猪附红细胞体荧光定量PCR检测方法的建立及应用

摘要: 根据GenBank已登录的猪附红细胞体（M.suis）推测的功能性蛋白基因ORF2序列设计引物和TaqMan荧光探针，以定量的10倍系列稀释含M.suis部分ORF2基因的T载体重组质粒（pGEX-T/M.suis）为标准品，进行荧光定量PCR扩增并制作了标准曲线，经对荧光定量PCR的反应条件进行优化，建立了M.suis的TaqMan荧光定量PCR检测方法（Taqman FQ-PCR）；对所建立的FQ-PCR检测方法进行了敏感性、特异性和重复性实验，并对疑似M.suis感染临床抗凝全血样品进行了检测应用。结果显示：标准曲线的曲线循环阈值与模板浓度有良好的线性关系，相关系数为0.998；所建立的FQ-PCR方法检测灵敏度可达10个拷贝/μL，比对照常规PCR灵敏度高100倍；FQ-PCR方法特异性高，对pGEX-T/M.suis重组质粒扩增呈现阳性反应曲线，而对8个对照细菌、病毒和寄生虫DNA扩增曲线均呈现阴性反应；对不同浓度的pGEX-T/M.suis重组质粒分别重复扩增2次，重复结果良好；用该方法对24份临床疑似M.suis感染样品进行了应用检测，结果有20份样品为阳性，阳性检出率高于常规PCR方法。

Establishment and Application of Real-time TaqMan-quantitative PCR Assay for Detection of Mycoplasma suis (Eperythrozoon suis)

Abstract: The Taqman Fluorescent probes and primers were designed and synthesized according to the conserved part of hypothetical functional protein gene of Mycoplasma suis (Eperythrozoon suis) available in GenBank，and then reaction parameters were optimized and standard carve was established based on the quantitative 10×series of dilutions recombinant M.suis plasmid （pGEX-T/M.suis）to develop a real-time TaqMan-fluorescent-quantitative PCR assay (FQ-PCR) of Mycoplasma suis. The sensitivity, specificity and repetition assay of FQ-PCR were tested, and 24 heparinized-blood samples taken from suspicious infected pigs basing on the clinical signs and microscope check were detected by using the established FQ-PCR assay comparing with the routine PCR method. The results indicated the cycle numbers of standard carve had a good linear relation with the template concentration, and the correlation coefficient was 0.998. The developed FQ-PCR assay could detect 1.0×101 copy/μL of plasmid DNA and its sensitivity was 100 times higher than that of the routine PCR. The sensitivity assay exhibited that the positive carves could be achieved from the pGEX-T/M.suis recombinant plasmid, whereas the negative carve were displayed from the genomic DNA of the other 8 kinds of pathogenic microorganism acting as the control. Three pGEX-T/M.suis recombinant plasmids samples with different concentrations were examined using the FQ-PCR repeated 2 times and the results indicated that the FQ-PCR was reproducible. The FQ-PCR was used to detect the DNA of 24 suspected heparinized-blood samples and 20 positive samples were displayed, which showed the better sensitivity than that of the routine PCR , with 19 positive samples from the same 24 suspected samples.