微小隐孢子虫RPA-CRISPR/Cas12a可视化检测方法的建立及应用

doi:10.16431/j.cnki.1671-7236.2023.12.002

摘要/Abstract

摘要： 【目的】建立一种快速、准确、灵敏的微小隐孢子虫可视化检测方法。【方法】本研究将重组酶聚合酶扩增(recombinase polymerase amplification,RPA)技术与CRISPR/Cas12a系统相结合,通过荧光检测法和侧流层析试纸条读取结果。针对微小隐孢子虫的小亚基核糖体RNA(small subunit ribosome RNA,SSU rRNA)基因设计和筛选crRNA和RPA引物,利用两种不同的单链DNA报告分子以荧光数值和侧流层析试纸条呈现检测结果。通过检测微小隐孢子虫和7个其他病原体评估RPA-CRISPR/Cas12a检测方法的特异性。通过梯度稀释的重组质粒和微小隐孢子虫基因组评估RPA-CRISPR/Cas12a检测方法的敏感性,并将该方法应用于临床样本的检测。【结果】本研究建立的RPA-CRISPR/Cas12a检测方法可在90 min内完成对样本中微小隐孢子虫的检测,与大肠杆菌、十二指肠贾第虫、华支睾吸虫、弓形虫、肝片吸虫、人五毛滴虫、沙门菌均无交叉反应,可特异性检测出微小隐孢子虫;敏感性试验结果显示,该方法的最低检测限为10个卵囊/mL。利用此方法对70份牛粪便样本和50份人粪便样本进行检测,经荧光检测法和侧流层析试纸条读取结果,微小隐孢子虫的阳性率分别为15.7%(11/70)和6.0%(3/50),与套式PCR检测结果完全一致。【结论】本研究建立的微小隐孢子虫RPA-CRISPR/Cas12a可视化检测方法具有高特异性和高灵敏度,且检测结果直观,不依赖昂贵的仪器设备,在微小隐孢子虫的现场检测和风险监测方面具有广阔的应用价值。

关键词: CRISPR/Cas12a; 微小隐孢子虫; 重组酶聚合酶扩增; 可视化检测

Abstract: 【Objective】 This study was aimed to developed a rapid,accurate,and sensitive visualization detection method for Cryptosporidium parvum (C. parvum). 【Method】 In this study,the recombinase polymerase amplification (RPA) was combined with the CRISPR/Cas12a system,and the results were read by the fluorescence reporter system and the lateral flow strip biosensor. The crRNA and RPA primers were designed and screened against the small subunit ribosome RNA (SSU rRNA) gene of C. parvum,and two different single-stranded DNA (ssDNA) reporters were used to present the detection results by fluorescence values and the LFS biosensor.The specificity of the RPA-CRISPR/Cas12a assay was verified by detecting C. parvum and 7 other pathogens.The sensitivity of the RPA-CRISPR/Cas12a detection method was evaluated by serially diluted recombinant plasmids and C. parvum genome,and the method was applied to clinical sample detection.【Result】 The RPA-CRISPR/Cas12a assay established in this study could detect C. parvum in samples within 90 min,and had no cross-reactivity with Escherichia coli,Giardia duodenalis,Clonorchis sinensis,Toxoplasma gondii,Fasciola hepatica,Pentatrimonas hominis,and Salmonella,the method could specifically detect C. parvum.The sensitivity test results showed that the minimum detection limit was 10 oocysts/mL.Using this method to detect 70 cattle feces samples and 50 human feces samples,the positive rate of C. parvum was 15.7% (11/70) and 6.0% (3/50) after reading the results by the fluorescent reporter system and lateral flow strips biosensor,which was completely consistent with the results of the nested PCR.【Conclusion】 The RPA-CRISPR/Cas12a assay for C. parvum had high specificity and sensitivity with intuitive results,without relying on expensive instrumentation,and had broad application value as a new strategy for more rapid and portable identification of C. parvum on-site and risk monitoring.

Key words: CRISPR/Cas12a; Cryptosporidium parvum; recombinase polymerase amplification; visual diagnostic method

中图分类号:

S852.72

王丽, 李珊, 李璐, 张西臣, 王晓岑, 李新, 张楠, 宫鹏涛. 微小隐孢子虫RPA-CRISPR/Cas12a可视化检测方法的建立及应用[J]. 中国畜牧兽医, 2023, 50(12): 4793-4804.

WANG Li, LI Shan, LI Lu, ZHANG Xichen, WANG Xiaocen, LI Xin, ZHANG Nan, GONG Pengtao. Establishment and Application of the RPA-CRISPR/Cas12a Visual Diagnostic Method for Cryptosporidium parvum[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(12): 4793-4804.

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