5  Advances in laboratory diagnosis
The development of rapid diagnosis is urgently needed during the early stages of an epidemic to enable community-based screening and consistent course development monitoring. During the early stage of the COVID-19 epidemic, diagnosis was based on symptoms and chest radiology. The majority of COVID-19 cases showed bilateral distribution of patchy shadows and ground-glass opacity on CT images. However, 17.9% of non-critically ill patients and 2.9% patients with severe illnesses showed no radiologic abnormality on hospital admission [39].
RT-PCR has been extensively deployed for the detection of SARS-CoV-2 RNA fragments, and has been recommended for the diagnosis of acute infection. However, false-negative results may be problematic, as they can jeopardize the whole community [40]. The fact that a patient who was RT-PCR negative developed clinical symptoms of COVID-19, suggesting that insufficient amounts of viral genome collection for amplification, or mutations in the NP and ORF of SARS-CoV-2, may lead to false-negative results [41].
Serological analysis is another typical method for COVID-19 diagnosis [42], [43], [44]. Accurate serological tests would enrich our understanding of the personal process of viral exposure, particularly in the monitoring of asymptomatic individuals. Nucleocapsid (N)- and S-specific immunoglobulin M (IgM) and IgG could be used for the detection of SARS-CoV-2 infection because of the progressively elevated titers after symptom onset (typically peaking at Days 7–10). The combined detection of N- and S-specific IgM and IgG may increase the detection rate at early stages. In fact, the combination of N- and S-induced IgM and IgG could be detected in up to 75% of infections within the first week of symptom onset [45]. The sensitivity of combined detection of N-IgM and N-IgG, or N-IgG and S-IgG, reached 94.7% within the second week [45]. At Week 3, S-IgG titers were significantly higher in non-intensive care unit (ICU) patients than in ICU patients [45]. Moreover, the expression level of N-IgG was significantly higher in ICU patients, although S-IgG titers were higher in patients with moderate illness. These findings provide hints for prognosis prediction [46].
To accelerate the clinical diagnostic testing of COVID-19 (particularly for population-based survey or point-of-care testing), a rapid, accurate, and portable detection method based on clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associate system (Cas) has been developed. The CRISPR/Cas system is an adaptive immune system in archaea and bacteria that defends against foreign genetic elements [47], [48]. CRISPR allows a programmable protein to attach onto the target site assisted by a guide RNA for cleavage of the target sequence [47], [49], [50], [51]. CRISPR/Cas12a-based detection has been established together with SARS-CoV-2-specific CRISPR RNAs (crRNAs) targeting the orf1a, orf1b, N, and E genes, and a single-stranded DNA (ssDNA) reporter labeled with a quenched green fluorescent molecule has been developed [48]. The fluorescent molecule is cleaved in the presence of a trace amount of SARS-CoV-2 sequences; more importantly, the results can be determined by the naked eye. This system also allows for simultaneous reverse transcription and isothermal amplification at a low temperature, independent of laboratory instruments, and thus could meet the urgent need for rapid diagnosis.