巨细胞病毒pp65抗原检测的新方法

目的传统的检测CMVpp65抗原的方法是间接免疫荧光,操作麻烦,步骤较多,整个实验需要6个多小时,而且只能定性。需要寻找一种简便的可以定量的方法来检测CMVpp65抗原阳性细胞。因此我们设计了流式的方法来检测CMVpp65抗原阳性细胞。方法我们用间接免疫荧光试剂盒中的一抗和购自invitrogen公司的二抗进行了流式标记移植病人外周血中的WBC,操作步骤明显简便了,只需不到2 h就可完成检测,而且可以定量检测CMVpp65抗原阳性细胞。同时我们用荧光定量PCR检测病人尿中的CMV基因的拷贝数。结果流式检测阳性率高的标本,用间接免疫荧光方法检测也为阳性,同时流式检测强阳性的病人尿用荧光定量PCR检测到CMV基因,表明病人处于CMV病毒血症。结论我们用间接免疫荧光试剂盒中的一抗和购自invitrogen公司的二抗进行流式标记,成功检测了移植病人的CMVpp65抗原阳性细胞的百分比,新方法可以定量,与荧光定量PCR方法检测病人尿中的CMV基因的结果符合率较高,可以推广应用。

New methods to detect cytomegalovirus pp65 antigen

The traditional method to detect CMVpp65 antigen positive cells is indirect immunofluorescence,which is only qualitative and the operation is troublesome.To find a simple quantitative method to detect CMVpp65 antigen positive cells,we used the primary antibodies from a kit and secondary antibodies from Invitrogen Company for flow cytometry.The steps were simplified,and antigen-positive cells can also be quantitative.At the same time CMV gene copy number in the urine of patients were detected by fluorescent quantitative PCR.The samples with high positive rate detected by flow cytometry were positive when detected by indirect immunofluorescence.And the patients with strong positive results by flow cytometry and their urine contained CMV genes by fluorescent quantitative PCR,indicating that the patients were in the CMV viremia.The percentage of CMVpp65 antigen positive cells of the transplant patients were successful detected.The new method is simplified,quantitative,and showed higher consistent with fluorescent quantitative PCR results.