中国畜牧兽医2023，Vol.50Issue(12)：4793-4804,12.DOI:10.16431/j.cnki.1671-7236.2023.12.002
微小隐孢子虫RPA-CRISPR/Cas12a可视化检测方法的建立及应用
Establishment and Application of the RPA-CRISPR/Cas12a Visual Diagnostic Method for Cryptosporidium parvum
摘要
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.关键词
CRISPR/Cas12a/微小隐孢子虫/重组酶聚合酶扩增/可视化检测Key words
CRISPR/Cas12a/Cryptosporidium parvum/recombinase polymerase amplification/visual diagnostic method分类
畜牧业引用本文复制引用
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):4793-4804,12.基金项目
长春市科技发展计划(21ZY24) （21ZY24）
国家重点研发计划(2021YFF0702900) （2021YFF0702900）
国家自然科学基金(31972704) （31972704）
