基于CRISPR/Cas12a-RT-RAA的猪繁殖与呼吸综合征病毒快速检测方法的建立

doi:10.16431/j.cnki.1671-7236.2024.08.002

摘要/Abstract

摘要： 【目的】本研究将CRISPR/Cas12a系统与反转录重组酶介导等温核酸扩增技术(RT-RAA)结合,拟建立一种基于CRISPR/Cas12a-RT-RAA的猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)快速检测方法。【方法】分析不同基因型PRRSV全基因组序列,在3'-UTR端保守区设计重组酶介导等温核酸扩增技术(RAA)引物、crRNA及DNA报告底物分子,筛选RT-RAA最佳引物对。表达纯化AsCas12a蛋白,并以质粒为标准品,经RT-RAA扩增后,将产物加入CRISPR/Cas12a系统,建立PRRSV的CRISPR/Cas12a-RT-RAA检测方法,分别以PRRSV、猪圆环病毒2型(PCV2)、猪流行性腹泻病毒(PEDV)、猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)核酸为模板验证建立方法的特异性,以不同浓度的pMD18T-PRRSV为模板验证建立方法的敏感性。以5.62×10⁶、5.62×10³、5.62拷贝数/μL的质粒为模板进行重复性试验,最后进行临床效果评价,将所建立的方法与实时荧光定量逆转录PCR方法进行比较。【结果】本研究成功表达并纯化获得具有良好剪切活性的AsCas12a蛋白,蛋白分子质量大小为196 ku;所建立的CRISPR/Cas12a-RT-RAA方法的检测下限可达5.62 拷贝/μL;该方法具有良好的特异性,可特异性检测出PRRSV,不能检出其他猪病病毒PCV2、PEDV、CSFV和PRV,并具有良好的重复性。应用该方法对121份猪组织样品进行检测,与实时荧光定量逆转录PCR的阳性符合率为93.75%,阴性符合率为99.05%,总符合率为99.17%。【结论】本研究成功建立基于CRISPR/Cas12a-RT-RAA的PRRSV快速检测方法,该方法特异性强、灵敏性高、重复性好且不依赖复杂设备,可在现场和基层实验室使用。

关键词: 猪繁殖与呼吸综合征病毒(PRRSV); 反转录重组酶介导等温核酸扩增技术(RT-RAA); CRISPR/Cas12系统

Abstract: 【Objective】 This study was aimed to establish a rapid detection method for Porcine reproductive and respiratory syndrome virus (PRRSV) based on the combination of CRISPR/Cas12a system and reverse transcription recombinase aided amplification (RT-RAA) technology.【Method】 The whole genome sequences of different genotypes of PRRSV were analyzed,and recombinase aided amplification (RAA) primers,crRNA and DNA reporter substrate molecules were designed in the conserved region of the 3'-UTR terminal,and the best primer pairs of RT-RAA were screened.AsCas12a protein was expressed and purified,and the plasmid was used as the standard product.After amplification by RT-RAA,the product was added to CRISPR/Cas12a system to establish the CRISPR/ Cas12A-RT-RAA detection method for PRRSV.Nucleic acids of PRRSV,Porcine circovirus type 2 (PCV2),Porcine epidemic diarrhea virus (PEDV),Classical swine fever virus (CSFV) and Porcine pseudorabies virus (PRV) were used as templates to verify the specificity of the established method,and different concentrations of pMD18T-PRRSV were used as templates to verify the sensitivity of the established method.The plasmids of 5.62×10⁶,5.62×10³ and 5.62 copies/μL were used as templates for repeat tests,and finally the clinical effect was evaluated,and the established method was compared with reverse transcription Real-time quantitative PCR.【Result】 This study successfully expressed and purified AsCas12a protein with good shear activity,with a molecular weight of 196 ku.The detection lower limit of the CRISPR/Cas12a RT-RAA method established could reach 5.62 copies/μL.This method had good specificity and could specifically detect PRRSV,but could not detect other porcine disease viruses such as PCV2,PEDV,CSFV and PRV.And it had good repeatability.This method was applied to detect 121 pig tissue samples,the positive conformity rate with reverse transcription Real-time quantitative PCR was 93.75%,and the negative conformity rate was 99.05%,with a total conformity rate of 99.17%.【Conclusion】 This study successfully established a rapid detection method for PRRSV based on CRISPR/Cas12a-RT-RAA.The method had strong specificity,high sensitivity,good repeatability,and did not rely on complex equipment.It could be used in both on-site and grassroots laboratories.

Key words: Porcine reproductive and respiratory syndrome virus (PRRSV); reverse-transcription recombinase aided amplification (RT-RAA); CRISPR/Cas12 system

中图分类号:

S852.65

于晶雪, 韦珊珊, 覃绍敏, 吴健敏, 杨丽华, 陈凤莲, 许力士, 秦树英, 华俊, 韦珏, 方芳, 刘金凤. 基于CRISPR/Cas12a-RT-RAA的猪繁殖与呼吸综合征病毒快速检测方法的建立[J]. 中国畜牧兽医, 2024, 51(8): 3237-3246.

YU Jingxue, WEI Shanshan, QIN Shaomin, WU Jianmin, YANG Lihua, CHEN Fenglian, XU Lishi, QIN Shuying, HUA Jun, WEI Jue, FANG Fang, LIU Jinfeng. Establishment of a Rapid Detection Method for Porcine Reproductive and Respiratory Syndrome Virus Based on CRISPR/Cas12a-RT-RAA[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(8): 3237-3246.

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