Reverse transcription polymerase chain reaction (RT-PCR) is one of the simplest virus detection methods and due to its simplicity; it is frequently used in clinical and research laboratories. In this molecular technique, ribonucleic acid (RNA) as genome template initially is converted into complementary deoxyribonucleic acid (cDNA) by reverse transcriptase enzyme and after that in a separate reaction, the cDNA will be amplified by DNA Taq polymerase enzyme leading to production of billions copies of amplicons [14]. Usually three primers are used in RT-PCR procedure including specific, random hexamer and oligo-dT primers. The random hexamer primer (sequence: NNNNNN) used for RT-PCR amplification is annealed to cell's mRNAs and pre-mRNAs and therefore, the synthesized cDNA usually is representative for cell total mRNAs [15]. The oligo-dT primer (T) _18-20n is bond to poly (A) sequence at the 3′ end of mRNAs, amplifies just cell's mRNAs and therefore it can only be used for recognition of cells' mRNAs. Viral RNAs are identified with random hexamer primer and the oligo-dT primer will not work due to lack of poly (A) sequence at the 3′ end of their RNA except some viruses [16, 17].

Although the RT-PCR method is a basic tool for several goals such as evaluation of gene expression, RNA virus detection and gene cloning, however, it has some disadvantages which cause its application to be limited in diagnosis of RNA viruses. These limitations include lack of ability to determine virus infectivity and inhibition by some compounds, leading to false negatives results [18]. According to published literatures, there are only limited reports for detection of COVID19 based on RT-PCR method.

Currently, real time RT-PCR is the reference and standard method for the diagnosis of active COVID-19 infections [19]. This method employs the viral RNA extracted from patients’ samples (e.g., nasopharyngeal/oropharyngeal/nasal swabs, tracheal aspirates, sputum, bronchoalveolar lavage (BAL), pleural fluid, or lung biopsies) to synthesize complementary DNA (cDNA), a reaction catalyzed by the reverse transcription (RT) enzyme, which then serves as a template to amplify target viral genes [19]. The genes encoding nucleocapsid (N), glycoprotein spike (S), RNA polymerase-dependent RNA (RdRp), and Helicase (Hel) are common target genes detected by COVID-19 real time RT-PCR assays [20].

Chan et al. employed a very sensitive, specific and innovative real time RT-PCR method for detecting the RdRp/Hel in the clinical samples of COVID-19 patients. In the recent study, the researchers investigated the reliability of real-time RT-PCR to detect the N, S, or RdRp/Hel genes of the SARS-Cov-2 virus. Of the three mentioned targets, COVID-19-RdRp/Hel assay represented the highest sensitivity by detecting the lowest amount of the virus. Among 273 samples from 15 patients with confirmed COVID-19 infection in Hong Kong, 28.2% were positive for both RdRp-P2 and COVID-19-RdRp/Hel. The COVID-19-RdRp/Hel test rendered positive results in 42 (29 (24.2%) respiratory and 13 (8.5%) non-respiratory) samples that were negative for the RdRd-P2. While the presence of other respiratory pathogens and human coronaviruses in cell culture and clinical samples did not interfere with COVID-19-RdRp/Hel assay, a cross-reactivity phenomenon was observed between the SARS-CoV-2 and RdRp-P2 assays in cell culture samples. This study showed that COVID-19-RdRp/Hel method was very sensitive and specific for detecting COVID-19 infection [20].

Nevertheless, real-time RT-PCR has also been associated with false-negative results in patients with established COVID-19 infection [21]. Li et al. conducted a study on a confirmed case with COVID-19 pneumonia who had several real-time RT-PCR negative tests. In this patient, while the first five real-time RT-PCR tests were negative for COVID-19, the sixth test delivered a positive result. This observation suggested that in the presence of supportive epidemiological evidence and the typical clinical and imaging manifestations of COVID-19 pneumonia, real-time RT-PCR should be serially repeated, and suspected patients must be treated in isolation [21].

In a study by Tao et al., the researchers examined the correlation between real-time RT-PCR results and chest CT scan findings in Chinese patients with COVID-19 infection. They showed that out of 1014 patients, while only 601 (59%) patients showed positive real-time RT-PCR results, 888 (88%) had characteristic chest CT scans. Considering real-time RT-PCR as a reference test, the sensitivity of CT scan for detecting COVID-19 infection was about 97%. In addition, 75% of the patients who had negative real-time RT-PCR results revealed positive chest CT scan findings. So, the results of this study indicated the high sensitivity of chest CT scan for detecting COVID-19 infection [22].

In another study conducted by Lan et al. to examine the applicability of real-time RT-PCR in Chinese patients recovered from COVID-19 infection, the results showed that four COVID-19 carriers who had the criteria of either quarantine discontinuation or hospital discharge (i.e., two consecutive negative real-time RT-PCR results and the lack of clinical symptoms) rendered positive real-time RT-PCR results within 5 to 13 days after the last negative test. The study's findings showed that real-time RT-PCR may also be associated with false negative results in coronavirus carriers [23]. Recently, Kasteren et al. compared several commercial COVID-19 detection kits and found that all of the studied kits are suitable for diagnostics of symptomatic COVID-19 patients [24]. They also recommended real-time RT-PCR kits from R-Biopharm AG, BGI, KH Medical manufacturers when the viral load in the specimen is low. The detection limits of studied kits are shown on Table 1.

Regarding high sensitivity, specificity, and reliability, as well as relatively rapid deliverance of results, it seems reasonable to use this technique to accurately detect the SARS-CoV-2 virus. In comparison with other diagnostic techniques, this method is considerably faster and has a lower potential to be affected by contaminations and errors. However, the test may rarely fail to discover a small ratio of infected people and carriers, so it is better to use other diagnostic methods along with this technique.

Nucleic acid amplification tests such as real-time reverse-transcription-polymerase chain reaction (RT-qPCR) have been rapidly developed to quantitatively detect the SARS-Cov-2 virus in different samples including nasopharyngeal swabs, sputum, alveolar lavage fluid, and blood. However, RT-qPCR requires fully-equipped laboratories and skilled staff.

Therefore, as RT-qPCR is a time-consuming and laborious process, an alternative specific nucleic acid amplification test, loop-mediated isothermal amplification (LAMP), has been developed to rapidly and efficiently amplify DNA under isothermal conditions [25, 26]. This approach uses a set of three to four specific primers targeting conserved sequences such as ORF 1ab. Furthermore, a DNA polymerase enzyme with chain displacement activity is employed to amplify the target DNA up to 109 copies which is accomplished at a constant temperature between 60 and 65°C in less than an hour. Out of the 4 designed primers in this method, two are “inner primers” (BIP and FIP) which synthesize new DNA strands [26]. The outer primers (B3 and F3) anneal to the template strand allowing the replication of the new DNA strand. Stem-loop DNAs with multiple inverted repeats are produced as the final products of the target DNA. The LAMP assay can be simply and rapidly (i.e. a one-step reaction) performed by minimally trained staff without requiring for well-equipped laboratories [27]. This method can be combined with reverse transcription reaction (i.e. RT-LAMP) to allow the direct detection of RNAs [28]. A multiplex LAMP assay was designed by Kim et al. for detecting SARS-CoV. Compared to real-time RT-PCR, it showed a detection limit of 10e5 RNA copies in each reaction [29]. This assay is a one-step nucleic acid amplification test that is used to diagnose infectious diseases caused by either bacteria or viruses. Similar to RT-qPCR, the RT-LAMP utilizes the reverse transcriptase enzyme to synthesize cDNA from RNA sequences and successfully detects pathogen-specific nucleic acids applying either pH-sensitive or bioluminescent indicators. Collectively, the LAMP technology seems to be a sensitive and stable method to rapidly detect viral pathogens. Accordingly, the method can be considered as a rapid and simple diagnostic test for infections caused by RNA-containing viruses such as the SARS-Cov-2. Nguyen et al. proposed using the LAMP method as a point-of-care test to detect zoonotic viruses [such as avian influenza virus (AIV)] and coronaviruses (such as the SARS-Cov2) [30].

In the recent decades, in addition to LAMP assay, several types of isothermal nucleic acid amplification techniques such as strand displacement amplification(SDA), signal mediated amplification of RNA technology(SMART), rolling circle amplification(RCA), isothermal multiple displacement amplifi- cation(IMDA), single primer isothermal amplification(SPIA), circular helicase-dependent amplification(HDA) and recom- binase polymerase amplification (RPA) have been introduced [31]. The mechanism of these procedures and their applications have been reviewed by Gill et al., comprehensively [32].

The reverse transcriptase-RPA (RT-RPA) method has been recently used to detect bovine coronavirus (BCoV) and the results of the study has shown that BCoV RT-RPA in comparison with real-time RT-PCR was more sensitive with detection limit of 19 copies RNA [33]. The authors of the study suggested that the RT-RPA method could be used in point-of-care diagnosis by applying a transferable fluorescence reading machine.

James et al. believe that RPA and also LAMP techniques have high potential performance to diagnose COVID-19 and it could also apply at community level [34]. It seems that applying the isothermal amplification methods to diagnose COVID-19 needs more researches to evaluate their sensitivities and specificities and furthermore, their reproducibility should be determined.

The RCA method also carries outs under isothermal condition without false-positive results and it needs minimal reagents for its performance. Recently, the method has been used for the detection of SARS-CoV in 7 clinical respiratory specimens and the results showed that the RCA technology was capable to amplify signals up to 109-folds in each amplification cycle [35].

NASBA is another isothermal amplification method for the detection of RNA targets. Three enzymes are involved in this method including T7 RNA polymerase, RNase H, and avian myeloblastosis virus reverse transcriptase. The method is a proven diagnostic tool and has more sensitivity than RT-PCR for detection of pathogens. Therefore, NASBA could be considered as a diagnostic tool for SARS-CoV-2. A study by Wat et al. used NASBA assay to detect respiratory viruses in nasal swabs which were taken from patients with cystic fibrosis. The results showed that out of 138 specimens, 46% samples were positive for respiratory viruses [36].

The patients infected by viruses develop antiviral antibodies during the acute phase of infections. Generally, IgM antibodies against several viruses become detectable within three days post-infection. The serological tests of COVID-19 infection measure the levels of serum antibodies resulted from the immune reaction against the SARS-COV2 virus. Compared to molecular methods, compatibility with moderate-quality samples and widely-available equipment (i.e., 96-well microplate ELISA automatic reader) is one of the main advantages of serological assays. Moreover, due to the high testing capacity of serological procedures, a larger ratio of febrile patients can be tested within a respectively short time.

The different types of serological tests include neutralization tests, immunofluorescent (IFA), enzyme-linked immunosorbent (ELISAs), and western blotting assays [37]. Using validated serological methods can improve the diagnostic process of COVID-19 infection, especially when there is a strong epidemiological evidence for the infection in the context of negative molecular tests and in both acute and recuperation phases [38].

Serological tests can be easily performed in hospital clinical laboratories, so they are much more widely used than molecular tests. A main target population for serological tests includes asymptomatic patients or those with mild COVID-19 infection who comprise a large portion of patients. This is particularly important as these individuals are not generally referred for molecular tests [38].

Although cross-reactions with other coronaviruses can be challenging, but new sensitive and specific commercial and non-commercial serological tests are being developed to resolve this problem [39, 40]. So far, multiple serological methods have been approved to diagnose COVID-19 infection. These include specific kits for detecting antibodies (IgM or IgG) against the whole virus by ELISA, chemiluminescence, or colloidal gold assays.

In addition to the nucleocapsid phosphoprotein (NP) which provides a sensitive antigen, other epitopes and specific molecules of the SARS-Cov2 virus can also be considered to develop serological assays.

Zhou et al. used the nucleocapsid (N) protein of the SARS-CoV Rp3 recovered from bats to detect IgG and IgM antibodies against the corresponding antigen in the SARS-Cov2 virus. This was due to the two antigens had a 92% similarity in their amino acid sequences. The applied antigen did not show cross-reactivity with antibodies against other human viruses than SARS. They assessed the levels of anti-viral antibodies in one patient at 7, 8, 9 and 18 days after the onset of the disease. There was a significant increasing trend in the titers of IgG and IgM antibodies over time, except that the titer of IgM showed a reduction in the last sample. They also evaluated anti-viral antibodies in samples from 5 out of 7 COVID-19 confirmed patients approximately 20 days after the onset of the disease. All the patients and healthy individuals’ samples rendered positive results for anti-virus IgG antibodies [41].

Shaoli et al. examined the epidemiological history and clinical features of seven patients with the novel coronavirus pneumonia in Lanzhou Pulmonary Hospital. After 7 days of the diagnosis, only one patient had negative results for anti-virus antibodies. This suggested that in patients who have a negative molecular test result but show supportive lung lesions, the screening of COVID-19 specific IgM antibodies can be of a great diagnostic value [42].

In order to identify IgM and IgG against the SARS-CoV-2, Haveri et al. examined serum samples from COVID-19 patients by IFA after 4, 9, 10, and 20 days of the onset of symptoms. While the antibodies were undetectable at the 4^th day, IgG titers rose to 80 and 1280, and IgM titers to 80 and 320 on the days of 9^th and 20^th, respectively [43].

In comparison with molecular methods, a study in China reported that the sensitivities of serum IgM and IgG antibodies for detecting COVID-19 infection were reported as 48.1% and 88.9%, respectively. Also, the specificities of these antibodies were 100% and 90.9%, respectively. The titer of IgM and its positivity rate showed no significant differences before and after recuperation [44]. The positivity rate of IgG was more than 90% showing no significant difference before and after recuperation.

To date, the FDA has approved only one serological test called the qSARS-CoV-2 IgG/IgM Rapid Test (Cellex Co.). This test which is planned to be used in clinical laboratories in the near future is based on the qualitative detection of IgM and IgG anti-SARS-CoV-2 antibodies in the plasma or serum samples of suspected COVID-19 patients [45].

Recently, Zhang et al have developed an ELISA assay for detection of IgG and IgM against SARS-CoV-2. The SARS-CoV-2 Rp3 nucleocapsid protein has been used in this assay. This protein has 90% amino acid sequence similarity to the other SARS-related viruses. They studied the samples of sixteen SARS-CoV-2 positive cases whose infection were confirmed by RT-PCR and reported the increase levels of these antibodies during the first 5 days after initiation of the symptom. It has been reported that antibodies were detectible in blood, respiratory, as well as fecal samples [46]. Applying two ELISA kits based on recombinant nucleocapsid protein (rN) and spike protein (rS) for detecting antibodies in COVID-19 patient’s sera showed that the sensitivity of the rS-based ELISA was significantly higher than that of the rN-based ELISA [47]

Western blotting is a precise technique used in molecular biology laboratories to identify proteins. In this technique, proteins within a mixture are initially separated through SDS-gel electrophoresis based on their molecular weights. In the next step, proteins resided on the gel are transferred on specific membranes (such as PVDF). Finally, the presence or absence of a target protein is confirmed through incubation of the membrane with a target-specific antibody [48-50].

In virology, western blot technique is widely used to identify viruses [51], and anti-viral antibodies [52, 53]. This technique can also serve as a confirmatory test to detect viral proteins or genes [54].

Generally, the applicability of western blot techniques depends on the availability of specific antibodies against target proteins. In fact, it is inapplicable to produce specific antibodies for all, especially unknown or unimportant, proteins. Unlike PCR-based methods, western blot techniques are relatively expensive limiting their wide application in clinical and research laboratories.

Despite these limitations, some advantages of western blot methods render them popular in many virology experimental fields. A western blot method has recently been developed to diagnose the coronavirus causing severe acute respiratory syndrome (SARS). In this study, a C-terminal fragment from the nucleocapsid (N) protein of the virus confirmed to have no cross-reactivity with the proteins derived from canine, pig, and chicken coronaviruses was used as a target to detect anti-SARS antibodies. The sensitivity and specificity of the method were obtained as 90.9% and 98.3%, respectively. The results of this western blot assay performed on 274 clinical samples showed strong correlations with IFA findings [55]. Nonetheless, a main disadvantage of western blot is that antibodies against pathogenic agents are not detectable in early or lag phases of infections. Generally, it takes at least 7 days for the body to develop antibodies against infectious agents or coronaviruses [56, 57].

The nucleocapsid (N) protein, as a highly conserved peptide among coronaviruses, seems to be an appropriate target for detecting these viruses in clinical samples. However, the extent of the humoral immune response against the various regions of this protein is variable. One study on human sera using rabbit polyclonal antibody described a great immunogenicity for the central region (amino acids of 174 to 300) of HCoV-OC43 nucleocapsid protein. On the other hand, variable immune responses were observed against the C or N-terminal domains of this protein in the course of HCoV-OC43 infection [58].

It seems that the nucleocapsid protein of coronaviruses is an acceptable target to be used in western blot techniques to diagnose infections by these viruses. For the COVID-19 infections; however, it is suggested to recruit a region with no or least cross-reactivity with proteins from other coronaviruses.