تشخيص ويروسSARS-CoV-2 در بيماران زير 45 سال با روش RT-qPCR و بررسي امكان تشخيص آن با روش RT-LAMP

Detection of SARS-CoV-2 virus in patients under 45 years of age by RT-qPCR and eva‎luation of its diagnosis possibility by RT-LAMP technique

زيست شناسي سلولي مولكولي و ميكروبيولوژي

The rapid spread of the SARS-CoV-2 virus throughout the world and its high transmission speed have created the need to expand various diagnostic methods. Different diagnostic methods are used to identify people infected with the virus. Some of these methods are based on the molecular amplification of one or more target genes of the virus. The most common method is RT-qPCR, which is considered as a standard diagnostic method. Of course, due to a series of disadvantages of this method, including the need for a thermocycler, other molecular methods such as isothermal amplification have also been considered, the most common of which is RT-LAMP. This method amplifies the target gene with the help of 4-6 primers at a constant temperature (unlike RT-qPCR). Along with these methods, rapid diagnostic kits have also been developed to identify people infected with SARS-CoV-2. These kits combine conventional immunology or molecular methods with LFA and make it easier to observe the results of these methods. In this research, with the help of two molecular methods, RT-qPCR and colorimetric RT-LAMP, the SARS-CoV-2 virus was detected in 34 nasal and pharyngeal swab samples.RdRp gene was detected by RT-qPCR method and ORF8 gene was detected by RT-LAMP method. Moreover, the colorimetric RT-LAMP was carried out on swab samples directly (without the RNA extraction process). Then strip tests were made based on gold nanoparticles and combined with the RT-LAMP method. In this method, two out of six primers of the ORF8 gene were labeled with biotin and Dig. Gold nanoparticles were conjugated with Anti-Dig antibodies. Anti-Dig antibody and streptavidin were loaded in control and test points, respectively. Without the need for RNA extraction, RT-LAMP reaction was performed on nasal and pharyngeal swab samples, and then the results were analyzed by two methods, gel electrophoresis, and LFA. Based on the results obtained from the comparison of RT-qPCR and RT-LAMP colorimetric results, this isothermal amplification method has a sensitivity of 96.67% and a specificity of 75% and showed an acceptable performance on samples without extraction. On the other hand, the RT-LAMP-LFA method could well distinguish positive and negative samples with 100% sensitivity and 77.27% specificity. In this method, only nasal and pharyngeal swab samples were heated for 10 minutes at 65 ºC and then used in the RT-LAMP reaction. In general, this isothermal amplification method in combination with LFA rapid detection method and without the need for RNA extraction process can be used as a diagnostic method for SARS-CoV-2.

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