Overview

Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP genes) Dx is an in vitro diagnostic test for the qualitative detection of SARS-CoV-2 specific RNA using a multiplexed TaqMan® fluorescence detection assay (FAM and HEX/VIC) based on the Charité/Berlin protocol. The assay is designed for use with RNA isolated from nasopharyngeal swabs, oropharyngeal swabs and saliva samples collected from individuals with clinical signs/symptoms related to SARS-CoV-2 infection for in vitro diagnostic use.

Product Description

Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP genes) Dx includes 2XOne-Step RT-PCR Master Mix and 2 primer/probe mixes, a positive control and a negative control (nuclease-free water). The first primer/probe mix is used for first line screening and contains the E gene/RP that targets the SARS-CoV-2, SARS-CoV and bat-SARS-related CoVs Envelope gene(E gene -FAM) in addition to the human RNase P transcript (RP-HEX/VIC) as an internal control target to monitor for PCR inhibition, and to validate the quality of the sample and the detection result. The second Primer/Probe Mix is only required as a confirmatory/discriminatory step with samples showing positive amplification of the E gene. This second Primer/Probe Mix is for the RdRP gene and detects two RNA-dependent RNA Polymerase (RdRP) targets where the first RdRP target is SARS-CoV-2 specific (FAM) while the second RdRP target is to detect SARS-CoV-2, SARS-CoV and bat-SARS-related CoVs (HEX). The provided E gene/RdRP/RP Positive Control contains an in vitro RNA transcript for the three SARS-related target genes: E gene, RdRP gene as well as the human RP gene (internal control).

Positive results are indicative of SARS-CoV-2 RNA detection, however clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out co-infection with other viruses and therefore the agent detected may not be the definite cause of disease. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Any negative results must be combined with clinical observations, patient history, and epidemiological information.

Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP genes) Dx is intended for use by professional users including clinical laboratory personnel experienced and trained in molecular biology techniques including real-time PCR and in vitro diagnostic procedures.

Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP genes) Dx was developed and validated to be used with the BioRad CFX96 Touch™ Real-Time PCR Detection System.

Details

Supporting Data

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Figure 1. Duplex TaqMan RT-PCR analytical sensitivity for each of the SARS-CoV-2 targets based on the Charité/Berlin protocol. The analytical sensitivity was determined by analyzing six serial dilutions (200,000 copies to 2 copies) of Norgen's positive control from the COVID-19 TaqMan RT-PCR Kit (E/RdRP gene)(Cat. TM67200). Five µL of template was used in a 20 µL of the one-step RT-PCR reaction and the arrow indicates a copy number of 10.

Table 1. To test the analytical specificity of Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP genes), the E/RdRP/RP Positive Control was used to test the kits specificity against RNA purified from other related pathogens. RdRP reactions were only prepared for samples required for confirmation and discriminatory detection based on the first gene detection result. Amplification was measured by real time RT-PCR.  As can be seen, only Norgen’s E/RdRP/RP Positive Control showed amplification for all genes. COVID-19 WA showed amplification of the E gene and the two RdRP gene targets. SARS-CoV showed amplification only with E gene and RdRP Discriminatory. None of the remaining pathogens showed amplification with any of the tested genes.

 

Figure 2. Clinical testing of the COVID-19 TaqMan RT-PCR Kit (E/RdRP gene)(Cat. TM67200) was conducted with contrived oropharyngeal swabs (circle) and nasopharyngeal swabs (cross).  Swab samples were collected from healthy donors and placed into preservative using Norgen’s Total Nucleic Acid Preservation Tubes (Cat. 69200).  To prepare the contrived positive samples, the preserved swab samples were spiked with 100,000 copies of positive control. Total viral RNA was then isolated using Norgen’s Total RNA Purification Kit (Cat. 17200).  Following RNA isolation, one-step RT-PCR for detection of the four targets was performed using Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP gene) (TM67200). 

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