In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis tests
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has rapidly spread through the entire world and has become the worst pandemic from December 2019 until now. The establishment of positive standards for molecular diagnostic test...
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| Format: | Article |
| Language: | English |
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Vietnam Ministry of Science and Technology
2022-12-01
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| Series: | Vietnam Journal of Science, Technology and Engineering |
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| Online Access: | https://vietnamscience.vjst.vn/index.php/vjste/article/view/1085 |
| _version_ | 1797937847995465728 |
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| author | Hang Thi Thu Dinh Mai Ngoc Nguyen Su Xuan Hoang |
| author_facet | Hang Thi Thu Dinh Mai Ngoc Nguyen Su Xuan Hoang |
| author_sort | Hang Thi Thu Dinh |
| collection | DOAJ |
| description |
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has rapidly spread through the entire world and has become the worst pandemic from December 2019 until now. The establishment of positive standards for molecular diagnostic testing for SARS-CoV-2 plays a critical role in development and assessment of diagnostic assays associated with the shortage of positive specimens and viral culture fluids. This study aims to establish a novel assay for in-vitro RNA fragment synthesis based on amplicons of self-priming PCR targeting envelope protein gene and RNA-dependent RNA polymerase (RdRp) gene of SARSCoV-2. The cDNA library of the targeted genes of SARS-CoV-2 was generated by using long-primers named sMn1 forward/reverse primer (E gene) and sMn2 forward/reverse primer (RdRp gene) for self-priming PCR assays. The synthesised amplicons that overlap the target sequence of the World Health Organization (WHO) assay were cloned into a pGEM-T easy vector, then transformed into E. coli competent cells by conventional methods. The recombinant plasmids were used as materials for in-vitro RNA transcription. Concentrations of the in-vitro transcribed RNA were 200-800 ng/µl with A260/A280 ratios of 2.0-2.2. Gel electrophoresis showed a single band of each RNA molecule with sizes of 216 and 214 bases for sMn1- E and sMn2- RdRp gene, respectively. Furthermore, we effectively evaluated the in-vitro transcribed RNA by a one-step, real-time RT-PCR assay according to the standard WHO protocol. The stability of in-vitro RNA over a 6-month storage period was then investigated. In conclusion, our assay for in-vitro synthesis of RNA fragments transcribed from self-priming amplicons were successfully established and thus these positive standards were useful for molecular diagnosing of SARS-CoV-2.
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| first_indexed | 2024-04-10T18:50:42Z |
| format | Article |
| id | doaj.art-b64325a7553e4599b3efb655a634bdbd |
| institution | Directory Open Access Journal |
| issn | 2525-2461 2615-9937 |
| language | English |
| last_indexed | 2024-04-10T18:50:42Z |
| publishDate | 2022-12-01 |
| publisher | Vietnam Ministry of Science and Technology |
| record_format | Article |
| series | Vietnam Journal of Science, Technology and Engineering |
| spelling | doaj.art-b64325a7553e4599b3efb655a634bdbd2023-02-01T08:10:37ZengVietnam Ministry of Science and TechnologyVietnam Journal of Science, Technology and Engineering2525-24612615-99372022-12-0164410.31276/VJSTE.64(4).60-63In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis testsHang Thi Thu DinhMai Ngoc NguyenSu Xuan Hoang Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has rapidly spread through the entire world and has become the worst pandemic from December 2019 until now. The establishment of positive standards for molecular diagnostic testing for SARS-CoV-2 plays a critical role in development and assessment of diagnostic assays associated with the shortage of positive specimens and viral culture fluids. This study aims to establish a novel assay for in-vitro RNA fragment synthesis based on amplicons of self-priming PCR targeting envelope protein gene and RNA-dependent RNA polymerase (RdRp) gene of SARSCoV-2. The cDNA library of the targeted genes of SARS-CoV-2 was generated by using long-primers named sMn1 forward/reverse primer (E gene) and sMn2 forward/reverse primer (RdRp gene) for self-priming PCR assays. The synthesised amplicons that overlap the target sequence of the World Health Organization (WHO) assay were cloned into a pGEM-T easy vector, then transformed into E. coli competent cells by conventional methods. The recombinant plasmids were used as materials for in-vitro RNA transcription. Concentrations of the in-vitro transcribed RNA were 200-800 ng/µl with A260/A280 ratios of 2.0-2.2. Gel electrophoresis showed a single band of each RNA molecule with sizes of 216 and 214 bases for sMn1- E and sMn2- RdRp gene, respectively. Furthermore, we effectively evaluated the in-vitro transcribed RNA by a one-step, real-time RT-PCR assay according to the standard WHO protocol. The stability of in-vitro RNA over a 6-month storage period was then investigated. In conclusion, our assay for in-vitro synthesis of RNA fragments transcribed from self-priming amplicons were successfully established and thus these positive standards were useful for molecular diagnosing of SARS-CoV-2. https://vietnamscience.vjst.vn/index.php/vjste/article/view/1085in-vitro transcribed RNAone-step real-time RT-PCRSARS-CoV-2self-priming |
| spellingShingle | Hang Thi Thu Dinh Mai Ngoc Nguyen Su Xuan Hoang In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis tests Vietnam Journal of Science, Technology and Engineering in-vitro transcribed RNA one-step real-time RT-PCR SARS-CoV-2 self-priming |
| title | In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis tests |
| title_full | In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis tests |
| title_fullStr | In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis tests |
| title_full_unstemmed | In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis tests |
| title_short | In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis tests |
| title_sort | in vitro synthesis of rna fragments specifying envelope protein gene and rdrp gene of sars cov 2 as positive standards for molecular diagnosis tests |
| topic | in-vitro transcribed RNA one-step real-time RT-PCR SARS-CoV-2 self-priming |
| url | https://vietnamscience.vjst.vn/index.php/vjste/article/view/1085 |
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