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 , RNA
The spectrum of emerging new diseases as well as re-emerging old diseases is broadening as infectious agents evolve, adapt, and spread at enormous speeds in response to changing ecosystems. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recent phenomenon and may take a while to understand its transmission routes from less traveled territories, ranging from fomite exposure routes to wastewater transmission. The critical challenge is how to negotiate with such catastrophic pandemics in high-income countries (HICs ~20% of the global population) and low-and middle-income countries (LMICs ~ 80% of the global population) with a total global population size of approximately eight billion, where practical mass testing and tracing is only a remote possibility, particularly in low-and middle-income countries (LMICs). Keeping in mind the population distribution disparities of high-income countries (HICs) and LMICs and urbanisation trends over recent years, traditional wastewater-based surveillance such as that used to combat polio may help in addressing this challenge. The COVID-19 era differs from any previous pandemics or global health challenges in the sense that there is a great deal of curiosity within the global community to find out everything about this virus, ranging from diagnostics, potential vaccines/therapeutics, and possible routes of transmission. In this regard, the fact that the gut is the common niche for both poliovirus and SARS-CoV-2, and due to the shedding of the virus through faecal material into sewerage systems, the need for long-term wastewater surveillance and developing early warning systems for better preparedness at local and global levels is increasingly apparent. This paper aims to provide an insight into the ongoing COVID-19 crisis, how it can be managed, and what measures are required to deal with a current global international public health concern. Additionally, it shed light on the importance of using wastewater surveillance strategy as an early warning practical tool suitable for massive passive screening, as well as the urgent need for microfluidic technology as a rapid and cost-effective approach tracking SARS-CoV-2 in wastewater.