Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA
文献类型：期刊论文
| 作者 | Feng, Wei; Peng, Hanyong; Xu, Jingyang; Liu, Yanming; Pabbaraju, Kanti; Tipples, Graham; Joyce, Michael A.; Saffran, Holly A.; Tyrrell, D. Lorne; Babiuk, Shawn |
| 刊名 | ANALYTICAL CHEMISTRY
 |
| 出版日期 | 2021-09-21 |
| 卷号 | 93期号:37页码:12808-12816 |
| ISSN号 | 0003-2700 |
| 英文摘要 | CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 degrees C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules. |
| WOS研究方向 | Chemistry, Analytical |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/45759]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 作者单位 | 1.Univ Alberta, Fac Med & Dent, Dept Lab Med & Pathol, Div Analyt & Environm Toxicol, Edmonton, AB T6G 2G3, Canada 2.Chinese Acad Sci, Res Ctr Ecoenvironm Sci, State Key Lab Environm Chem & Ecotoxicol, Beijing 100085, Peoples R China 3.Alberta Precis Labs, Prov Lab Publ Hlth ProvLab, Calgary, AB T2N 4W4, Canada 4.Univ Alberta Hosp, Prov Lab Publ Hlth, Alberta Precis Labs, Edmonton, AB T6G 2J2, Canada 5.Univ Alberta, Fac Med & Dent, Li Ka Shing Inst Virol, Dept Med Microbiol & Immunol, Edmonton, AB T6G 2E1, Canada 6.Canadian Food Inspect Agcy, Natl Ctr Foreign Anim Dis, Winnipeg, MB R3E 3M4, Canada |
| 推荐引用方式 GB/T 7714 | Feng, Wei,Peng, Hanyong,Xu, Jingyang,et al. Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA[J]. ANALYTICAL CHEMISTRY,2021,93(37):12808-12816. |
| APA | Feng, Wei.,Peng, Hanyong.,Xu, Jingyang.,Liu, Yanming.,Pabbaraju, Kanti.,...&Le, X. Chris.(2021).Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA.ANALYTICAL CHEMISTRY,93(37),12808-12816. |
| MLA | Feng, Wei,et al."Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA".ANALYTICAL CHEMISTRY 93.37(2021):12808-12816. |
入库方式： OAI收割
来源：生态环境研究中心
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