Sensitive SARS-CoV-2 detection, air travel Covid-19 testing, variant determination and fast direct PCR detection, using ddPCR and RT-qPCR methods

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monitoring in air traffic is important in the prevention of the virus spreading from abroad. The gold standard for SARS-CoV-2 detection is RT-qPCR; however, for early and low viral load detection, a much more sensitive method, such as droplet digital PCR (ddPCR), is required. Our first step was to developed both, ddPCR and RT-qPCR methods, for sensitive SARS-CoV-2 detection. Analysis of ten swab/saliva samples of five Covid-19 patients in different stages of disease showed positivity in 6/10 samples with RT-qPCR and 9/10 with ddPCR. We also used our RT-qPCR method for SARS-CoV-2 detection without the need of RNA extraction, obtaining results in 90-120 minutes. We analyzed 116 self-collected saliva samples from passengers and airport staff arriving from abroad. All samples were negative by RT-qPCR, while 1 was positive, using ddPCR. Lastly, we developed ddPCR assays for SARS-CoV-2 variants identification (alpha, beta, gamma, delta/kappa) that are more economically advantageous when compared to NGS. Our findings demonstrated that saliva samples can be stored at ambient temperature, as we did not observe any significant difference between a fresh sample and the same sample after 24 hours (p = 0.23), hence, saliva collection is the optimal route for sampling airplane passengers. Our results also showed that droplet digital PCR is a more suitable method for detecting virus from saliva, compared to RT-qPCR.

Comparison of the methods for isolation and detection of SARS-CoV-2 RNA in municipal wastewater

“…Thresholding was carried out by using QuantaSoft Software manually at the lowest amplitude that captures true negative clusters based on the signals of the negative control and positive control samples. The results were reported as positive when at least five copies of each viral gene ( RdRp, E ) occurred ( 15 ). Data are interpreted as copies per reaction according to previous works evaluating the presence of SARS-CoV-2 in samples by RT-ddPCR ( 16 ).…”

Comparison of L452R mutation variant diagnosis in SARS-COV-2 PCR positive samples with two different qPCR kits

“…While commercial qPCR tests used in daily practice e-ISSN: in the diagnosis of SARS-CoV-2 virus detect some gene regions of the virus, they can not accuretely respond to the issues such as prolonged positivity, disease stage, reinfection and clinical condition of the patient [3]. Diagnostic tests with the qPCR method developed for the SARS-CoV-2 infection detection is one of the most important mechanisms that helps limiting the spread of the virus along with the measures to be taken by monitoring it [4]. SARS-CoV-2 qPCR kits basically targets "hemagglutinin-esterase" HE, "open reading frame 1" ORF1, "envelope glycoproteins spike" S, "RNA-dependent RNA polymerase" RdRp, "helicase" Hel, "nucleocapsid protein targets" N, "envelope" E, and "transmembrane" M [5].…”