重组酶聚合酶扩增结合CRISPR-Cas12a快速检测十足目虹彩病毒1方法的建立

Rapid detection of decapod iridescent virus 1 by recombinase polymerase amplification combined with CRISPR-Cas12a

摘要:

[背景] 十足目虹彩病毒1（Decapod Iridescent Virus 1，DIV1）可感染南美白对虾、中国对虾和日本对虾等，是危害对虾养殖业的主要病毒之一。当前高效、快速、简便地检测对虾是否感染DIV1是减少其发生和危害的重要途径。[目的] 将成簇的规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats，CRISPR）及其相关蛋白12a （CRISPR Associated Protein 12a，Cas12a），即CRISPR-Cas12a系统，与重组酶聚合酶扩增（Recombinase Polymerase Amplification，RPA）技术相结合，建立快速检测DIV1的方法（RPA-Cas12a），并探讨该方法在实际样本检验中的应用价值。[方法] 通过提取DIV1 DNA，设计其RPA引物、crRNA及报告探针，优化并建立RPA结合CRISPR-Cas12a的快速检测方法，进一步分析该方法检测DIV1的灵敏度与特异性，并比较建立的方法与qPCR法的一致性。[结果] 建立的RPA-Cas12a快速检测方法可在40 min内实现对虾样本DNA中DIV1的检测，检测限为10 copies/reaction。用该方法分别对对虾白斑综合征病毒（White Spot Syndrome Virus，WSSV）、传染性皮下及造血组织坏死病毒（Infectious Hypodermal and Hematopoietic Necrosis Virus，IHHNV）、虾肝肠胞虫（Enterocytozoon Hepatopenaei，EHP）及DIV1进行检测，结果仅DIV1发生特异性反应，而WSSV、IHHNV和EHP的检测结果均为阴性。采用RPA-Cas12a检测61份实际样本，结果均与qPCR检测法的阳性检出率一致。[结论] 建立的RPA-Cas12a方法检测十足目虹彩病毒1具有快速、简便、灵敏度高且特异性强的特点，为十足目虹彩病毒1的快速检测提供了新的工具。

Abstract:

[Background] Decapod iridescent virus 1 (DIV1), which can infect Penaeus vannamei, Penaeus chinensis, Penaeus japonicus, etc., is one of the main viruses harmful to the prawn aquaculture. Currently, the efficient, rapid, and simple detection of whether the prawn is infected with DIV1 is an effective way to reduce the occurrence and damage of the virus. [Objective] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas12a), termed CRISPR-Cas12a system, was combined with recombinase polymerase amplification (RPA) to establish a method RPA-Cas12a for the rapid detection of DIV1, and then the application value of this method in detecting actual samples was assessed. [Methods] We extracted the DNA of DIV1, and designed the RPA primers of DIV1, crRNA, and reporting probes to establish the rapid detection method. Subsequently, we analyzed the sensitivity and specificity of this method in detecting DIV1 and compared the consistency of the established method with qPCR. [Results] RPA-Cas12a can detect the DIV1 DNA samples within 40 min and had the sensitivity of 10 copies/reaction. This method only detected DIV1 while showed negative results for white spot syndrome virus, infectious hypodermal and hematopoietic necrosis virus, and Enterocytozoon hepatopenaei. Moreover, the RPA-Cas12a method revealed consistent positive rate with qPCR in detecting 61 actual samples. [Conclusion] The RPA-Cas12a method established in this study is rapid, simple, sensitive, and specific, and can serve as a new tool for the rapid detection of DIV1.