key: cord-1008309-0oak9ggm
authors: Sheikhzadeh, Elham; Eissa, Shimaa; Ismail, Aziah; Zourob, Mohammed
title: Diagnostic techniques for COVID-19 and new developments
date: 2020-07-14
journal: Talanta
DOI: 10.1016/j.talanta.2020.121392
sha: f6042f8ede1473b31e45f877675aa39dbeaa99e1
doc_id: 1008309
cord_uid: 0oak9ggm

COVID-19 pandemic is a serious global health issue today due to the rapid human to human transmission of SARS-CoV-2, a new type of coronavirus that causes fatal pneumonia. SARS -CoV-2 has a faster rate of transmission than other coronaviruses such as SARS and MERS and until now there are no approved specific drugs or vaccines for treatment. Thus, early diagnosis is crucial to prevent the extensive spread of the disease. The reverse transcription-polymerase chain reaction (RT-PCR) is the most routinely used method until now to detect SARS-CoV-2 infections. However, several other faster and accurate assays are being developed for the diagnosis of COVID-19 aiming to control the spread of infection through the identification of patients and immediate isolation. In this review, we will discuss the various detection methods of the SARS-CoV-2 virus including the recent developments in immunological assays, amplification techniques as well as biosensors.

Moreover, mutations lead to faster transport of viruses from animals to humans and humans to humans.

Mutations in the ORF8 region at 28144 and the ORF1B region at 8872 were reported in the early phase of the SRAS-CoV-2 epidemic [23].

Patients with COVID-19 showed a similar pattern of viral load change to those with influenza, and different from SARS and MERS. In SARS and MERS viral load reached the maximum value about 10 days after the beginning of symptoms [24] While in SARS CoV-2, high viral loads in the upper respiratory tract and as a consequence high risk of transmission were reported in the early days from the onset of symptom. Moreover, the RT-PCR test revealed low levels of virus in the upper respiratory tract even after the disappearance of symptoms [17] . Another feature of SARS-CoV-2 is the higher viral load reported in elder people [25] As of June 29, 2020, the disease has infected over 10.1 million people worldwide leading to around 502 K deaths [26] . Because of the many asymptomatic cases and poor testing, it is expected that the total number of identified COVID-19 infections worldwide is underestimated. These asymptomatic individuals pose a serious risk because they are capable of further spreading of the disease [25] . Moreover, most of the symptoms of COVID-19 are similar to those of normal influenza and cold. Therefore, it is highly important to early and accurate diagnosis the infected individuals to prevent the extensive spread of this fatal disease. Particularly, the identification of the COVID-19 patients in early stages will allow the physicians to help them before developing serious complications. Developing fast and reliable screening tools for COVID-19 will also help to identify negative people and avoid unnecessary quarantine that negatively impacted social life and caused a serious economic crisis. A schematic diagram that showed the importance of fast detection and isolation of infected cases is shown in Figure 1 . In this report, we discuss various existing diagnostic methods for COVID-19 as well as ongoing developments and innovations such as point-of-care (POC) diagnostic tests and biosensors. Different diagnostic methods for detection SARS-CoV-2 is shown in Figure 2 . respectively. The strip test was checked with patient fingerstick blood, vein blood and plasma and it showed 100% uniformity and demonstrated the applicability of test for POC measurements. The test produced false-negative results likely due to the low amount of IgM and IgG or variation in the immune response of different people. Moreover, the IgM antibody level reduces after two weeks of infection [30] . The schematic of these methods were illustrated in Figure 3A .IgG/IgM Rapid Test Cassette for COVID-19 which is available on the market from Zhejiang Orient Gene Biotech Co Ltd was developed for the detection of SARS-CoV-2 specific antibodies. Control experiments of 80 samples were negative for IgM but only one case provided false-positive result. There were no positive results for IgM and IgG for 6-12 months babies. When people were divided into two groups (9-17 days) and (18-29 days) after the beginning of infection, both groups showed more positive IgG results. The assay showed a sensitivity of 69% and 93.1% for IgM and IgG, respectively as well as 100% specificity and 99.2% sensitivity for both antibodies [15] . A fluorescence immunochromatographic assay was also applied in the detection of SARS-CoV-2 in which control and test lines were modified with goat anti-rabbit IgG antibody and mouse antinucleocapsid protein, respectively. Carboxylate-modified polystyrene Europium (III) chelate microparticles with the anti-nucleocapsid protein of SARS-CoV-2 monoclonal antibody M4 or rabbit IgG were added to conjugation pad.

Capturing nucleocapsid protein by the antibody in the test and control lines caused the appearance of the fluorescent band which was measured by fluorescence analyzer. 100 nasal swab samples of healthy individuals were used to evaluate the Cut-off value of the assay. Diluted nasopharyngeal swab or urine samples were poured into the sample well and the strips were read after ten minutes. The positive results were obtained by analyzing the value of the sample against the cut-off value. The samples were also tested with RT-PCR. There were 208 positive results from 239 patients. Among 208 people with positive RT-PCR results, 141 cases have shown antigen positive results (68%). All the negative samples with RT-PCR were also negative with the ICA test. 14/19 patients with positive results had antigen in their urine samples. One person showed antigen after 3 days of fever. The authors proposed determining N antigen in urine samples to check for the kidney failure of patients [42] . The efficiency of Coris COVID-19 Ag Respi-Strip as a frontline test for SARS-CoV2 has been investigated in nasopharyngeal samples. The assay showed 30.2% (32/106) sensitivity and 100% specificity among positive RT-PCR samples. Viral load of around 1.7 × 10 5 copies mL -1 caused a higher detection rate while 9.4 × 10 3 copies mL -1 showed a great decrease in sensitivity of the test [43] .

Sensitivity and specificity of the immunological assays are considered imperative factors in the practical application of these methods. For the detection of SARS-CoV-2, immunological assays mostly utilized S, N and receptor-binding domain (RBD) proteins as targets. S protein is essential for the attachment of the virus to host cells while RBD of S protein plays the role of mediator with angiotensin-converting enzyme 2 (ACE2) [44] . The S protein antigen showed higher interference with the S protein SARS-CoV than MERS-CoV. But S1 subunit protein has only shown cross-reactivity with SARS-CoV. The presence of a highly conserved S2 subunit domain in coronavirus is probably the cause of this effect. Developed methods were more specific with the S1 subunit. RBD region inside the S protein has also shown cross-reactivity between SARS-CoV and SARS-CoV-2 [27, 45] . The N based -ELISA method has shown good specificity and sensitivity to detect SARS-CoV-2. Three ELISA methods that utilized RDB, N, or S1 protein were compared. Among them, RDB and N-protein based methods showed more sensitivity than S1 in patients with mild sickness. Comparison between IgA and IgM ELISA demonstrated that the former was more sensitive and the later was more specific [45] PCR result is considered positive if the Ct value was less than 40 [48] . This value is usually decreased in the third week of infection and may not be detected later. Ct values of extremely sick patients who were hospitalized are lower than patients with mild symptoms and may remain positive after 3 weeks of sickness. PCR positivity decreases more slowly in sputum and can be positive while nasopharyngeal swab is negative. Positive RT-PCR results were observed in stool (55 of 96) patients beyond nasopharyngeal swab during 4-11days and was not correlated to the severity of the disease [48] . RT-PCR test of COVID 19 can give positive results one day before starting symptoms but in most cases, patients were not identified before the onset of symptoms due to low viral load

RT-PCR methods are generally designed to amplify S, E, N, RdRp and ORF1a/b genes while ORF1a/b and E genes were used more frequently [44] . Orf1ab and N genes are regularly utilized for SRAS-CoV-2 in China while N1, N2 and N3 genes and E, N and RdRp genes are mostly applied in US CDC and Europe, respectively [47] .

There is still a lack of information about the variety of genetic SARS-CoV-2 in humans and animals. Therefore, two RT-PCR assays that can detect multiple coronaviruses in the subgenus of Sarbecovirus were developed [49] .

These 1-step qRT-PCR assays have identified two different regions of the viral genome; ORF1ab and N. The study was applied to SARS-CoV-2 and SARS coronaviruses while RNA of SARS coronavirus was used as a positive control. Moreover, the RT-PCR products of SARS coronavirus produced by the ORF1b and N gene assays were cloned into plasmids. Because of the application of DNA plasmids as positive standards, the assay has realized a limit of detection of 10 copies per reaction. Control samples were completely negative and real samples from two infected patients had shown positive results. The authors have recommended the N gene for screening and the ORF1b gene for confirming the results. These assays were capable of achieving a wide dynamic range [49] . Spin column-and poly amino ester magnetic nanoparticle (pcMNPs) extraction method was utilized in the conventional RT-PCR and direct RT-PCR amplification of the SARS-CoV-2 virus. Direct RT-PCR was applied with magnetic nanoparticles coated with poly amino ester. The magnetic nanoparticles were synthesized with co-precipitation reaction and hydrolysis of TEOS/APTES. Then NH 2 -MNP reacted with the prepared polymer to form poly amino ester through a Michael addition reaction. Direct RNA extraction protocol has shown nearly 100% RNA extraction efficiency in serum samples and provided high-purity products without interference with the PCR reagents. Using this method, lysis and binding steps were combined and the pcMNP was applied in the RT-PCR system directly.

The pcMNPs had superb viral RNA binding ability that provided high sensitivity (10 copies) and a wide linear range (up to 10 5 copies). This method can be coupled with automated nucleic acid extraction systems. It is also adaptable to isothermal amplification methods and can be used in POC devices [50] . Detection of SARS-CoV-2 in saliva samples was investigated with both RT-PCR and viral culturing methods. 91.7% (11 of the 12) patients have shown positive results. However, salivary RNA levels were then decreased after hospitalization. The viral culturing method has shown the presence of live viruses in the saliva of 3 patients. The collection of saliva samples from patients has advantages in diagnosis. It is a non-invasive method and samples can be collected outside the hospital by nonexperts [51] . A comparative RT-PCR test was performed with the nasopharyngeal swab and saliva samples (n=53) to detect RdRp, E and N genes of SARS-CoV-2. The method showed 89 and 77% sensitivity for the nasopharyngeal swabs and saliva samples, respectively. There was no significant variation between nasopharyngeal swabs and saliva specimens but nasopharyngeal swabs were nearly 10% more sensitive than saliva. When specimens were collected in later times of illness, there was a greater difference in sensitivity between nasopharyngeal swabs and saliva samples likely due to the lower load of virus in this stage. Saliva can be replaced with nasopharyngeal swabs when a person cannot bear collecting nasopharyngeal swabs especially when viral concentration is higher in the upper respiratory tract. The nasopharyngeal swab should be checked as a second specimen in patients with a high index of clinical suspicion and their saliva is negative [52] .Three new real-time RT-PCR assays for RdRp/helicase (Hel), S and N genes of SARS-CoV-2 have been developed. Compared with the reported RdRp-P2 assay which is used in more than 30 European laboratories, the lowest detection limit was achieved by the RdRp/Hel assay which was 1.8 TCID50 mL -1 and 11.2 RNA copies/reaction with genomic RNA and in vitro RNA transcripts, respectively. 28.2% test results from people confirmed with COVID-19 were positive by both the RdRp/Hel and RdRp-P2 assays. The SARS-CoV-2 RdRp/Hel assay was positive for people whose RdRd-P2 test results were negative. The SARS-CoV-2-RdRp/Hel assay was specific and there was no interference with HCoVs and other respiratory pathogens in cell culture and clinical samples [53] . Corman et al. [54] have designed a workflow for the detection of SARS-CoV-2 with the help of synthetic nucleic acid technology in the case that the virus isolates or real patient samples are not available. They proposed using E gene assay as the first-line screening tool and confirming the test results with the RdRp gene assay. RdRp gene test with dual-color detection was capable to distinguish SARS-CoV-2 from SARS-CoV. They obtained the best results with the E gene and RdRp gene (LOD of 3.2 and 3.7 copies/reaction, respectively), while the N gene was less sensitive. They also evaluated the LOD for in-vitro transcribed RNA that was identical to the sequence of SARS-CoV-2. The obtained LODs were 3.9 and 3.6 copies / reaction for E gene and did not respond to SARS-CoV RNA. Endemic human coronaviruses (HCoV); 229E, NL63, OC43 and HKU1, as well as MERS-CoV had no interference with their results [54] . RT-PCR tests were performed to detect the presence of the virus in anal swabs and blood samples in the cases where they were not identified in oral swabs. It should be noted that patients may still carry the virus despite that their swab results were negative. The researchers also investigated the presence of IgM and IgG in patients by ELISA methods after 10 days of medical treatment. Both antibodies were low or unnoticeable on the first day of sampling. On day 5 both antibodies were detected in all patients [55] . RT-PCR was used to test samples collected from four medical staff that two of them were already recovered from sickness and another two who showed negative results at the beginning. In all cases, positive results were obtained after 5 to 13 days. This study suggested revising the guidelines for the discharge of infected people from hospitals or home lockdown [56] . Wang et al. [57] have investigated 205 patients with 1070 various samples including pharyngeal swabs, blood, sputum, feces, urine, and nasal samples. The positive rate was 14 of 15 (93%) in bronchoalveolar lavage fluids, 72 of 104 (72%) for sputum, 5of 8 (63%) for nasal swabs, 6 of 13 (46%) for fibrobronchoscope brush biopsy, 126 of 398 (32%) in pharyngeal swabs, 44 of 153 (29%) in feces, and 3 of 307 (1%) in blood. All urine samples were negative. Nasal swabs showed the highest mean cycle threshold 24.3 (1.4 × 10 6 copies mL -1 ) while other samples had 30 (<2.6 × 10 4 copies mL -1 ) [57] .Tear and conjunctival secretions were studied with RT-PCR and viral culture methods to trace the existence of the SARS-CoV-2 virus. Viral RNA was only found in one person who had conjunctivitis symptoms [58] . In another study, throat swab or sputum samples were investigated with RT-PCR method and the results demonstrated that sputum samples produced more positive results. Thus, they concluded that sputum can be used instead of throat swab in patients who produced sputum [59] . infection. They found that both methods showed superior sensitivity (91.9%) in contrast to 78.2% for RT-PCR and 66.7% for CT scans alone. They reported that RT-PCR was more important in the detection of mild infection. They have also emphasized the application of stool samples for RT-PCR as an indicator to enhance the diagnosis rate and the discharge from hospitals [61] . N-gene-specific qRT-PCR was applied to evaluate the viral load of SARS-CoV-2 in another study [62] . It was reported that the viral loads in throat swabs and sputum have reached the maximum level (10⁴ to 10⁴ copies mL -1 ) after 5-6 days from the beginning of the infection, while for SARS the peak was reached after 10 days. The viral loads were between 641-1.34× 10¹¹ copies mL -1 with a median value of 7·99 ×10⁴ in throat samples and 7·52 × 10⁴ in sputum samples. However, stool samples showed less amount of viral load (550-1.21 × 10⁴ copies mL -1 ). Wikramaratna et al. [63] have investigated the public data from patients who had RT-PCR positive results at least one time. They concluded that the probability of positive test reduced if tests were performed at later date after symptom appearance and the nasal samples had more positive results than throat samples [63] .

Some German researchers reported that the Real Star kit had better sensitivity and higher efficiency. [64] J.LeBlanc et al have evaluated RT-PCR tests in Canadian Laboratories. Their LODs were ranging from 3.4 to 4.5 log 10 copies mL -1 ) with was consistent with other reports. They also suggested that the detection of more than one target has improved the diagnosis of the virus in low viral load [65] . Diagnostic methods that are applied in China for SARS-CoV-2 detection were summarized in reference [66] .

AusDiagnostics Multiplex-tandem PCR (MT-PCR) assay which includes two tandem amplification steps were also applied for the detection of SARS-CoV-2. The first amplification step (enrichment) utilized a specific outer primer with fewer numbers of PCR cycles. In the second amplification step, the target region within the product from the first step was amplified by inner primers. 7839 samples were analyzed with this method and 127 samples were detected positive. Comparative analysis with State Reference Laboratory showed 118/127 (92.9%) consistency. After investigation of discrepancies, 125/127 (98.4%) positive results were obtained and this method has demonstrated reliable diagnosis for SARS-CoV-2 [67] . Protocols established in various countries for RT-PCR available on the WHO website. CDC website also reported protocols for the United States. The WHO protocols, available PCR commercial kits and serological test kits were summarized in reference [28] .

Real-time nanopore target sequencing (NTS) and amplification methods were employed for the simultaneous detection of SARS-CoV-2 and 10 other respiratory viruses in 6 -10 h with LOD of 10 copies mL -1 with at least 1 h sequencing data. Oxford nanopore sequencer is a small device that can be coupled with a personal computer for data processing. In this method, 11 virulence-related and specific gene fragments of ORF1ab of SARS-CoV-2 were amplified with an in-house primer panel. Then, the amplified fragments were sequenced on a nanopore platform. A comparative study of approved qPCR kits and NTS method with samples from patients have shown that NTS provided more positive results. The system has shown two orders of magnitudes more sensitivity than qPCR as well as specificity against other mutated nucleic acid sequences or various respiratory virus infections in the samples [68] . Total RNA sequencing was carried out by the Shotgun metatranscriptomics method. The obtained data were utilized for phylogenetic analysis and were assigned to subclade in New York subway samples [69].

Isothermal nucleic acid amplification is a technique that is used to amplify nucleic acids at constant temperature avoiding the complex requirement of the regular PCR that needs changing multiple temperatures in each cycle [70, 71] Several isothermal nucleic acid amplification techniques have been previously developed for the detection of SARS-CoV such as transcription-mediated amplification (TMA), Loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA) and clustered regularly interspaced short palindromic repeats (CRISPR). In the reverse transcription LAMP method for SARS-CoV detection [72] , four primers were used to enhance the sensitivity of the assay. The amplification product in the LAMP method can be detected by measuring the turbidity of the solution or the fluorescence of an intercalating dye. Moreover, unpurified samples can be applied in LAMP directly [73] . This method is a rapid and cost-effective way for virus detection but is limited only to one sample per run.

RT-LAMP was carried out in one step at 63 ⁴ within 30 minutes to detect SARS-CoV-2. The optical density at 400 nm and color change from orange to green were used to detect amplification. The assay was capable to identify ORF1ab gene, E gene and N gene simultaneously with accuracy rates of 99%, 98.5%, and 92.3%, respectively.

ORF1ab and N genes showed higher specificity and sensitivity. Both RT-LAMP and RT-PCR had similar specificity of 99% for evaluating 208 clinical samples and sensitivity in 20 fold diluted samples with LOD of 1000 copies mL -1 . In this method, three gene amplifications were combined to prove the presence of SARS-CoV-2. The technique was specific because of using six to eight primers to distinguish eight different regions on the target DNA [6] . SARS-CoV-2 virus from purified RNA or cell lysis was visually detected with the LAMP method. The LAMP method was performed with 5 full primers sets targeting SARS-CoV-2 RNA with amplicon regions designed to the 3. Novel developed technologies for SARS-CoV-2 detection As described above, the RT-PCR and immunological assays are currently the most widely used methods for the diagnosis of COVID 19. However, these methods require trained personnel to perform. Moreover, PCR takes up to few days to obtain the results. Immunological assays require complex production of antibodies and recombinant proteins. Therefore, there is a trend to produce novel faster, lower cost, more reliable diagnostic methods for the detection of SARS-CoV-2.

Biosensors are bioanalytical devices that combine the selectivity features of a biomolecule with the sensitivity of a physicochemical transducer [87] . They can be a fast and reliable alternative for clinical diagnosis, real-time detection and routine measurements [68] . There are various types of biosensors that have been previously applied for the diagnosis of infectious diseases [88] .

LSPR is an optical phenomenon produced when light waves are trapped in conductive nanoparticles which are smaller than the wavelength of light. The incident light and surface electrons in the conduction band interact to produce coherent localized plasmon oscillation. The resonance frequency is sensitive to local changes like the variation in refractive index and molecular binding [89] .

Dual-functional plasmonic biosensor utilizing plasmonic photothermal (PPT) effect and localized surface plasmon resonance (LSPR) sensing transduction were applied for the detection of various viral sequences including RdRp-COVID, ORF1ab COVID, and E genes from SARS-CoV-2. The converted PPT heat energy, in the proximity of gold nanoislands, provided a stable heat source to enhance the in situ hybridization of RdRp of SAR S-CoV-2 and its complementary DNA. The slope of the photothermal enhanced LSPR curve was higher than the system without the photothermal effect. The proposed sensor was capable to discriminate between SARS-CoV and SARS-CoV-2 viruses. Without the assistance of the photothermal unit, a false positive response signal was obtained for the RdRp-SARS sequence. The sensor has shown a LOD of 0.22 pM [90] . The schematic procedure for this biosensor is depicted in Fig.4a. 

The FET transducer is based on modulation of carrier mobility across a biased semiconductor due to the electrostatic field. The gate surface of FET is covered with a layer that can be modified with biomolecules for selective detection of targets [91].

Graphene FET was decorated with an antibody of SARS-CoV-2 spike S1 subunit protein (CSAb) or angiotensinconverting enzyme 2 (ACE2) to detect SARS-CoV-2 spike protein S1. The binding of the S1 protein that possesses a slightly positive charge with the CSAb/ACE2 receptors on the graphene surface changed the conductance/resistance in graphene-FET which was considered the basis of the detection. CSAb modified graphene-FET exhibited better sensitivity due to the higher affinity of this antibody. The proposed sensor showed a LOD of 0.2 pM [92]. The FET system has detected SARS-CoV-2 based on the changes in channel surface potential and its effect on the electrical response. As discussed previously, the S protein is an excellent antigen because it is a major transmembrane protein of the virus and it shows amino acid sequence diversity among coronaviruses. The FET sensor was capable to detect S1 protein down to 1 fg mL -1 and 100 fg mL -1 in PBS and clinical samples, respectively. Moreover, the FET sensor determined SARS-CoV-2 with LOD of 1.6×10 1 pfu mL -1 and 2.42 × 10 2 copies mL -1 in culture medium and clinical samples, respectively. The biosensor was also able to discriminate the SARS-CoV-2 antigen protein from the MERS-CoV protein which indicated good selectivity of this platform [93] . The schematic procedure for FET biosensor was illustrated in Fig.4b. 

A membrane-engineered kidney cell modified with the SARS-CoV-2 SpikeS1 antibody via electro-insertion was applied to detect the SARS-CoV-2 S1 antigen. The potential of the membrane is changed by the interaction of the antibody with the target protein. The device was fabricated on 8 gold screen printed electrodes which were covered by polydimethylsiloxane (PDMS) layer with eight wells. Suspension of the modified membrane was added to PDMS well, followed by the addition of protein solution and signal measurement with a potentiometer. The sensor has achieved an excellent detection limit of 1fg mL -1 with a wide linear range of 10 fg to 1µg mL -1 [94]. Table 1 summarizes different methods for SARS-CoV-2 detection. 

We discussed different molecular and serological methods for the detection of SARS-CoV-2. RT-PCR can provide good sensitivity and specificity and the results can be obtained in a few hours. It can detect viral DNA in respiratory samples, saliva, blood, urine and stool. However, RT-PCR has some drawbacks including the need for expensive thermocycler and professional staff to perform the assay and interpret results. Moreover, the standard control has an important role in the accuracy of the results and false-negative results can be obtained due to sample degradation, time and quality of sample collection and the low efficiency of some test kits. LAMP methods have comparable sensitivity to RT-PCR and high specificity. However, some kits showed lower sensitivity. It can be performed in 30 min using a crude sample that allows their possible integration in POC tests. CRISPR method has been also developed for SARS CoV-2 detection showing high sensitivity and specificity. It can be performed in 1h and can be coupled with Lateral flow assay. There is no need for expensive thermocycler for LAMP and CRISPR. Lateral flow assay is an easy method to apply with the ability to obtain results in 15 min by non-professional personnel in blood or serum samples. Moreover, antibodies are less affected by storage, transport and sample collection. It has the disadvantage of prolonged time of antibody production. ELISA is easy to perform but, like Lateral flow assay, cannot be used for early detection. However, it can be used to check the immunity of healthcare staff and for the investigation of herd immunity.

In the future, we expect that other easier and more mature molecular systems like LAMP and CRISPR as well as biosensor platforms will replace RT-PCR. More studies for the comparison between newly developed methods in terms of sensitivity, reproducibility, reliability, and robustness are still needed. Moreover, developed techniques that can analyze samples from different routes should be combined with oral swabs detection to validate the results for making an informed decision to discharge people from hospitals or home quarantine. Saliva, sputum, posterior Oropharyngeal can replace the nasopharyngeal samples that can be less invasive and less dangerous for healthcare staff. As current tests are not sufficient to distinguish infected people in public areas, there is still an urgent need to produce POC devices that can detect infections on-site without the need for professionally trained personnel. CRISPR-Cas12DETECTR lateral flow assay for SARS-CoV-2 with permission ref [85] . they discharge from the hospital or have a first negative test. 

COVID-19): A literature review

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Rapid colorimetric detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform: iLACO

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Era of molecular diagnosis for pathogen identification of unexplained pneumonia, lessons to be learned

SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge

Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR

A novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2

coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform

Development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2

Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay

RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2

Multiple-centre clinical evaluation of an ultrafast single-tube assay for SARS-CoV-2 RNA

The authors extend their appreciation to the Deputyship for Research& Innovation, Ministry of Education in Saudi Arabia for funding this research work through project number 492.