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目的建立肠病毒71型(EV71)空斑检测方法,为EV71生物学特性及疫苗的研究提供手段。方法将不同稀释度的5株EV71分别接种至单层RD细胞和Vero细胞,加入甲基纤维素覆盖,计数空斑,并计算病毒滴度。通过3次连续检测,验证空斑法的精密性,并对空斑法与微量细胞病变法检测病毒滴度的结果进行比较。结果采用RD细胞和Vero细胞,通过空斑法对EV71病毒滴度进行检测,96h后均能规律地出现边缘整齐、清晰的空斑,RD细胞和Vero细胞检测的空斑直径分别为1～2mm和0.5～1mm。连续3次重复试验结果显示,试验间变异系数的平均值Vero细胞为2.52%,RD细胞为4.49%。空斑法与微量细胞病变法比较,其检测结果具有良好的线性相关性,相关系数为r=0.972,P=0.006。结论已建立了精密性好、出斑规律的EV71病毒空斑检测方法。
Objective To establish an EV71 plaque assay to provide a means for the study of the biological characteristics and vaccines of EV71. Methods Five strains of EV71 with different dilutions were inoculated into monolayers of RD cells and Vero cells respectively. The cells were covered with methylcellulose and counted for plaques. The virus titer was calculated. The accuracy of plaque assay was verified by three consecutive tests, and the results of plaque assay and micro-cytopathic assay for virus titer were compared. Results RD cells and Vero cells were used to detect the titer of EV71 virus by plaque method. After 96 hours, the neat and clear plaques with regular edges appeared. The diameters of plaques detected by RD cells and Vero cells were 1 ~ 2mm And 0.5 ~ 1mm. The results of three repeated experiments showed that the average coefficient of variation among trials was 2.52% for Vero cells and 4.49% for RD cells. The results of plaque assay showed that there was a good linear correlation between the results of cytometry and cytometry. The correlation coefficient was r = 0.972, P = 0.006. Conclusion The EV71 virus plaque detection method has been established with good precision and spot pattern.