ABSTRACT
This paper reports the design, development, and testing of a novel, yet simple and low-cost portable device for the rapid detection of SARS-CoV-2. The device performs loop mediated isothermal amplification (LAMP) and provides visually distinguishable images of the fluorescence emitted from the samples. The device utilises an aluminium block embedded with a cartridge heater for isothermal heating of the sample and a single-board computer and camera for fluorescence detection. The device demonstrates promising results within 20 min using clinically relevant starting concentrations of the synthetic template. Time-to-signal data for this device are considerably lower compared to standard quantitative Polymerase Chain Reaction(qPCR) machine (~10-20 min vs. >38 min) for 1 × 102 starting template copy number. The device in its fully optimized and characterized state can potentially be used as simple to operate, rapid, sensitive, and inexpensive platform for population screening as well as point-of-need severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) detection and patient management.
ABSTRACT
Novel corona virus SARS-CoV-2, causing coronavirus disease 2019 (COVID-19), has become a global health challenge particularly for developing countries like Pakistan where overcrowded cities, inadequate sanitation, little health awareness and poor socioeconomic conditions exist. The SARS-CoV-2 has been known to spread primarily through direct contact and respiratory droplets. However, detection of SARS-CoV-2 in stool and sewage have raised the possibility of fecal-oral mode of transmission. Currently, quantitative reverse-transcriptase PCR (qRT-PCR) is the only method being used for SARS-CoV-2 detection, which requires expensive instrumentation, dedicated laboratory setup, highly skilled staff, and several hours to report results. Considering the high transmissibility and rapid spread, a robust, sensitive, specific and cheaper assay for rapid SARS-CoV-2 detection is highly needed. Herein, we report a novel colorimetric RT-LAMP assay for naked-eye detection of SARS-COV-2 in clinical as well as sewage samples. Our SARS-CoV-2 RdRp-based LAMP assay could successfully detect the virus RNA in 26/28 (93%) of RT-PCR positive COVID-19 clinical samples with 100% specificity (n = 7) within 20 min. We also tested the effect of various additives on the performance of LAMP assay and found that addition of 1 mg/ml bovine serum albumin (BSA) could increase the sensitivity of assay up to 101 copies of target sequence. Moreover, we also successfully applied this assay to detect SARS-CoV-2 in sewage waters collected from those areas of Lahore, a city of Punjab province of Pakistan, declared as virus hotspots by local government. Our optimized LAMP assay could provide a sensitive first tier strategy for SARS-CoV-2 screening and can potentially help diagnostic laboratories in better handling of high sample turnout during pandemic situation. By providing rapid naked-eye SARS-CoV-2 detection in sewage samples, this assay may support pandemic readiness and emergency response to any possible virus outbreaks in future.
Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , SARS-CoV-2/isolation & purification , Sewage/virology , COVID-19/virology , COVID-19 Testing , Colorimetry , Feces/virology , Humans , Mass Screening , Pakistan/epidemiology , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics , Sensitivity and SpecificityABSTRACT
This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng µL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.