| 摘要: |
| 【目的】建立一种快速、灵敏的舒伯特气单胞菌（Aeromanas schubertii）检测方法。【方法】以舒伯特气单胞菌rpoD (RNA polymerase sigma-70 factor)为靶标基因，设计特异性引物及 TaqMan探针，建立舒伯特气单胞菌的TagMan实时荧光定量PCR检测方法，同时验证该方法的特异性、灵敏度、重复性，并用该方法对50份临床样品进行检测，验证其应用效果。【结果】含舒伯特气单胞菌rpoD基因的质粒拷贝数与循环阈值（ Ct）的标准曲线相关系数为1.000，扩增效率为94.705%。实时荧光定量PCR检测方法只对舒伯特气单胞菌的靶基因检测结果呈阳性，对嗜水气单胞菌(Aeromanas hydrophila)、维氏气单胞菌(Aeromonas veronii)、温和气单胞菌(Aeromonas sobria)、诺卡氏菌(Nocardia seriolae)等其他14种致病菌的检测结果均为阴性，具有高度特异性；对舒伯特气单胞菌重组质粒和纯培养细菌的检出灵敏度分别为4.76拷贝/μL和15 CFU/mL；批内和批间重复性试验变异系数分别为0.95%和1.05%。50份临床样品检测结果显示，该方法阳性样品检出率为22%，高于传统细菌分离方法（10%）的检出率。【结论】建立的舒伯特气单胞菌实时荧光定量PCR检测方法特异性强、灵敏度高、重复性好，适用于舒伯特气单胞菌的快速诊断检测和流行病学调查。 |
| 关键词: 舒伯特气单胞菌 rpoD基因 实时荧光定量 PCR |
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| 基金项目:2017年度广东省渔港建设和渔业发展专项资金（鱼病防治）项目；中国水产科学研究院基本科研业务费专项（2017HY-ZC0405） |
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| Development and application of a real-time PCR assay for detection of Aeromanas schubertii |
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LIU Lihui,HU Lei,YANG Yuanyuan,et al
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| Abstract: |
| 【Objective】This study established a sensitive protocol for the detection of Aeromanas shuberti.【Method】The TaqMan real-time PCR was developed with a pair of primers and TaqMan probe targeting the conserved region of A. schubertii rpoD gene.The specificity,sensitivity and repeatability of the method were verified,and 50 clinical samples were tested to verify its application.【Result】The correlation efficiency for standard curves of Ct value and plasmid copy number was 1.000 and the amplification efficiency was 94.705%.The assay was highly specific for amplification from A.schubertii,and had no detection of other 14 pathogenic bacteria including Aeromanas hydrophila，Aeromonas veronii，Aeromonas sobria and Nocardia seriolae.The detection limits of recombinant plasmid and pure culture bacteria were 4.76 copies/μL and 15 CFU/mL,respectively.The variation efficiencies in inter-batch repeatability test and intra-batch repeatability test were 0.95% and 1.05%.The positive rate of 50 clinical samples detected by the established method was 22%,which was higher than that of the traditional method (10%).【Conclusion】The developed real-time PCR assay was specific,sensitive,repeatable and suitable for epidemiological investigation,prevention and control of A. schubertii. |
| Key words: Aeromanas schubertii rpoD gene real time PCR |
