Analytical and clinical evaluation of a heat shock SARS-CoV-2 detection method without RNA extraction for N and E genes RT-qPCR

Abstract

Background : The COVID-19 pandemic has caused significant supply shortages worldwide for SARS-CoV-2 molecular diagnosis, like RNA extraction kits. Objective : The aim of our study was to evaluate the clinical performance and analytical sensitivity of a simple SARS-CoV-2 diagnosis protocol based on heat shock without RNA extraction using both "CDC" (N gene) and "Charite" (E gene) RT-qPCR protocols. Results : 1,036 nasopharyngeal samples, 543 of them SARS-CoV-2 positive, were analyzed. The heat shock method correctly identified 68,8% (232/337) and 89.4% (202/226) SARS-CoV-2 positive samples for N gene and E gene, respectively. Analytical sensitivity was assessed for heat shock method using the CDC RT-qPCR protocol, obtaining sensitivity values of 98,6%, 93,3% and 84,8% for limit of detection of 100.000, 50.000 and 20.000 viral RNA copies/mL of sample. Conclusions : Our findings show that a simple heat shock SARS-CoV-2 RT-qPCR diagnosis method without RNA extraction is a reliable alternative for potentially infectious SARS-CoV-2 positive patients. This affordable protocol can help to overcome the cost and supply shortages for SARS-CoV-2 diagnosis, especially in developing countries. In Ecuador, it has been used already by laboratories in the public health system for more than 100.000 specimens.

Diagnostic performance of a colorimetric RT -LAMP for the identification of SARS-CoV-2: A multicenter prospective clinical evaluation in sub-Saharan Africa

“…Furthermore, the RT-LAMP assay does not require expensive equipment, such as a thermal cycler with real-time fluorescence measurement, because readout of the RT-LAMP can be simply colorimetric, with a pH dependent shift of colour for a positive reaction [8] . Also the step of RNA extraction, common to both RT-PCR and RT-LAMP, could be skipped both from swabs or directly from saliva with minimal loss of sensitivity and a great advantage in terms of savings as several reports have been proposing [ [9] , [10] , [11] , [12] , [13] , [14] ]. Colorimetric RT-LAMP represents a formidable alternative to RT-PCR, but comprehensive clinical studies are still required for evidence-based decision-making towards its broad implementation across geographical settings.…”

High SARS-CoV-2 infection rates and viral loads in community-dwelling individuals from rural indigenous and mestizo communities from the Andes during the first wave of the COVID-19 pandemic in Ecuador

“…Nasopharyngeal swabs were collected on a 0.5-mL TE pH 8 buffer for SARS-CoV-2 diagnosis by RT-qPCR, following an adapted version of the CDC protocol as it has been previously described by our laboratory. In brief, the CDC RT-qPCR protocol is based on N1 and N2 probes to detect SARS-CoV-2 and RNase P as an RNA extraction quality control ( 20 – 28 ). In addition, negative controls (TE pH 8 buffer) were included as a control for carryover contamination, one for each set of RNA extractions, to guarantee that only true positives were reported.…”

Advancements in Testing Strategies for COVID-19

“…Moreover, a longer time is needed for nucleic acid isolation, which is unfavorable for rapid diagnosis. Several methods such as BSA-based protocol [ 248 ], heat shock-based [ 249 ] and acid pH method [ 250 , 251 ] are taken to conduct the RNA isolations of SARS-CoV-2. The low viral loads hinder the accurate screening of the viral genome that cause false-negative testing [ 252 , 253 , 254 , 255 ].…”