Detecting and typing target DNA with a novel CRISPR-typing PCR (ctPCR) technique
Detecting and typing target DNA with a novel CRISPR-typing PCR (ctPCR) technique
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使用新型 CRISPR 分型 PCR (ctPCR) 技术检测目标 DNA 并进行分型
DOI：
10.1016/j.ab.2018.09.012
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发表时间：
2018
影响因子：
2.9
通讯作者：
Wang Jinke
中科院分区：
文献类型：
--
作者：
Zhang Beibei;Xia Qiang;Wang Qiao;Xia Xinyi;Wang Jinke
本研究开发了一种基于CRISPR的DNA检测和分型新方法，命名为ctPCR 3.0，代表CRISPR-或Cas9/sgRNA分型PCR，版本3.0。该技术仅在一个均相步骤中检测靶DNA：定量PCR（qPCR）扩增Cas9/sgRNA切割的DNA样品。通过将Cas9和sgRNA直接添加到qPCR反应中，并在qPCR程序之前进行额外的等温孵育，可以在短短2小时内均匀检测靶DNA。在整个检测过程中不需要打开检测管，ctPCR 3.0可以像传统的qPCR检测一样检测目标DNA。通过对10种高危型人乳头瘤病毒（HPV）L1基因的克隆检测，验证了该技术的有效性。该技术还成功地检测了两种高危型HPV，HPV 16和HPV 18的L1和E6-E7基因，在两种HPV阳性宫颈癌细胞HeLa和SiHa的基因组DNA中。最后，ctPCR 3.0方法通过成功地检测许多临床样本中的HPV进行了验证。通过进行这些检测，本研究因此提供了一种新的基于CRISPR的DNA检测和分型平台以及一种即用型HPV临床检测技术。该平台在临床诊断中具有广泛的应用前景。
This study develops a new method for detecting and typing the interested DNAs based on CRISPR, which was named as ctPCR3.0, representing CRISPR- or Cas9/sgRNA-typing PCR, version 3.0. This technique detects target DNA in just one homogeneous step: quantitative PCR (qPCR) amplifying the Cas9/sgRNA-cleaved DNA samples. By directly adding Cas9 and sgRNA into the qPCR reaction and giving an additional isothermal incubation before qPCR program, the target DNA can be homogeneously detected in as few as 2 h. Without opening the detecting tube in the whole detection process, ctPCR3.0 can be used to detect target DNA as the traditional qPCR detection. The technique was fully verified by detecting the cloned HPV L1 genes of 10 high-risk human papillomavirus (HPV) subtypes. The technique also successfully detected the L1 and E6-E7 genes of two highest-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells, HeLa and SiHa. Finally, the ctPCR3.0 method was validated by successfully detecting HPVs in many clinical samples. By performing these detections, this study thus provides a new CRISPR-based DNA detection and typing platform and a ready-to-use HPV clinical detection technique. The platform has wide application in clinical diagnosis.