Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2.
TL;DR: In this paper, single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation are presented. But they are not suitable for screening at the point of need, and in resource-poor settings.
Abstract: Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.
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TL;DR: In this article, the authors reported fabricated fluorine doped tin oxide (FTO) electrodes/gold nanoparticles (AuNPs) complex coupled with in-house developed SARS-CoV-2 spike S1 antibody (SARS CoV-1 Ab) to measure the response with Cyclic Voltammetry (CV) and Differential Pulse Voltamermetry (DPV).
40 citations
TL;DR: In this paper , the authors reported an assay for rapid COVID-19 testing and its implementation in a prototype microfluidic device called DISCoVER (for diagnostics with coronavirus enzymatic reporting), which involves extraction-free sample lysis via shelfstable and low-cost reagents, multiplexed isothermal RNA amplification followed by T7 transcription, and Cas13-mediated cleavage of a quenched fluorophore.
Abstract: Rapid nucleic acid testing is central to infectious disease surveillance. Here, we report an assay for rapid COVID-19 testing and its implementation in a prototype microfluidic device. The assay, which we named DISCoVER (for diagnostics with coronavirus enzymatic reporting), involves extraction-free sample lysis via shelf-stable and low-cost reagents, multiplexed isothermal RNA amplification followed by T7 transcription, and Cas13-mediated cleavage of a quenched fluorophore. The device consists of a single-use gravity-driven microfluidic cartridge inserted into a compact instrument for automated running of the assay and readout of fluorescence within 60 min. DISCoVER can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva with a sensitivity of 40 copies μl–1, and was 94% sensitive and 100% specific when validated (against quantitative PCR) using total RNA extracted from 63 nasal-swab samples (33 SARS-CoV-2-positive, with cycle-threshold values of 13–35). The device correctly identified all tested clinical saliva samples (10 SARS-CoV-2-positive out of 13, with cycle-threshold values of 23–31). Rapid point-of-care nucleic acid testing may broaden the use of molecular diagnostics. A COVID-19 test implemented in an automated microfluidic device and leveraging isothermal RNA amplification followed by T7 transcription and Cas13-mediated cleavage of a quenched fluorophore rapidly detects SARS-CoV-2 RNA in saliva samples.
34 citations
TL;DR: The LAMP approach presents promising results to monitor exposed individuals and also improves screening efforts in potential ports of entry and the effectiveness of LAMP to the q‐RT‐PCR is compared.
Abstract: Coronavirus disease 2019 or COVID‐19 caused by novel coronavirus/severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2 or 2019‐nCoV) is an ongoing pandemic that has emerging global effects and requires rapid and reliable diagnostic testing. Quantitative reverse transcription‐polymerase chain reaction (q‐RT‐PCR) is the gold standard method for SARS‐CoV‐2 detections. On the other hand, new approaches remedy the diagnosis difficulties gradually. Reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) as one of these novel approaches may also contribute to faster and cheaper field‐based testing. The present study was designed to evaluate this rapid screening diagnostic test that can give results in 30–45 min and to compare the effectiveness of LAMP to the q‐RT‐PCR. The 30 randomly chosen patient samples were generated by nasopharyngeal swabs with a portion of the SARS‐CoV‐2 nucleic sequence. The sample of quantification cycle (Cq) values was tested using RT‐LAMP as well as by conventional q‐RT‐PCR. The patient samples were tested with four different kits (SENSObiz COVID‐19 [SARS‐CoV‐2] LAMP Assay, the QIAseq DIRECT SARS‐CoV‐2 kit, Biospeedy SARS‐CoV‐2 Variant Plus kit, and CoVirion‐CV19‐2 SARS‐CoV‐2 OneStep RT‐PCR kit) and two different PCR devices (GDS Rotor‐Gene Q Thermocycler and Inovia Technologies GenX series). Based on 30 patient samples, the positive/negative ratio (P/N) was 30/0 as Biospeedy and Covirion (positivity 100%), 28/2 as Qiagen kit (positivity 93.3%) for the samples studied on the Inovia device while the same samples on the Rotor‐Gene device were 30/0 as Biospeedy and Covirion (positivity 100%), 29/1 as Qiagen kit at the first day (96.7%). On the fifth day, the samples were studied in the Inovia device and the respective results were obtained: 27/3 as Biospeedy (positivity 90%), 16/14 as Qiagen (positivity 53.3%), 28/2 as Covirion kit (positivity 93.3%). When these samples were studied in the Rotor‐Gene device, it was 29/1 in Biospeedy and Covirion (positivity 96.7%), 19/11 in the Qiagen kit (positivity 63.3%). When these samples were compared with the LAMP method it was found to be 19/11 (positivity 63.3%) on the first day and 18/12 (positivity 60%) on the fifth day. SARS‐CoV‐2 test studies will contribute to a proactive approach to the development of rapid diagnosis systems. The LAMP approach presents promising results to monitor exposed individuals and also improves screening efforts in potential ports of entry.
20 citations
17 citations
TL;DR: In this work, virus detection tests were evaluated from the perspective of the COVID-19 pandemic, and the requirements to develop a target product profile for a platform for virus detection were generalized.
Abstract: Coronavirus disease 2019 (COVID-19) is a transmitted respiratory disease caused by the infection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although humankind has experienced several outbreaks of infectious diseases, the COVID-19 pandemic has the highest rate of infection and has had high levels of social and economic repercussions. The current COVID-19 pandemic has highlighted the limitations of existing virological tests, which have failed to be adopted at a rate to properly slow the rapid spread of SARS-CoV-2. Pandemic preparedness has developed as a focus of many governments around the world in the event of a future outbreak. Despite the largely widespread availability of vaccines, the importance of testing has not diminished to monitor the evolution of the virus and the resulting stages of the pandemic. Therefore, developing diagnostic technology that serves as a line of defense has become imperative. In particular, that test should satisfy three criteria to be widely adopted: simplicity, economic feasibility, and accessibility. At the heart of it all, it must enable early diagnosis in the course of infection to reduce spread. However, diagnostic manufacturers need guidance on the optimal characteristics of a virological test to ensure pandemic preparedness and to aid in the effective treatment of viral infections. Nanomaterials are a decisive element in developing COVID-19 diagnostic kits as well as a key contributor to enhance the performance of existing tests. Our objective is to develop a profile of the criteria that should be available in a platform as the target product. In this work, virus detection tests were evaluated from the perspective of the COVID-19 pandemic, and then we generalized the requirements to develop a target product profile for a platform for virus detection.
13 citations
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TL;DR: Detailed virological analysis of nine cases of coronavirus disease 2019 (COVID-19) provides proof of active replication of the SARS-CoV-2 virus in tissues of the upper respiratory tract.
Abstract: Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity—but also aided in the control—of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6–8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples—in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19. Detailed virological analysis of nine cases of coronavirus disease 2019 (COVID-19) provides proof of active replication of the SARS-CoV-2 virus in tissues of the upper respiratory tract.
5,840 citations
TL;DR: The serial respiratory viral load of SARS-CoV-2 in posterior oropharyngeal saliva samples from patients with COVID-19, and serum antibody responses from patients infected with severe acute respiratory syndrome coronavirus 2 are ascertained.
Abstract: Summary Background Coronavirus disease 2019 (COVID-19) causes severe community and nosocomial outbreaks. Comprehensive data for serial respiratory viral load and serum antibody responses from patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not yet available. Nasopharyngeal and throat swabs are usually obtained for serial viral load monitoring of respiratory infections but gathering these specimens can cause discomfort for patients and put health-care workers at risk. We aimed to ascertain the serial respiratory viral load of SARS-CoV-2 in posterior oropharyngeal (deep throat) saliva samples from patients with COVID-19, and serum antibody responses. Methods We did a cohort study at two hospitals in Hong Kong. We included patients with laboratory-confirmed COVID-19. We obtained samples of blood, urine, posterior oropharyngeal saliva, and rectal swabs. Serial viral load was ascertained by reverse transcriptase quantitative PCR (RT-qPCR). Antibody levels against the SARS-CoV-2 internal nucleoprotein (NP) and surface spike protein receptor binding domain (RBD) were measured using EIA. Whole-genome sequencing was done to identify possible mutations arising during infection. Findings Between Jan 22, 2020, and Feb 12, 2020, 30 patients were screened for inclusion, of whom 23 were included (median age 62 years [range 37–75]). The median viral load in posterior oropharyngeal saliva or other respiratory specimens at presentation was 5·2 log10 copies per mL (IQR 4·1–7·0). Salivary viral load was highest during the first week after symptom onset and subsequently declined with time (slope −0·15, 95% CI −0·19 to −0·11; R2=0·71). In one patient, viral RNA was detected 25 days after symptom onset. Older age was correlated with higher viral load (Spearman's ρ=0·48, 95% CI 0·074–0·75; p=0·020). For 16 patients with serum samples available 14 days or longer after symptom onset, rates of seropositivity were 94% for anti-NP IgG (n=15), 88% for anti-NP IgM (n=14), 100% for anti-RBD IgG (n=16), and 94% for anti-RBD IgM (n=15). Anti-SARS-CoV-2-NP or anti-SARS-CoV-2-RBD IgG levels correlated with virus neutralisation titre (R2>0·9). No genome mutations were detected on serial samples. Interpretation Posterior oropharyngeal saliva samples are a non-invasive specimen more acceptable to patients and health-care workers. Unlike severe acute respiratory syndrome, patients with COVID-19 had the highest viral load near presentation, which could account for the fast-spreading nature of this epidemic. This finding emphasises the importance of stringent infection control and early use of potent antiviral agents, alone or in combination, for high-risk individuals. Serological assay can complement RT-qPCR for diagnosis. Funding Richard and Carol Yu, May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Michael Tong, Marina Lee, Government Consultancy Service, and Sanming Project of Medicine.
2,778 citations
TL;DR: Two 1-step quantitative real-time reverse-transcription PCR assays can achieve a rapid detection of 2019n-CoV in human samples, thereby allowing early identification of patients.
Abstract: BACKGROUND: A novel coronavirus of zoonotic origin (2019-nCoV) has recently been identified in patients with acute respiratory disease. This virus is genetically similar to SARS coronavirus and bat SARS-like coronaviruses. The outbreak was initially detected in Wuhan, a major city of China, but has subsequently been detected in other provinces of China. Travel-associated cases have also been reported in a few other countries. Outbreaks in health care workers indicate human-to-human transmission. Molecular tests for rapid detection of this virus are urgently needed for early identification of infected patients. METHODS: We developed two 1-step quantitative real-time reverse-transcription PCR assays to detect two different regions (ORF1b and N) of the viral genome. The primer and probe sets were designed to react with this novel coronavirus and its closely related viruses, such as SARS coronavirus. These assays were evaluated using a panel of positive and negative controls. In addition, respiratory specimens from two 2019-nCoV-infected patients were tested. RESULTS: Using RNA extracted from cells infected by SARS coronavirus as a positive control, these assays were shown to have a dynamic range of at least seven orders of magnitude (2x10-4-2000 TCID50/reaction). Using DNA plasmids as positive standards, the detection limits of these assays were found to be below 10 copies per reaction. All negative control samples were negative in the assays. Samples from two 2019-nCoV-infected patients were positive in the tests. CONCLUSIONS: The established assays can achieve a rapid detection of 2019n-CoV in human samples, thereby allowing early identification of patients.
1,058 citations
TL;DR: The Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal zoonotic pathogen that was first identified in humans in Saudi Arabia and Jordan in 2012.
1,030 citations
TL;DR: It is demonstrated that effective screening depends largely on frequency of testing and speed of reporting and is only marginally improved by high test sensitivity, and should prioritize accessibility, frequency, and sample-to-answer time.
Abstract: The COVID-19 pandemic has created a public health crisis. Because SARS-CoV-2 can spread from individuals with presymptomatic, symptomatic, and asymptomatic infections, the reopening of societies and the control of virus spread will be facilitated by robust population screening, for which virus testing will often be central. After infection, individuals undergo a period of incubation during which viral titers are too low to detect, followed by exponential viral growth, leading to peak viral load and infectiousness and ending with declining titers and clearance. Given the pattern of viral load kinetics, we model the effectiveness of repeated population screening considering test sensitivities, frequency, and sample-to-answer reporting time. These results demonstrate that effective screening depends largely on frequency of testing and speed of reporting and is only marginally improved by high test sensitivity. We therefore conclude that screening should prioritize accessibility, frequency, and sample-to-answer time; analytical limits of detection should be secondary.
891 citations