ABSTRACT
The ongoing Corona virus disease (COVID-19) outbreak has become a huge global health concern. Here, we reported a novel detection platform based on the loop-mediated isothermal amplification (LAMP), termed real-time reverse transcription LAMP (rRT-LAMP) and applied it for the diagnosis of COVID-19 (COVID-19 rRT-LAMP). rRT-LAMP integrates reverse transcription, LAMP amplification, restriction endonuclease cleavage and real-time fluorescence detection into one-pot reaction, and facilitates the diagnosis of COVID-19 at 64°C for only 35 min. The ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2 were detected for diagnosing COVID-19. The limit of detection (LoD) of COVID-19 rRT-LAMP assay was 14 copies (for each marker) per vessel, and no positive results were obtained from non-SARS-CoV-2 templates. To demonstrate its feasibility, a total of 33 oropharynx swab samples collected from COVID-19 patients also were diagnosed as SARS-CoV-2 infection using COVID-19 rRT-LAMP protocol. No cross-reactivity was yielded from 41 oropharynx swab samples collected from non-COVID-19 patients. These data suggesting that the COVID-19 rRT-LAMP assay is a potential detection tool for the diagnosis of SARS-CoV-2 infection in clinical, field and disease control laboratories, and will be valuable for controlling the COVID-19 epidemic.
ABSTRACT
BACKGROUND: The prognostic role of the interval between disease onset and hospital admission (O-A interval) was undetermined in patients with the coronavirus disease 2019 (COVID-19). METHODS: A total of 205 laboratory-confirmed inpatients admitted to Hankou hospital of Wuhan from January 11 to March 8, 2020 were consecutively included in this retrospective observational study. Demographic data, medical history, laboratory testing results were collected from medical records. Univariate and multivariate logistic regression models were used to evaluate the prognostic effect of the O-A interval (≤7 versus >7 days) on disease progression in mild-to-moderate patients. For severe-to-critical patients, the in-hospital mortality and the length of hospital stay were compared between the O-A interval subgroups using log-rank test and Mann-Whitney U test, respectively. RESULTS: Mild-to-moderate patients with a short O-A interval (≤7 days) are more likely to deteriorate to severe-to-critical stage compared to those with a long O-A interval (>7 days) [unadjusted odds ratio =2.93, 95% confidence interval (CI), 1.32-6.55; adjusted odds ratio =3.44, 95% CI, 1.20-9.83]. No association was identified between the O-A interval and the mortality or the length of hospital stay of severe-to-critical patients. CONCLUSIONS: The O-A interval has predictive values for the disease progression in mild-to-moderate COVID-19 patients. Under circumstances of the specific health system in Wuhan, China, the spontaneous healthcare-seeking behavior is usually determined by patients' own heath conditions. Hence, the O-A interval can be reflective of the natural course of COVID-19 to some extent. However, our findings should be validated further in other cohorts and in other health systems.
ABSTRACT
The ongoing global pandemic (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a huge public health issue. Hence, we devised a multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (mRT-LAMP-LFB) for diagnosing COVID-19. Using two LAMP primer sets, the ORF1ab (opening reading frame 1a/b) and N (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified in a single-tube reaction, and detected with the diagnosis results easily interpreted by LFB. In presence of FITC (fluorescein)-/digoxin- and biotin-labeled primers, mRT-LAMP produced numerous FITC-/digoxin- and biotin-attached duplex amplicons, which were determined by LFB through immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the test line of LFB) and biotin/treptavidin interaction (biotin on the duplex and strptavidin on the polymerase nanoparticle). The accumulation of nanoparticles leaded a characteristic crimson band, enabling multiplex analysis of ORF1ab and N gene without instrumentation. The limit of detection (LoD) of COVID-19 mRT-LAMP-LFB was 12 copies (for each detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. The analytical sensitivity of SARS-CoV-2 was 100% (33/33 oropharynx swab samples collected from COVID-19 patients), and the assay's specificity was also 100% (96/96 oropharynx swab samples collected from non-COVID-19 patients). The total diagnostic test can be completed within 1 h from sample collection to result interpretation. In sum, the COVID-19 mRT-LAMP-LFB assay is a promising tool for diagnosing SARS-CoV-2 infections in frontline public health field and clinical laboratories, especially from resource-poor regions.
Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Biosensing Techniques , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Biosensing Techniques/statistics & numerical data , COVID-19 , COVID-19 Testing , China/epidemiology , Clinical Laboratory Techniques/instrumentation , Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/statistics & numerical data , Coronavirus Infections/epidemiology , Equipment Design , Feasibility Studies , Humans , Limit of Detection , Molecular Diagnostic Techniques , Multiplex Polymerase Chain Reaction/methods , Multiplex Polymerase Chain Reaction/statistics & numerical data , Nanoparticles , Nanotechnology , Nucleic Acid Amplification Techniques , Pneumonia, Viral/epidemiology , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and SpecificityABSTRACT
Given the scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, known as 2019-nCov) infection (COVID-19), the ongoing global SARS-CoV-2 outbreak has become a huge public health issue. Rapid and precise diagnostic methods are thus immediately needed for diagnosing COVID-19, providing timely treatment and facilitating infection control. A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) coupled with nanoparticles-based biosensor (NBS) assay (RT-LAMP-NBS) was successfully established for rapidly and accurately diagnosing COVID-19. A simple equipment (such as heating block) was required for maintaining a constant temperature (63 C) for only 40 min. Using two designed LAMP primer sets, F1ab (opening reading frame 1a/b) and np (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified and detected in a one-step and single-tube reaction, and the detection results were easily interpreted by NBS. The sensitivity of SARS-CoV-2 RT-LAMP-NBS was 12 copies (each of detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. Among clinically diagnosed COVID-19 patients, the analytical sensitivity of SARS-CoV-2 was 100% (33/33) in the oropharynx swab samples, and the assay's specificity was also 100% (96/96) when analyzed the clinical samples collected from non-COVID-19 patients. The total diagnosis test from sample collection to result interpretation only takes approximately 1 h. In sum, the RT-LAMP-NBS is a promising tool for diagnosing the current SARS-CoV-2 infection in first line field, public health and clinical laboratories, especially for resource-challenged regions.