ABSTRACT
Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of seven wastewater samples from COVID-19 hotspots. RTLAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best target gene for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be the best target gene for SARS-CoV-2 detection. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except microchannels which subjected to RT-LAMP for targeting N region after RNA extraction (yellow color) in 6 out of 7 samples. This study shows that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips. This study brings the novelty involving the use of wastewater samples for detection of SARS-CoV-2 without previous virus concentration and with/without RNA extraction.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Wastewater , COVID-19/diagnosis , COVID-19 Testing , Point-of-Care Systems , Microfluidics , Sensitivity and Specificity , RNAABSTRACT
Here, we report the coding-complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate obtained from a nasopharyngeal swab from the first patient with COVID-19 in Gilgit, Pakistan.
ABSTRACT
Coronavirus disease-19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is now a pandemic threat. This virus is supposed to be spread by human to human transmission. Cellular angiotensin-converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2 which is identical or similar in different species of animals such as pigs, ferrets, cats, orangutans, monkeys, and humans. Moreover, a recent study predicted that dogs might be secondary hosts during the evolution of SARS-CoV-2 from bat to human. Therefore, there is a possibility of spreading SARS-CoV-2 through domestic pets. There are now many reports of SARS-CoV-2 positive cases in dogs, cats, tigers, lion, and minks. Experimental data showed ferrets and cats are highly susceptible to SARS-CoV-2 as infected by virus inoculation and can transmit the virus directly or indirectly by droplets or airborne routes. Based on these natural infection reports and experimental data, whether the pets are responsible for SARS-CoV-2 spread to humans; needs to be deeply investigated. Humans showing clinical symptoms of respiratory infections have been undergoing for the COVID-19 diagnostic test but many infected people and few pets confirmed with SARS-CoV-2 remained asymptomatic. In this review, we summarize the natural cases of SARS-CoV-2 in animals with the latest researches conducted in this field. This review will be helpful to think insights of SARS-CoV-2 transmissions, spread, and demand for seroprevalence studies, especially in companion animals.