人冠状病毒NL63超速离心富集方法的建立及优化

Establishment and optimization of an ultracentrifugation enrichment method for human coronavirus NL63

目的建立并优化人冠状病毒NL63参考株(HCoV-NL63-NC_005831)的体外富集方法。方法以恒河猴肾细胞系(LLC-MK2)培养HCoV-NL63,以离心力135000×g、分别离心病毒培养上清4 h、6 h、8 h、10 h和12 h以富集病毒,并以商品化PEG沉淀试剂盒富集方法作为对照。采用实时荧光定量PCR(qRT-PCR)、半数组织培养感染剂量(TCID50)和空斑实验对原始病毒液、富集病毒液分别进行病毒核酸和病毒生物活性定量检测,评价病毒富集效果;通过透射电镜负染观测病毒富集前后的形态。结果经层超速离心法及经PEG沉淀法分别进行体积100∶1富集处理,两种方法获得的富集病毒液相较于原液的病毒核酸浓度及活病毒浓度均有提高(P<0.001)。超速离心方法富集前后,电镜下观察到的病毒颗粒呈正常的负染形态;4 h、6 h、8 h、10 h和12 h超速离心后活病毒浓度平均分别是原病毒液的(7.35±1.62)倍、(13.98±1.71)倍、(36.36±10.41)倍、(48.16±9.38)倍、(54.26±7.02)倍,PEG沉淀后活病毒浓度是原病毒液的(3.39±0.16)倍,经超速离心法获得的富集病毒液的核酸浓度及活病毒浓度显著高于PEG沉淀法(P<0.05);8 h、10 h、12 h超速离心后病毒浓度显著高于4 h和6 h(P<0.05),但8 h、10 h、12 h之间病毒含量差异无统计学意义(P>0.05)。结论超速离心法相较于商品化的PEG沉淀法能更有效富集HCoV-NL63病毒;离心力为135000×g时,选择8 h的超离时间可获得较优的体外富集效果。

Objective To establish and optimize the in vitro enrichment method for human coronavirus NL63 reference strain(HCoV-NL63-NC_005831).Methods HCoV-NL63 was cultured in rhesus monkey kidney cell line(LLC-MK2).Then at 135000×g,the virus culture supernatant was centrifuged for 4 h,6 h,8 h,10 h and 12 h to enrich the virus,and compared with the enrichment method of commercial PEG Precipitation Kit.Real time reverse transcription polymerase chain reaction(qRT-PCR),TCID50 assay and plaque assay were used to quantitatively detect the viral nucleic acid and viral biological activity of the original virus solution and the enriched virus solution respectively.The virus morphology before and after enrichment was observed after negative staining under a transmission electron microscope.Results After 100∶1 volume enrichment by ultracentrifugation and PEG precipitation,both of the concentration of virus nucleic acid and live virus were higher than that of the original virus solution(P<0.001),and before and after the enrichment of the two method,the normal negative staining morphology of virus particles can be observed under the electron microscope.After 4 h,6 h,8 h,10 h and 12 h ultracentrifugation,the concentration of live virus was(7.35±1.62)times,(13.98±1.71)times,(36.36±10.41)times,(48.16±9.38)times,(48.16±9.38)times and(54.26±7.02)times that of the original virus solution,respectively;the concentration of live virus after PEG precipitation was(3.39±0.16)times that of the original virus solution,and the nucleic acid concentration and live virus concentration of concentrated virus solution obtained by ultracentrifugation were significantly higher than those obtained by PEG precipitation(P<0.05).After 8 h,10 h or 12 h ultracentrifugation,the virus concentration was significantly higher than that of 4 h or 6 h(P<0.05).However,there was no significant difference in virus content between 8 h,10 h and 12 h(P>0.05).Conclusions Compared with commercial PEG precipitation method,ultracentrifugation method can enrich HCoV-NL63 virus more effectively;when the relative centrifugal force is 135000×g,the better enrichment effect in vitro can be obtained by selecting the super separation time of 8 h.