快速检测十足目虹彩病毒1 RPA-LFD方法的建立及初步应用

Rapid visual detection of DIV1 by combinase recombinase polymerase amplification with lateral flow dipstick

为建立一种高效、快速、简便的十足目虹彩病毒1(DIV1)检测方法,本研究以DIV1 ATPase基因为检测靶标,设计特异性引物和探针,采用方阵法优化反应温度和时间,结果显示,重组酶聚合酶扩增技术(RPA)最优反应温度为37℃,时间为15 min,结果观察时间为5 min,检测过程总时长为20 min,初步建立了DIV1的重组酶聚合酶扩增技术结合侧向流试纸条方法(RPA-LFD)。提取其他7种常见虾类病原基因组与DIV1基因组,采用该方法检测,分析其特异性;构建重组质粒标准品p UC57-DIV1,10倍倍比稀释后采用该方法检测,分析其灵敏性;以3种不同浓度的重组质粒标准品为模板进行批间、批内重复性试验。结果显示,该方法能特异性检测DIV1,与虾类其他常见易感病原均无交叉反应;其对重组质粒标准品的检测限为1×10^(1)拷贝/μL;批内和批间重复性试验的检测结果均一致,重复性好。利用该方法与已发表的荧光定量PCR(qPCR)同时检测15份感染DIV1的罗氏沼虾样品及40份临床样品,结果显示,该方法的阳性检测率为69.09%(38/55),阴性率为30.91%(17/55),与q PCR检测结果一致,二者的阳性符合率、阴性符合率、总符合率均为100%。综上所述,本研究建立的RPA-LFD方法检测DIV1具有快速、简便、灵敏度高且特异性强的特点,且不需要精密昂贵的仪器设备,为基层实验室及现场检测提供了可行技术手段。

In order to establish an efficient,rapid,and simple method for detecting decapod iridovirus 1(DIV1),the DIV1 ATPase gene was used as the detection target in this study.Specific primers and probes were designed,and the reaction time and temperature were optimized by the square array method.The results showed that the optimal reaction temperature was 37℃,the reaction time was 15 minutes,the result observation time was 5 minutes,and the total duration of the entire detection process was 20 minutes.The recombinase polymerase amplification technology combined with the lateral flow test strip method(RPA-LFD)of DIV1 was preliminarily established.Seven other common shrimp pathogen genes and the DIV1 gene were extracted and detected using this method to analyze their specificity;a recombinant plasmid standard pUC57-DIV1 was constructed and detected after 10-fold dilution to analyze its sensitivity;recombinant plasmid standards prepared at different times were used as templates for inter-batch and intra-batch repeatability tests.The specificity test revealed that the DIV1 could be specifically detected,and had no cross reaction with other common susceptible pathogens of shrimp.The sensitivity test indicated that the detection limit for DIV1 was 1×10^(1) copies/μL.Moreover,the repeatability test showed that the detection results within and between three batches of plasmid standards with three concentrations were consistent,indicating good reproducibility of this method.We applied this method and the published fluorescence quantitative PCR method to simultaneously detect 15 samples of Macrobrachium rosenbergii infected with DIV1 and 40 clinical samples.The result showed that the positive detection rate of this method was 69.09%(38/55),and the negative rate was 30.91%(17/55),which is consistent with the results of fluorescence quantitative PCR detection.The positive,negative,and total coincidence rates of the two methods were 100%.In summary,the recombinase polymerase amplification technology established in this study combined with the lateral flow test strip method for detecting decapod iridovirus 1 is rapid,simple,high sensitive,and highly specific,and does not require precise and expensive equipment,providing a feasible technical method for grassroots laboratories and on-site detection.