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https://pubmed.ncbi.nlm.nih.gov/33593370
Results: The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 …
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Affiliations
1 Department of Urology, International Cancer Center, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen, 518039, China.
2 International Cancer Center, Shenzhen University School of Medicine, Shenzhen, 518060, China.
3 The First Affiliated Hospital of Shantou University, Shantou, 515041, China.
4 Guangdong Key Laboratory of Systems Biology and Synthetic Biology for Urogenital Tumors, Shenzhen, 518035, China.
5 Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai, 200240, China.
6 Yuebei Second People's Hospital, Shaoguan, 512000, Guangdong, China. lidechangsg@sina.com.
7 Department of Urology, International Cancer Center, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen, 518039, China. pony8980@163.com.
8 International Cancer Center, Shenzhen University School of Medicine, Shenzhen, 518060, China. pony8980@163.com.
9 The First Affiliated Hospital of Shantou University, Shantou, 515041, China. pony8980@163.com.
10 Guangdong Key Laboratory of Systems Biology and Synthetic Biology for Urogenital Tumors, Shenzhen, 518035, China. pony8980@163.com.
Yangyang Sun et al. J Transl Med. 2021.
Affiliations
1 Department of Urology, International Cancer Center, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen, 518039, China.
2 International Cancer Center, Shenzhen University School of Medicine, Shenzhen, 518060, China.
3 The First Affiliated Hospital of Shantou University, Shantou, 515041, China.
4 Guangdong Key Laboratory of Systems Biology and Synthetic Biology for Urogenital Tumors, Shenzhen, 518035, China.
5 Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai, 200240, China.
6 Yuebei Second People's Hospital, Shaoguan, 512000, Guangdong, China. lidechangsg@sina.com.
7 Department of Urology, International Cancer Center, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen, 518039, China. pony8980@163.com.
8 International Cancer Center, Shenzhen University School of Medicine, Shenzhen, 518060, China. pony8980@163.com.
9 The First Affiliated Hospital of Shantou University, Shantou, 515041, China. pony8980@163.com.
10 Guangdong Key Laboratory of Systems Biology and Synthetic Biology for Urogenital Tumors, Shenzhen, 518035, China. pony8980@163.com.
Background: COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.
Methods: We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.
Results: The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input.
Conclusions: The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.
Keywords: COVID-19; CRISPR/Cas; RT-RPA; SARS-CoV-2 assay.
The authors declare that they have no competing interests.
An OR-DETECTR platform for detection…
An OR-DETECTR platform for detection of SARS-CoV-2. a Schematic of OR-DETECTR testing SARS-CoV-2…
An OR-DETECTR platform for detection of SARS-CoV-2. a Schematic of OR-DETECTR testing SARS-CoV-2 workflow. RNA extraction from pharyngeal swab can be used as an input to OR-DETECTR (One-tube detection platform combined RT-RPA based preamplification and CRISPR/Cas12a based DETECTR), which is visualized by a fluorescent reader. The RT-RPA mix is located at the bottom of the centrifuge tube, while the DETECTR mix is located in the transparent tube lid. b Genome map showing primers and crRNAs. RT-RPA primers are indicated by black arrows which partially overlap with rRT-PCR primers of RdRp gene (WHO) or N gene (China CDC). Two crRNAs are indicated by light blue curves. c, d crRNA specificity. The crRNA targeting RdRp gene is designed to detect SARS-CoV-2 and SARS-CoV, which cause severe respiratory infection presented as fever, cough and dyspnea. e, f The crRNA targeting N gene was specific for SARS-CoV-2. A positive result requires the detection of the SARS-CoV-2 viral N gene target. Dots represent identical nucleotides compared to the sequence of RPA amplicon, (“–”) means sequence gaps are not covered by oligonucleotides. Black shadows represent primer binding sequences and blue shadows represent crRNA recognition sequences. “+” means positive and “–” means negative. Fold change value was used to normalize the fluorescence signal values. Fold change (FC) = (F (PC)-B (PC)) / (F (NC)-B (NC)), F = fluorescence signal value of 19 min, B = fluorescence signal value of 0 min, PC = positive control, NC = negative control. The result was shown as mean ± SD, N = 3. ***P < 0.01
OR-DETECTR assay for SARS-CoV-2 in…
OR-DETECTR assay for SARS-CoV-2 in standard RNA and clinical oropharyngeal swab samples. a…
OR-DETECTR assay for SARS-CoV-2 in standard RNA and clinical oropharyngeal swab samples. a Fold change data (OR-DETECTR) on RdRp gene and CT values (rRT-PCR) on Orf1ab of patient samples. There were 14 RNA samples from 14 suspected COVID-19 patients. N50S was one RNA mixed sample from 40 fever patients. The CT value of each RNA sample was larger than 40 and undetected. The blue pentagrams (star) represent the CT values of the samples on Orf1ab gene, and the blue pentagrams exceeding the coordinate axis represent the CT values of the samples on Orf1ab gene greater than 40. The black columns represent the FC values of samples on RdRp gene, and the cutoff was 1.6. b Fold change data (OR-DETECTR) and CT values (rRT-PCR) on the N gene of suspected COVID-19 patient samples. The samples were the same as a. The red pentagrams (star) represent the CT values of the samples on the N gene, and the red pentagrams exceeding the coordinate axis represent the CT values of the samples on the N gene greater than 40. The black columns represent the FC values samples on the N gene, and the cutoff of the N gene was 2.6. c, d LoD for OR-DETECTR assay. SARS-CoV-2 RdRp and N gene standard RNA mix were used as the samples for evaluating LoD of OR-DETECTR assay. Standard RNA mix is serially diluted, and there were 7 replicates per dilution. e Visual OR-DETECTR assay by test card with lateral flow strip. Standard RNA mix is serially diluted, and there were 2 replicates per dilution
Brandsma E, Verhagen HJMP, van de Laar TJW, Claas ECJ, Cornelissen M, van den Akker E. Brandsma E, et al. J Infect Dis. 2021 Feb 3;223(2):206-213. doi: 10.1093/infdis/jiaa641. J Infect Dis. 2021. PMID: 33535237
Nawattanapaiboon K , Pasomsub E , Prombun P , Wongbunmak A , Jenjitwanich A , Mahasupachai P , Vetcho P , Chayrach C , Manatjaroenlap N , Samphaongern C , Watthanachockchai T , Leedorkmai P , Manopwisedjaroen S , Akkarawongsapat R , Thitithanyanont A , Phanchana M , Panbangred W , Chauvatcharin S , Srikhirin T . Nawattanapaiboon K , et al. Analyst. 2021 Jan 21;146(2):471-477. doi: 10.1039/d0an01775b. Epub 2020 Nov 9. Analyst. 2021. PMID: 33165486
Zhang WS, Pan J, Li F, Zhu M, Xu M, Zhu H, Yu Y, Su G. Zhang WS, et al. Anal Chem. 2021 Mar 2;93(8):4126-4133. doi: 10.1021/acs.analchem.1c00013. Epub 2021 Feb 11. Anal Chem. 2021. PMID: 33570401 Free PMC article.
Rahman MR, Hossain MA, Mozibullah M, Mujib FA, Afrose A, Shahed-Al-Mahmud M, Apu MAI. Rahman MR, et al. Biomed Pharmacother. 2021 Aug;140:111772. doi: 10.1016/j.biopha.2021.111772. Epub 2021 May 27. Biomed Pharmacother. 2021. PMID: 34062417 Free PMC article. Review.
Palaz F, Kalkan AK, Tozluyurt A, Ozsoz M. Palaz F, et al. Clin Biochem. 2021 Mar;89:1-13. doi: 10.1016/j.clinbiochem.2020.12.011. Epub 2021 Jan 9. Clin Biochem. 2021. PMID: 33428900 Free PMC article. Review.
Zhang H, Pajer P, Kudr J, Zitka O, Neužil P. Zhang H, et al. Biotechniques. 2021 Aug 16:10.2144/btn-2021-0040. doi: 10.2144/btn-2021-0040. Online ahead of print. Biotechniques. 2021. PMID: 34392709 Free PMC article. No abstract available.
Chong ZX, Liew WPP, Ong HK, Yong CY, Shit CS, Ho WY, Ng SYL, Yeap SK. Chong ZX, et al. Pathol Res Pract. 2021 Jul 24;225:153565. doi: 10.1016/j.prp.2021.153565. Online ahead of print. Pathol Res Pract. 2021. PMID: 34333398 Free PMC article. Review.
Zhan Y, Li XP, Yin JY. Zhan Y, et al. Int J Biol Sci. 2021 May 13;17(8):2080-2088. doi: 10.7150/ijbs.60655. eCollection 2021. Int J Biol Sci. 2021. PMID: 34131407 Free PMC article. Review.
Ali J, Elahi SN, Ali A, Waseem H, Abid R, Mohamed MM. Ali J, et al. Nanomaterials (Basel). 2021 May 18;11(5):1328. doi: 10.3390/nano11051328. Nanomaterials (Basel). 2021. PMID: 34069866 Free PMC article.
Kostyusheva A, Brezgin S, Babin Y, Vasilyeva I, Glebe D, Kostyushev D, Chulanov V. Kostyusheva A, et al. Methods. 2021 Apr 9:S1046-2023(21)00099-2. doi: 10.1016/j.ymeth.2021.04.007. Online ahead of print. Methods. 2021. PMID: 33839288 Free PMC article.
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