate COVID‐19 from other acute respiratory tract infections via clinical symptoms because those who are infected display a wide range of symptoms. An effective, point‐of‐care (POC) diagnostic tool could mitigate healthcare system strain, protect healthcare professionals, and support quarantine efforts. We believe that a POC tool can be developed that would be rapid, easy to use, and inexpensive. It could be used in the home, in resource‐limited areas, and even in clinical settings. In this article, we summarize the current state of POC tools and propose an all‐in‐one, highly sensitive POC assay that integrates antibody detection, protein detection, and serum cytokine detection to diagnose COVID‐19 infection. We believe this article will provide insight into the current state of POC diagnostics for COVID‐19, and promote additional research and tool development that could be exceptionally impactful. Gammacoronavirus, and some subgenera and species. 1 Coronaviruses are so named because they have characteristic club-shaped spikes projecting from their surface that resemble a solar corona when viewed with an electron microscope. 2 Three coronaviruses belonging to the genus Betacoronavirus, severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2, have been responsible for outbreaks of severe human respiratory tract infection over the past 20 years. [3] [4] [5] [6] First discovered in 2002, SARS-CoV causes the deadly disease known as SARS. 5, 7 This disease spread quickly from China to more than 30 countries, resulting in more than 8,000 cases and 700 deaths over 2 years. 5, 7 Initially spread from camels to humans, MERS-CoV causes the disease known as MERS. MERS was first observed in Saudi Arabia in 2012, and has since spread to several other countries. 5 December of 2019. 9 This virus, also named Coronavirus-19 (COVID- 19) after the year in which it was discovered, has spread globally and resulted in the ongoing 2019-20 coronavirus pandemic. 10 The diseases caused by these three coronaviruses display similar clinical symptoms including fever, dyspnea, malaise, myalgia, headache, and cough. 11 These viruses are notorious for their rapid progression toward acute respiratory failure that can be fatal for a significant number of infected patients. 11, 12 It is difficult for clinicians to distinguish them from each other based simply on their clinical presentations. The standard diagnostic tools for SARS, MERS, and COVID-19 are summarized in Table 1 . Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) is the gold standard approach for detecting the presence of viral RNA from respiratory specimens. [13] [14] [15] [16] It can only be effectively used for disease diagnosis during the acute phase of infection, after which viral titers drop below the limit of detection. The advantages of qRT-PCR include its high sensitivity and specificity for disease diagnosis. However, the accuracy of this approach relies on quality sample collection and transportation, 15 and it can only be carried out by highly skilled technicians using expensive instrumentation. In addition, limitations in testing capacity and the availability of reagents render qRT-PCR impractical for widespread application, a problem that is especially acute given the current pandemic. 14 A serological assay is used to detect previous infection in people who have developed antibodies after exposure to the virus. 14, 16, 17 It is a straightforward method for estimating the prevalence of a disease in a population by identifying individuals who have recovered from an infection and have established an immune response. This method is always employed during the convalescent phase of the disease but is not characteristically an element of early-stage infection diagnosis because of the lag time associated with the adaptive immune system in the production of specific antibodies against the virus. Serological assays have played an important role in examining SARS epidemiology 18 and other coronavirus outbreaks. 19 Several serological assay approaches exist including enzyme-linked immunosorbent assay (ELISA), immunofluorescent assay (IFA), and neutralization tests. The characteristic sensitivity and specificity of each approach varies. 14, 17 Another approach for disease diagnosis is virus isolation in cell culture. 14 Table 2 . The receptor-binding spike protein allows the virus to infect cells, 23 and mediates receptor binding and membrane fusion, which determines host tropism and transmission capabilities. 23 Compared to all previously described SARS-related coronaviruses (SARSr-CoVs), the S gene of SARS-CoV-2 is divergent with <75% nucleotide sequence similarity. 24 The other three structural proteins are more conserved than the spike protein and essential for general coronavirus function. 22 For detecting the presence of novel infectious diseases, the gold standard method has been the use of qRT-PCR for the detection of 29 Saliva has also been approved as a noninvasive specimen for detecting SARS-CoV-2. 30 Several companies in other countries have developed novel molecular POC assays for COVID-19. One example is the VitaPCR™ COVID-19 Assay produced by a Singapore company, Credo Diagnostics Biomedical. 46 This assay can provide results in 20 min and has obtained a CE mark. Another well-known test is the Vivalytic COVID-19 test (Bosch, Germany), which delivers results in less than 2.5 hr using multiplex PCR and μArray-detection to identify SARS-CoV-2. 47 The abovementioned serological POC diagnostics are designed to detect antibodies in serum. An advantage of some serological POC diagnostics is that they can be performed on blood samples obtained by fingerstick rather than venipuncture. These methods are inexpensive, easy to use, and can provide results within approximately 20 min. The travel restrictions and social distancing policies recommended by the CDC have left many people around the world confined to their homes. 48 Because of this, self-administered testing can be effective. One diagnostic kit that merits particular note is the "Pixel" COVID-19 At-Home Test (LabCorp, NC) authorized by the US FDA on April 21, 2020. 49 This kit allows people to collect a nasal swab sample at home and ship it back to the lab. This breakthrough innovation is an ideal diagnostic approach that is especially well suited for the COVID-19 pandemic. As it does not require an in-person visit to a medical professional, this collection method can protect frontline healthcare staff from exposure to symptomatic patients and conserve valuable time and personal protective equipment (PPE) resources. After LabCorp received a green light for their first at-home kit, 49 the US FDA approved the use of 10 additional at-home collection kits for COVID-19 as of June 30, 2020 (Table 4 ). 50-59 These kits allow people to self-collect nasal swab or saliva samples outside of a healthcare setting and transported it to the manufacturer's testing laboratory. All of these tests employ PCR to detect the presence of SARS-CoV-2 RNA, and can provide results within 72 hr. While these kits are promising, their application is still limited. First, because all collected samples using these kits must be transported to the manufacturer's laboratory for analysis, they take longer to provide results compared to POC assays conducted in healthcare facilities. Second, all of these at-home kits are designed to detect SARS-CoV-2 RNA during early-stage infection, but they are not used to determine the presence of antibodies. Kim's group at Korea Basic Science Institute also proposed a fieldeffect transistor-based biosensing device with a specific antibody against SARS-CoV-2-spiked protein for detecting SARS-CoV-2 in clinical NP swab specimens. 64 This device detected target SARS-CoV-2 antigen protein with a limit of detection of 1 fg/ml which is low enough for practical use. In addition to this new device, the targeted spike protein used in this technique also aroused our interest. Clustered regularly interspaced short palindromic repeat (CRISPR) CRISPR-Cas9 was adapted from a naturally occurring genome editing system in bacteria. The bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays that allow the bacteria to "remember" the viruses (or closely related ones). If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses' DNA, and then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus. The CRISPR-Cas9 system works similarly in the laboratory. In this approach, a small piece of RNA is created with a short "guide" sequence that attaches to a specific target sequence of DNA in a genome. The RNA also binds to the Cas9 enzyme. As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cas12) can also be used. 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