Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs

Abstract

The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.

Rapid Detection of SARS-CoV-2 RNA in Human Nasopharyngeal Specimens Using Surface-Enhanced Raman Spectroscopy and Deep Learning Algorithms

“…Purified IVT RNA was eluted in nuclease-free water, adjusted to a final concentration of 10 9 copies/mL in nuclease-free water, and stored at −80 °C. 9 …”

“…The SARS-CoV-2 pandemic and COVID-19 disease have led to an unprecedented burden on worldwide healthcare. This has driven researchers to develop rapid and reliable sensors to aid SARS-CoV-2 diagnostics. − Among various diagnostic strategies, − viral nucleic acid detections, such as reverse-transcription polymerase chain reaction (RT-PCR), , reverse-transcription loop-mediated isothermal amplification, − and clustered regularly interspaced short palindromic repeats, , have become the mainstream technology. Several RT-PCR-based assays are the most common and reliable method for viral diagnostics and are considered the “gold standard” for selectivity and sensitivity.…”

Diagnostic assay and technology advancement for detecting SARS-CoV-2 infections causing the COVID-19 pandemic

“…Recently, Jones and colleagues developed an RT-LAMP assay for SARS-CoV-2 and SARS-CoV-2 variant RNA amplification detection directly from nasopharyngeal swab samples taken from patients. The advantage of this method was there was no requirement to isolate the RNA from the specimen [73]. The RCA assay was used to detect SARS-CoV both in liquid and solid phases [74].…”

Validation of the RT-LAMP assay in a large cohort of nasopharyngeal swab samples shows that it is a useful screening method for detecting SARS-CoV-2 and its VOC variants

“…This aims to genotype the virus and, in the future, provide an alternative approach for detecting SARS-CoV-2 and performing genomic surveillance both in symptomatic and asymptomatic individuals, even employing a variety of samples (74-77). Unfortunately, few studies report RT-LAMP validation in samples based on variants propagated in Vero cells (78), or the RT-LAMP assay is focused on validation with a VOC variant (79). We propose that validating and using RT-LAMP to identify infected patients could make this assay a very important tool for epidemiological and genomic surveillance of SARS-CoV-2 in laboratories where the necessary infrastructure is not available, even in saliva samples that have turned out to be a useful type of sample for the diagnosis of SARS-CoV (80).…”