Razi Journal of Medical Sciences

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The recent SARS-CoV-2 pandemic is the hottest topic of the scientific world all around the globe. Regarding the fast-spreading nature of the infectious agent and the involvement of all countries, it seems necessary to take fundamental practical actions to restrain the spread of this deadly viral disease. The first and likely most important action could be breaking the chain of infection between susceptible hosts which needs the performance of intensive fast and accurate screening tests for specific detection of the virus and infected individuals. This review focuses on different influential aspects of performing a molecular diagnostic test using standard Real-Time PCR assay to detect SARS-CoV-2. Practical hints for performing an accurate test and overcoming potential technical problems that might affect the final result are discussed as well. Moreover, pre-analytical aspects of the test, including standard sampling, sample transfer, and genome extraction are taken into consideration. Briefly, both nasopharyngeal and oropharyngeal samples were taken in the virus transport medium (VTM), highly recommended to be kept in the cold chain. After primary checks regarding sampling criteria, all samples are aliquoted in screw-capped tubes. This step is considered with the highest potential for contamination and the risk of infection for laboratory technicians. So, it should be performed in biosafety level 2 laminar flow cabinets. Aliquoted samples are then sent to the genome extraction unit. Genomic RNA extraction is performed by different methods, including phenol-chloroform precipitation, silica-based membrane isolation, and magnetic beads. Although precipitation-based methods, mostly performed using TRIzol are associated with higher yields of RNA, the presence of impurities such as proteins, salts, polyphenols, and polysaccharides in the final product is lead to reduced success in the following molecular experiments. Column-based methods, although more expensive, are more user-friendly and result in a more pure product. Extracted RNA is subjected to one-step real-time PCR assay. To perform real-time PCR, it is highly recommended to prepare a reaction mix for all the samples plus one (n+1) which results in the optimum use of kit contents, homogenous solution for all samples, and reduced chance of contamination. Moreover, several control reactions are needed to be included in the assay including negative control, positive control, and no-template control (NTC). Currently, available diagnostic kits of SARS-CoV-2 use mostly the TaqMan probe strategy to detect different viral genes (mostly, E, N, RdRP). Moreover, failure in sampling is investigated through analysis of the amplification of an endogenous internal control gene (mostly RNase P). Based on the manufacturers’ instructions, the thermal profile is set in the real-time PCR instrument. This profile commonly includes a hold step for reverse transcription of RNA in ~50 ͦ C for 10-30 minutes and another hold step for cDNA pre-denaturation in 95 ͦ C for 1-15 minutes. Amplification cycles (not less than 40 cycles) are then applied via the denaturation step and annealing/extension step. It is very important to pay attention to the acquisition of fluorescence in different kits, because they may use different channels to read the data in pre-defined instruments. At the end of the test, the threshold cycle (Ct) is defined by the user in the exponential phase of the test, and all the curves which hit the threshold before cycle 40 can be considered positive if the shape of the curve is sigmoidal. Analysis and report of the results are dependent on the accuracy of the test which is deduced from the results of negative control, positive control, and NTC. The main drawback of the real-time PCR assay to test SARS-CoV-2 infection is in the pre-analytical step in which inappropriate sampling may lead to false-negative results. Moreover, the course of the disease in the time of sampling, sampling area, the shipment of the sample, quality of the extraction kit as well as quality of the detection kit is all contributed in the final result of the virus detection.resistance characteristics are determined and appropriate antibiotics are prescribed.

Keywords: SARS-CoV-2, COVID-19, RT-PCR

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