Volume 30, Issue 2 (4-2023)                   RJMS 2023, 30(2): 75-83 | Back to browse issues page

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Elsagh A, Ghavamipour lahij H, Pormehr M, Ghaderi Aslshabestari M. Design and Production of Viral RNA Extraction Kit with Emphasis on Coronavirus. RJMS 2023; 30 (2) :75-83
URL: http://rjms.iums.ac.ir/article-1-7144-en.html
Assistant Professor of Physical Chemistry, Department of Chemistry, North Tehran Branch, Islamic Azad University, Tehran, Iran , a_elsagh@iau-tnb.ac.ir
Abstract:   (674 Views)
Background & Aims: Chirgwin et al. (1979) were the first authors who proposed the ribonucleic acid (RNA) isolation technique. Without denaturation of the structure of nucleic acid, this method breaks the cell membrane using guanidinium thiocyanate and 2-mercaptoethanol (β-met). The extraction is carried out using ethanol or ultracentrifuge, and cesium chloride gradient. This method is time-consuming, inefficient, and involves toxic and incompatible compounds for the user. Thus, the quality and efficiency of the process of extraction were developed over time and through conducting more studies. Chomczynski and Sacchi (1987) improved the method suggested by Chirgwin et al. and proposed a single-step extraction process called the AGPC method, and it used acid guanidinium, phenol, and chloroform. The advantage of this method is its higher purity and efficiency, plus this process of isolation does not include RNA fragmentation. This method has been developed and using silica columns together with magnetic beads resulted in a revolution in the process of isolating nucleic acids. Extraction of viral nucleic acid includes high-purity DNA and RNA, determining sensitivity, and genome viral load within a short process is quite crucial for the majority of downstream molecular and medical studies such as sequencing, real-time PCR, PCR for research or diagnostic purposes. Efficient protocols for the extraction of nucleic acids are the prerequisite of these methods. As already mentioned the most available protocols are time-consuming and have limitations regarding some samples or disparate types of nucleic acids. An optimal protocol must provide a high sensitivity concerning the extraction of the viral genome from a broad spectrum of samples together with a shorter scientific time and lower price. The majority of methods up to the present time have been using precipitation steps to obtain pure nucleic acids from the extracts. New protocols use the binding capacity of the nucleic acid extract to the silica column. Vogelstein and Gillespie were the first scholars to describe the absorption of nucleic acid to the surface of silica in the presence of great concentrations of chaotropic salts. They used pieces of DNA recovered from agarose gel using silica powder. This technology is developed over time and now selective binding of DNA or RNA through silica gel surfaces is modified and binding and wash buffers have been optimized to provide the maximum separation and isolation capacity in nucleic acids. After the initial lysis of the sample, the respective nucleic acid bound to the membrane is regulated. Polysaccharides and proteins in the extract fail to function properly and lose their ability to bind to the membrane due to the destruction of the molecular structure. Finally, nucleic acid bounded to the column is desalinated by alcohol and washed, and then, separated from the column by elution buffer. One of the limitations of the process of extraction is separating various types of nucleic acids from different viral resources. For instance, Coronavirus contains an RNA genome and on the other hand, the samples are collected from the patient’s nose and throat, which requires an efficient and extremely sensitive procedure for extracting the genome of this virus. Given that, Tehran Cavosh Clon (former Sinaclon), a knowledge-based complex, proposed an optimal method for spearing viral RNA with high efficiency and free from toxic materials in accordance with conducted studies. The present research reported the results of the clinical studies on the basis of European Pharmacopoeia standards.
Methods: Viral RNA was extracted from HCV, HIV, and COVID-19 samples. Sensitivity, specificity, verification, and stability tests were carried out according to the European pharmacopeia standard. Furthermore, Tehran Cavosh Clon’s viral extraction kit (SinaPure Viral) was compared with extraction kits of QIA Gen and GeneAll companies, as valid and the most common brands in the market. Finally, the results of PCR and Real-Time tests by these kits were compared. Serums infected with HCV, HIV, and COVID-19 swab nasopharyngeal were provided from Keyvan Laboratory. The reference viral genome extraction kits (QIA Gen:CAT:52904; GeneAll: CAT:128-150), and HCV, HIV, and COVID-19 diagnostic kits (GenproofHIV1/ISEX/050:HCVD/ISEX/050; CMV/ISEX/050; Sansure Biotech: S3104E) Were purchased from the respective agencies.  Chemicals used in the kit structure were purchased from Sigma Company.
Kit comprises lysis buffer, precipitation buffer, wash buffer I, wash buffer II, and elution buffer, carrier RNA, and binding columns. First, 400 µl lysis buffer together with 5-6 µl of carrier RNA were added to microtubes containing the samples and they were vortexed for 20 seconds. Then, 300 µl of the precipitation buffer was added to the above solution and vortexed for 5 seconds. The final solution was transferred to the column and centrifuged at 12,100 x g,13,000 rpm, for 1 minute. It must be noted that the transferred solution must be discarded. In the next step, 400 µl of wash buffer I was poured on the column, and centrifuged, at the aforesaid revolutions for 1 minute. The transferred solution was discarded. 400 µl of wash buffer II was transferred to the above column, and centrifuged, at the aforesaid revolutions for 1 minute. The transferred solution was discarded. Finally, without adding any solution, the collector column was centrifuged for 2 minutes. Then, it was transferred to 1.5 ml sterile microtubes and 50 µl elution buffer was added to it. It was placed at 55 degrees centigrade for 3 to 5 minutes. Upon the final centrifuge, the viral RNA was separated from the column with elution buffer. The extracted nucleic acid was collected. This product is used for diagnostic tests.
The data were statistically analyzed using SPSS, 18. The diagrams were illustrated using Excel 2010. The One-way ANOVA test was carried out. The means were compared using Duncan's test with a significance level of P<0.05.
Results: Table 1 shows the results of comparing the sensitivity test of SinaPure viral extraction kit with the QIA Gen and GeneAll regarding samples with HIV, HCV, and COVID-19. According to these results, the obtained Ct for LOD of HIV samples indicated no significant difference between GeneAll and SinaPure viral kits. While there was a significant difference between the QIA Gen and the aforesaid kits. The results of the sensitivity test of samples with HCV and COVID-19 manifested a similar pattern. The only significant difference between the QIA Gen and SinaPure viral with respect to the amount of LOD.
Table 1 shows the results of the specificity of the SinaPure viral kit on different viral, bacterial, plant, and animal samples. Per the results, except for viruses with RNA such as HCV, HIV, and COVID-19, no positive response was observed in none of the viruses with DNA such as HSV, EPV, HPV, CMV, HBV, plus gram-negative bacteria, (Ecoli), gram-positive bacteria (Staphylococcus aureus), plant samples (wheat), and animal sample (CHO Cell). This confirms SinaPure Viral’s ability in extracting the genome of viruses with RNA.
SinaPure Viral confirmation kit was compared to QIA Gen and GeneAll kits in high, medium, and law counts of HCV and HIV serums, plus Swab Nasopharyngeal for COVID-19. The results of disparate counts of HIV-positive samples using all three kits demonstrated no significant difference (Table 2). On the other hand, the same pattern was observed in HCV samples (Table 3). Table 4 shows the results for the COVID-19 samples. Even though kits manifested no significant difference in the high and medium counts, in lower counts of the virus, QIA Gen had a significant difference with SinaPure Viral and GeneAll. It can be due to the difference in the sensitivity of sampling since in the lower loads of virus there is a possibility of making mistakes by the sample collector. On the other hand, the lack of significant difference between the HIV and HCV samples in disparate counts confirms this.
Tables 5, 6, and 7 show the results of investigating the stability of the SinaPure Viral kit in the time intervals of 6, 12, and 18 months. Per these results, SinaPure Viral kit manifested no significant difference with QIA Gen and GeneAll in the time intervals of 6 and 12 months. Even after after18 months, the SinaPure Viral kit manifested a significant difference with reference kits in low viral counts in all samples. These high count viruses did not demonstrate this reduction of efficiency. The possible cause of reduction of kit efficiency could be the location of storing carrier RNA after the first time they were used. Taking into account the considerable role of carrier RNA in the viral genome precipitation, after preparing the carriers RNA solution, its storage condition is extremely effective on the stability of RNA.
Conclusion: In accordance with the results, the pharmacopoeia standard of the kit designed by Tehran Cavosh Clon corresponded to reference kits, i.e. QIA Gen and GeneAll. Given that, this kit is comparable to reference kits and it can be considered as a valid replacement for foreign kits in the market.
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Type of Study: Research | Subject: Microbiology

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