不同的核酸提取方法在布鲁氏菌检测中的应用评价

Application of different nucleic acid rapid extraction method in the detection of Brucella abortus evaluated

目的比较不同核酸提取方法对布鲁氏菌检出率的影响,为布鲁氏菌核酸快速提取检测提供技术参考。方法分别使用磁珠法、直接裂解法和高温裂解法对感染布鲁氏菌患者的20例全血样本、10例关节腔及浆膜腔积液样本进行核酸提取,然后进行实时荧光RT-PCR检测,采用SPSS 22.0对结果进行统计分析,评价该核酸快速提取试剂的DNA提取效率。结果磁珠法阳性检出率为100%,Ct值中位数为29.17(21.19~31.33),批内变异系数(CV)最高为1.33%。直接裂解法阳性检出率为63.33%,Ct值中位数为33.29(28.69~34.54),批内CV最高为1.28%。高温裂解法阳性检出率为100%,Ct值中位数为30.26(26.63~33.85),批内CV最高为1.21%。高温裂解法和磁珠法检出率比较差异无统计学意义(P>0.05),直接裂解法检出率显著低于磁珠法和高温裂解法(P<0.05)。结论核酸快速提取试剂在高温裂解条件下可保证核酸提取效率和重复性稳定,具有操作简单、检测时间短、降低实验室生物安全风险等优势,可以应用于布鲁氏菌的核酸检测。

Objective To compare the effects of different nucleic acid extraction methods on the detection rate of Brucella,and to provide technical reference for the rapid extraction and detection of Brucella abortus nucleic acid.Methods Magnetic bead method,direct cracking method and high temperature cracking method were used to extract nucleic acid from 20 whole blood samples and 10 joint cavity and serosal cavity effusion samples of Brucella infected patients in our hospital,and then real-time fluorescence RT-PCR was performed to evaluate the DNA extraction efficiency of this rapid nucleic acid extraction reagent.Results The positive detection rate of magnetic bead method was 100%,the median Ct value was 29.17(21.19~31.33),and the maximum intra-batch coefficient of variation was 1.33%.The positive detection rate of direct cracking method was 63.33%,the median Ct value was 33.29(28.69~34.54),and the maximum coefficient of variation was 1.28%.The positive rate of high temperature cracking method was 100%,the median Ct value was 30.26(26.63~33.85),and the coefficient of variation in batch was the highest 1.21%.There was no significant difference in the detection rate between high temperature cracking method and magnetic bead method(P>0.05),but the detection rate of direct cracking method was significantly lower than that of magnetic bead method and high temperature cracking method(P<0.05).Conclusion The nucleic acid rapid extraction reagent can ensure the efficiency and repeatability of nucleic acid extraction under the condition of high temperature cracking,and has the advantages of simple operation,shorten detection time,and reduce laboratory biosafety,so it can be applied to Brucella nucleic acid detection.