PMC:7102556 / 2607-7639 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7102556","sourcedb":"PMC","sourceid":"7102556","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7102556","text":"1 Rapid identification of an emerging coronavirus\nIdentification of pathogens mainly includes virus isolation and viral nucleic acid detection. According to the traditional Koch’s postulates, virus isolation is the “gold standard” for virus diagnosis in the laboratory. First, viral culture is a prerequisite for diagnosing viral infections. A variety of specimens (such as swabs, nasal swabs, nasopharynx or trachea extracts, sputum or lung tissue, blood and feces) should be retained for testing in a timely manner, which gives a higher rate of positive detection of lower respiratory tract specimens. Then, immunological methods – including immunofluorescence assay, protein microarray, direct fluorescent antibody assay, MAb-based rapid NP (nucleocapsid protein) detection, semiconductor quantum dots, and the microneutralization test – which measure binding between the antigen from the whole virus or protein of the coronavirus and corresponding antibody, are easy to operate rapidly but have a lower sensitivity and specificity [3,4]. In addition, other immunological methods, including microneutralization ppNT assay (pseudo-particle neutralization) are highly sensitive and specific by using the gene coding for the coronavirus spike protein [5,6]. In the case of 2019-nCoV, viral research institutions can conduct preliminary identification of the virus through the classical Koch’s Postulates or observing its morphology through an electron microscopy [7]. Serology could also be used to identify the virus when 2019-nCoV-associated antigens and monoclonal antibodies are developed in the future [[7], [8], [9]]. All the examples above are traditional virus detection methods.\nViral nucleic acids can also be used for early diagnosis. The following are some of the new coronavirus detection methods. Polymerase chain reaction (PCR) is a molecular biological diagnosis technology based on the sequence of nucleic acids. The full gene sequence of 2019-nCoV has now been obtained [10], so patients who are suspected of being infected with 2019-nCoV [8] can be diagnosed by pan-coronavirus PCR for virus identification [11]. Reverse transcription polymerase chain reaction (RT-PCR) is a technology combining RNA reverse transcription (RT) with polymerase chain amplification (PCR) of cDNA. A duplex RT-PCR assay can be used to detect SARS-CoV and MERS-CoV using pUC57SARS-pS2 and pGEM-MERSS2 as templates, respectively [12]. Also, samples collected from the upper respiratory tract (oropharyngeal and nasopharyngeal) and lower respiratory tract (endotracheal aspirate, expectorated sputum, or bronchoalveolar lavage) of suspected 2019-nCoV patients can be diagnosed by RT-PCR [8]. Reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR), quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), Real-time RT-PCR (rtRT-PCR), and one-step rtRT-PCR were all further optimized [[13], [14], [15], [16]]. These optimized RT-PCR methods were used to detect the MERS-CoV envelope gene (upE) and the open reading frame 1a (ORF1a) or open reading frame 1b (ORF1b) genes separately. However, rtRT-PCR was used to identify 2019-nCoV through preliminary and validation detection of its E gene, RNA-dependent RNA polymerase (RdRp) gene, and N gene [17].\nThere are other molecular-based detection techniques in addition to RT-PCR and similar optimized detection techniques. For example, reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an RNA amplification technique that detects the N gene of MERS-CoV and the ORF1a gene [18]. One-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP) is the optimized RT-LAMP [19], while RT-LAMP-VF is the deformation of RT-LAMP [20], which is the combination of reverse transcription loop-mediated isothermal amplification and vertical flow visualization strips. Both are used to detect the N gene of MERS-CoV, making detection easier, faster, more efficient and highly specific. Besides these three methods, reverse transcription recombinase polymerase amplification assay (RT-PRA) is also used to identify MERS-CoV [21].\nFinally, the following multiplex tests can detect both coronaviruses and other viruses. MCoV-MS (multiplexed CoV mass spectrometry) uses an array matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system to accurately identify known human coronaviruses (hCoVs) and to provide phylogenetic evidence for emerging unknown hCoVs [22]. Another new test method, arch-shaped multiple-target sensor, is used to amplify the target for rapid identification of pathogens in clinical samples [23]. The method can detect hCoVs, and Zika and Ebola viruses. The last one, the paper-based colorimetric assay, uses Pyrrolidinyl Peptide Nucleic Acid-induced silver nanoparticles (AgNPs) aggregation of pathogen DNA testing [24]. The color change of AgNPs can distinguish between MERS-CoV, Mycobacterium tuberculosis (MTB), and human papillomavirus 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