Analytical and Clinical Validation for RT-qPCR detection of SARS-CoV-2 without RNA extraction

Background The recent COVID-19 pandemic has posed an unprecedented challenge to laboratory diagnosis, based on the amplification of SARS-CoV-2 RNA. With global contagion figures exceeding 4 million persons, the shortage of reagents for RNA extraction represents a bottleneck for testing globally. We present the validation results for a RT-qPCR protocol without prior RNA extraction. Because of its simplicity, this protocol is suitable for widespread application in resource-limited settings. Methods Optimal protocol was selected by comparing RT-qPCR performance under a set of thermal (65°, 70°, and 95° for 5, 10, and 30 minutes) and amplification conditions (3 or 3,5 uL loading volume; 2 commercial RT-qPCR kits with limit of detection below 10 copies/sample) in nasopharyngeal swabs stored at 4°C in sterile Weise’s buffer pH 7.2. The selected protocol was evaluated for classification concordance with the standard protocol (automated RNA extraction) in 130 routine samples and in 50 historical samples with Cq values near to the clinical decision limit. Results Optimal selected conditions were: Thermal shock at 70° C for 10 minutes, loading 3.5 ul in the RT-qPCR. Prospective evaluation in 130 routine samples showed 100% classification concordance with the standard protocol. The evaluation in historical samples, selected because their Cqs were at the clinical decision limit, showed 94% concordance with our confirmatory-gold standard which includes manual RNA extraction. Conclusions These results validate the use of this direct RT-qPCR protocol as a safe alternative for SARS CoV-2 diagnosis in case of a shortage of reagents for RNA extraction, with minimal clinical impact.