The most reliable test for the detection of SARS-CoV-2 virus is performed by the Real Time PCR method, providing the ability to perform a result between 12 to 28 hours.
The procedure initially involves obtaining a nasal / pharyngeal smear, from which total genomic RNA is extracted using the MagCore® Viral Nucleic Acid Extraction Kit (High Sensitivity) Code 203 (RBC Bioscience) in an automated MagCore HF16 isolation system according to manufacturer's instructions. The sample is then subjected to the One Step RT Reverse Transfer Reaction Process (One Step RT - Real Time PCR) using the 2019-nCoV CDC qPCR Probe Assay (CDC EUA kit, IDT) primers. analyzed below) according to the CDC guidelines. The primer-tracer mixture "targets" two genomic regions of the N (N1 and N2) gene of the virus encoding "Nucleocapsid protein" and the "human RNase P" (RP) gene, which is an indicator of sample quality as well as and a confirmatory indicator that the process of extracting genetic material from the sample has been achieved. The One Step RT - Real Time PCR process is performed on a LightCycler 480 (Roche Life Science) platform.
The limit of detection of the present test, as performed in the Neoscreen laboratory as determined using sequential dilutions of the positive control 2019-nCOV_N-Positive Control (REF: 225507240), is 20 copies / ul. It is characterized by a high degree of specificity and exclusivity for the SARS-CoV-2 virus according to in silico analyzes as well as a study of application of the test to samples carrying other pathogens (such as MERS-coronavirus, SARS-coronavirus, influenza H1N1), making this method as the most reliable for virus detection.
But how effective is the 2019-nCoV CDC qPCR Probe Assay (CDC EUA kit, IDT)?
Testing of 2071 samples of the respiratory system yielded 2021 negatives, 49 positives and only 1 as invalid (it was a pharyngeal / laryngeal smear). 17 of the 49 positive samples were further tested by viral genome sequencing and the initial finding of the 2019-nCoV CDC qPCR Probe Assay test was confirmed (100% agreement). Study of 117 respiratory samples with the independent application of the 2019-nCoV CDC qPCR Probe Assay and corresponding test with different genomic regions of the virus as targets (N4 & N5) showed 100% agreement of the two tests in both the samples that were characterized as negative and in them which were characterized as positive, finally finding the clinical performance of the kit and the RT-PCR method as extremely specific and effective.