| 实验动物常见支原体荧光定量PCR检测方法建立 |
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| 引用本文： | 辛闻婷,杨媛媛,王馨宇,吴 东,李灵恩. 实验动物常见支原体荧光定量PCR检测方法建立[J]. 实验动物科学, 2019, 36(5): 60 |
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| 作者姓名： | 辛闻婷 杨媛媛 王馨宇 吴 东 李灵恩 |
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| 摘 要： | 目的 建立能对实验动物常见支原体进行检测的荧光定量PCR方法。方法 针对多种大、小鼠易感支原体的16 s rRNA基因中较保守的区域设计1对引物和探针，建立荧光定量PCR方法并检测其特异性、灵敏性及重复性。结果 该方法在模板浓度为5×100～5×106 copies/μL之间呈良好的线性关系(R2=0.9972)且扩增效率为98.11%；特异性强，能够区分检测金黄色葡萄球菌等常见的病原菌；灵敏性高，检测下限为5 copies/μL，比常规PCR的检测灵敏性高100倍；重复性好，对不同浓度模板进行组内和组间重复性试验的变异系数均低于2%。用荧光定量PCR和普通PCR方法对不同来源不同类型的120份临床样品进行检测，结果表明小鼠支原体检测阳性率分别为3.33%(2/60)、1.67%(1/60)，细胞样品支原体检测阳性率均为5%(3/60)。结论 本研究建立的荧光定量PCR方法快速、特异强、灵敏性高、重复性好，可用于实验动物常见支原体的快速诊断。
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| 关 键 词： | 支原体 实验动物 荧光定量PCR |
Establishment of Real-time Fluorescent Quantitative PCR to Detect Various Mycoplasma Commonly Found in Laboratory Animal |
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| Abstract: | Objective This study aimed at developing a new real-time fluorescent quantitative PCR method for detecting various Mycoplasma commonly found in laboratory animal.Method In this study,a pair of primers and a Taqman probe were designed based on conserved region of 16 s rRNA gene from various Mycoplasma which sensitive to mice and rats,and the amplification efficiency,sensitivity,specificity and repetition of this method were evaluated with standard plasmid.Result The result showed the standard curve had a good linear relationship (R2=0.9972),and the amplification efficiency was 98.11%.The lowest detection limitation of this method was 5 copies/μL,which was about 100 times higher than the conventional PCR.And this real-time fluorescent quantitatiuve PCR method had a strongly specificity for distinguishing the other 8 pathogenic strains.Moreover,it showed the excellent reproducibility with the coefficient of variation of Intra-assay and inter-assay less than 2%.Using this real-time fluorescent quantitative PCR and conventional PCR to detect a batch of clinical samples,the result showed that the positive rate of samples from mice was 3.33% (2/60)and 1.67%(1/60)respecyively,and the positive rate of both method of samples from cells was 5% (3/60).Conclusion In summary,the real-time fluorescent quantitative PCR method developed in this study was fast,specific and sensitive,and could be applied in detection for Mycoplasma of laboratory animal. |
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| Keywords: | Mycoplasma laboratory animal Real-time fluorescent quantitative PCR |
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