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一种由一氧化辅助的数字CRISPR/Cas12a试验,用于在样本中选择性检测活细菌

Weihong Yin¹, Kai Hu¹, Yunxing Yang²

¹Research Centre for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, Zhejiang 310027, P. R. China.

Analytical chemistry

|November 1, 2024

概括

一种使用一氧化和CRISPR/Cas12a技术的新测定方法允许快速,灵敏地检测活体大肠杆菌O157:H7 (大肠杆菌O157:H7) 细菌. 这种方法可以区分活细胞和死细胞,改善食品安全和临床诊断.

科学领域:

分子生物学分子生物学
食品安全食品安全
临床诊断临床诊断临床诊断

背景情况:

大肠杆菌O157:H7 (大肠杆菌O157:H7) 是一种重要的食物传播病原体.
准确检测活体大肠杆菌O157:H7对于公共卫生和食品质量至关重要.
现有的方法可能难以区分活细胞和死细胞.

研究的目的:

开发一种快速,灵敏的检测方法来检测活体大肠杆菌O157:H7.7.
选择性地识别可活性的细菌,尽量减少死细胞的干扰.
为了能够在数字微流体芯片上绝对量化非放大核酸.

主要方法:

采用了一种由一氧化辅助的非放大数字CRISPR/Cas12a测定方法.
使用Propidium monoazide将核酸从死菌中排除在外.
测试是在微流体数字芯片上进行的,以实现绝对量化.

主要成果:

该方法实现了对活体大肠杆菌O157:H7的选择性检测,消除了98%的死亡细菌核酸干扰.
检测在120分钟内完成,不需要标准曲线.
获得了1.2 × 10^3 CFU/mL的高灵敏度,结果与盘子计数有很强的相关性 (皮尔森的r = 0.9490).

结论:

一氧化辅助的数字CRISPR/Cas12a检测方法为检测活体大肠杆菌O157:H7提供了灵敏和快速的方法.
这种技术有效地区分活菌和死菌,提高了诊断的准确性.
该测试显示了实时应用在食品安全监测和临床诊断中的强大潜力.

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相关概念视频

CRISPR and crRNAs

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...