摘要
为建立一种富集水中鲤春病毒血症病毒(SVCV)的技术,本研究利用SVCV多克隆抗体制备免疫磁珠,通过对磁珠耦联抗体量的优化,确定该免疫磁珠有效富集SVCV的最低浓度,建立一种富集SVCV的免疫磁珠分离技术,并联合荧光定量RT-PCR(RT-qPCR)技术对水中SVCV进行检测.结果显示,1 mg磁珠与75 μg SVCV多克隆抗体耦联时,耦联的抗体量最大,耦联率为98.48％;耦联后的免疫磁珠对SVCV(200 μL,浓度为1.54×106拷贝/μL)的富集效率最高为91.56％.以该多克隆抗体量制备的免疫磁珠富集10倍倍比稀释的SVCV(3.54×106拷贝/μL～3.54×103拷贝/μL),以确定其有效富集SVCV的最低浓度.结果显示,1 mg免疫磁珠能够使200 μL病毒液(3.54× 103拷贝/μL)富集率达到61.58％.表明该免疫磁珠能够有效富集SVCV.在免疫磁珠分离技术联合RT-qPCR技术检测水样品中SVCV(浓度约为1.16×104拷贝/μL)的结果显示,免疫磁珠组富集SVCV核酸量是裸磁珠组富集SVCV核酸量的1.77×103倍,是未处理组富集SVCV核酸量的1.68×103倍,免疫磁珠组富集SVCV均极显著高于裸磁珠组和未处理组(P＜0.01),裸磁珠组富集的SVCV与未处理组则无显著差异(P＞0.05).本研究首次建立了一种能够有效富集水中SVCV的方法,为SVCV的监测提供了技术手段.
Abstract
In order to establish a method for enriching spring viremia of carp virus(SVCV)in water,the immunomagnetic beads(IMBs)conjugated with polyclonal antibody against SVCV were prepared.By optimizing the amount of antibodies,the minimum concentration of the immunomagnetic beads for effectively enriching SVCV was determined.After enriched by the IMBs,SVCV was detected by the reverse-transcription quantitative PCR(RT-qPCR).The results showed the optimal amount of the conjugated antibody was 75μg,and the coupling rate was 98.48％.1mg IMBs which conjugated with 75μg polyclonal antibodies has the greatest efficiency(91.56％)to capture SVCV(200 μL,1.54×106 copies/μL).10-fold dilutions of SVCV(3.54×106 copies/μL)were used to determine the minimum concentration that could be enriched by IMBs.The result suggested that 1 mg IMBs enabled 61.58％enrichment of 200 μL virus solution(3.54× 103 copies/μL).The IMBs separation technology combined with RT-qPCR technology was used to detect SVCV in water samples(the concentration is approximately 1.16×104 copies/μL).The results showed that the amount of SVCV nucleic acid enriched by IMBs was significantly(1.77 × 103 times)higher than that in bare beads,and also significantly(1.68 × 103 times)higher than that in untreated virus liquid(P＜0.01).There was no significant difference between the bare magnetic beads treatment group and untreated virus liquid(P＞0.05).This study established a method that can effectively enrich SVCV in water for the first time,providing a technical means for SVCV monitoring.
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