Kit TAAG X30 Respiratory

PROCEDURE

a.- RNA is extracted and purified from respiratory samples nasopharyngeal, oropharyngeal and nasal swabs) using the RNA extraction kit: PureRNA Extraction (TAAG Genetics). The PureRNA Extraction kit is based in spin-column RNA purification.

b.- Previously purified RNA is reverse-transcribed into cDNA, and is then put through to real-time PCR. This real-time PCR is based in TaqMan technology. RT-PCR reaction is a multiple real-time PCR reaction, which simultaneously amplifies and detects both the N1 region and the RP gene, using the probes FAM and ROX, respectively

c.- In case of a weak positive result, it has to be confirmed with a second real-time PCR reaction, which detects the N2 region of the N gene using the FAM probe.

d.- TAAG X30 Respiratory kit has been validated in several real-time PCR systems: Aria Mx Real-time PCR system (Agilent), StepOnePlus Real-time PCR system (Thermo Fisher) and CFX96 Deep Well real-time PCR system (Bio-Rad).

KIT PERFORMANCE

Detection limit

The limit of detection (LoD) of the kit was estimated by inoculating serial dilutions of quantified RNA from SARS-CoV-2 virus into nasopharyngeal swab samples which had negative results. As a result, it was concluded that the concentration at which over 95% of the samples gave a positive result, was of 50 genomic copies / µL. LoD was verified by analyzing 20 additional samples of negative nasopharyngeal swab samples inoculated with 50 genomic copies / µL of viral RNA. Results are shown in table 1

Clinical evaluation

The clinical evaluation of TAAG X30 Respiratory was performed by inoculating quantified viral RNA from SARS-CoV-2 in negative nasopharyngeal swab samples. Altogether, 30 negative and 30 positive samples were analyzed. From the 30 positive samples, 24 of them were inoculated with 50 genomic copies / µL (1X LoD), 3 with 500 genomic copies / µL (10X LoD) and the final 3 were inoculated with 5000 genomic copies / µL (100X LoD). The results are shown in table 2.

Comparative Analysis

For this study, 768 samples were analyzed using both the kit recommended by the CDC (CDC 2019-nCoV real-time RT-PCT diagnostics panel) and TAAG X30 Respiratory in parallel.

The procedure is as follows:

a.- From each sample, viral RNA was extracted using the kit TAAG VRE RNA (TAAG Genetics)

b.- 5 µL of each sample were loaded into separate reaction tubes; containing both the CDC PCR mix and the nPLEX-SARS-CoV-2 PCR mix, respectively

c.- Then, a PCR program was carried for both mixes in a CFX96 Deep Well real-time PCR system (Bio-Rad).

d.- After completion, and following both the CDC and TAAG X30 Respiratory interpretation guidelines, results from both experiments were compared against each other.

From the analysis, it was obtained that both methods have a very strong correlation (table 3). If we consider the kit recommended by the CDC as the “gold standard”, TAAG X30 Respiratory kit has a 100% of sensitivity and a 98% of specificity.