美洲型猪繁殖与呼吸综合征病毒实时荧光RT-PCR定量检测试剂盒测量不确定度研究

Study on the Uncertainty of Real-time PCR Kits for Quantitative Detection of PRRSV of American Type

为填补测量不确定度在兽医领域病原学检测的空白,建立猪繁殖与呼吸综合征病毒(PRRSV)实时荧光定量RT-PCR检测试剂盒测量不确定度评估模型,对市售的3种美洲型PRRSV实时荧光RT-PCR检测试剂盒进行了测量不确定度评估,评估了移液器、温度、重复性测量因素对试验结果产生的不确定度影响。研究数据表明:移液器与重复性测量对试验结果产生的不确定度影响较大,应予以重视,需规范操作;建立的3种试剂盒测量不确定度模型缩小了试剂盒对可疑样品的判定区域,对样品临界参考值的检测结果判定具有实际意义。通过比对4个厂家的核酸提取试剂,筛选出了提取效率最高的试剂盒。本研究建立的PRRSV实时荧光RT-PCR定量检测试剂盒测量不确定度模型为兽医实验室检测结果不确定度评估及应用奠定了基础。

In order to fill in the blank of the measurement uncertainty in the field of veterinary etiology,an evaluation model of measurement uncertainty for real-time PCR kits for quantitative detection of PRRSV was established,and three kinds of commercial RT-PCR kits(detecting PRRSV strain of American type)were evaluated,including the influence of pipette,temperature and repeated measurement on test results. It was found that the test results were greatly influenced by the pipette and repeated measurement,which should be paid attention to,and the operations should be standardized;the determination area of the kits for suspected samples was reduced,which contributed to the determination of the critical reference value of samples. The kit with highest extraction efficiency was selected through comparing the nucleic acid extraction reagents produced by four manufacturers. In conclusion,a foundation was hereby laid for evaluation and application of measurement uncertainty in veterinary laboratories by the model established in this study.