荧光定量PCR检测嗜吞噬细胞无形体

目的采用新型TaqMan-MGB探针建立检测嗜吞噬细胞无形体的实时荧光定量PCR(quantitativereal-timePCR)方法。方法依据gltA基因序列设计嗜吞噬细胞无形体特异引物和探针,以克隆的嗜吞噬细胞无形体gltA基因片段作DNA模板,在荧光定量PCR检测仪(ABI7900HT)上建立实时荧光定量检测方法。结果建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.996);与套式PCR相比较,荧光定量PCR检测的灵敏度是其100倍。用荧光定量PCR检测其它相关立克次体和细菌DNA样本,检出结果几乎为0;对荧光定量PCR检测重复性进行分析,变异系数(CV)批内和批间误差在0~2.1%之间,证明该荧光定量PCR具有种特异性和良好的重复性。用荧光定量PCR检测体疑染嗜吞噬细胞无形体的10份蜱和30份小鼠脾脏标本,结果与套式PCR检测结果有密切相关性,但是定量PCR检测敏感性和准确率均高于套式PCR。结论本研究建立的检测嗜吞噬细胞无形体的实时荧光定量PCR具有很高的特异性和敏感性,特别适合检出样本中微量嗜吞噬细胞无形体。

Development of a quantitative real-time polymerase chain reaction assay specific for Anaplasma phagocytophila

According to the gltA gene sequences specific for Anaplasma phagocytophila,a pair of primers and one TaqMan-MGB probe were designed. And a quantitative real-time polymerase chain reaction (RtPCR) was developed with primers, probe, and template (gltA gene DNA fragment of A. phagocytophila). The standard curve was established with thegltA gene DNA in DNA sequence detection system (ABI 7900HT) and the relationship between the values of threshold cycle (C_ t) and the DNA copy number was found to be linear (r=0.996). The sensitivity of RtPCR was about 100 times higher than that of the nested PCR used to detect homologous DNA, accompanied by high species-specificity and good repeatability. This RtPCR was used to detect ticks and splenic samples of wild mice in forest and the positive and negative results were corresponded with that detected by nested PCR specific for the pathogen; however, it was more sensitive and reliable than nested PCR. These results suggest that the quantitative real-time PCR is highly specific and sensitive for detection of A. phagocytophila. It is very useful for detection of tiny DNA of A. phagocytophila in samples of animals infected with A. phagocytophila.