| 定量PCR快速鉴别念珠菌种方法的建立 |
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| 引用本文： | 刘建钗,柳焕章,刘彦威,张鹤平,宋阔阔,李聚芹,乔铁库,吴志勇. 定量PCR快速鉴别念珠菌种方法的建立[J]. 中国兽医科学, 2019, 0(1): 58-66 |
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| 作者姓名： | 刘建钗 柳焕章 刘彦威 张鹤平 宋阔阔 李聚芹 乔铁库 吴志勇 |
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| 作者单位： | 河北工程大学生命科学与食品工程学院河北省动物原食品安全检测技术工程实验室蛋鸡高效养殖岗;河北工程大学园林与生态工程学院;高邑县农牧局;武邑县农牧局;邯郸市畜牧技术推广站 |
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| 基金项目： | 河北省科技支撑项目(17226612D,HBCT2018150204);邯郸市科技计划项目(1622201050-2) |
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| 摘 要： | 由于禽类念珠菌种类多样,具有条件致病和感染症状非典型的特性,在临床上区分定植和感染状态并确诊何种念珠菌感染是困难的。为了快速准确地检测禽类念珠菌感染和鉴别病原种类,本研究借助Primer Express 6.0软件设计目标念珠菌基因组DNA的种特异引物,采用real-time qPCR技术,通过检测不同念珠菌种的菌株培养物而建立鉴别方法,并用人工感染鸡的血液和肝脏样本对建立的方法进行检验。结果,成功找出三种念珠菌(白色念珠菌、光滑念珠菌和热带念珠菌)的3对特异引物和1对共用引物;特异引物分别形成相应种的特异熔解曲线,共用引物形成对应于3个种且明显分开的三条熔解曲线,与参照病原菌无交叉反应;该方法对念珠菌样本检测最低限度可达1×10~1copies/L以下,敏感度高于常规PCR约100倍以上;同种念珠菌不同模板浓度Tm值不变,同一浓度3个重复C_t值的变异系数小于2.5%,说明离散度较低,重复性好。所建方法可以通过对感染鸡的血液和肝脏样本的检测确定目标念珠菌感染和病原种类,检测周期约2~3 h。综上,通过适当的引物设计,采用定量PCR技术可以在一定范围内快速、准确、定量地检测和鉴别感染样本中的目标念珠菌种类。
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| 关 键 词： | 特异引物 念珠菌 定量PCR 熔解曲线 快速检测 |
Rapid identification for the species of Candida by real-time qPCR |
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| LIU Jian-chai,LIU Huan-zhang,LIU Yan-wei,ZHANG He-ping,SONG Kuo-kuo,LI Ju-qin,QIAO Tie-ku,WU Zhi-yong. Rapid identification for the species of Candida by real-time qPCR[J]. Chinese Veterinary Science, 2019, 0(1): 58-66 |
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| Authors: | LIU Jian-chai LIU Huan-zhang LIU Yan-wei ZHANG He-ping SONG Kuo-kuo LI Ju-qin QIAO Tie-ku WU Zhi-yong |
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| Affiliation: | (The Efficient BreedingPostfor LayingHens ,Hebei Province Animal-derived Food Safety Testing Technology and Engineering Laboratory ,College of Life Science and Food Engineering,Hebei University of Engineering,Handan 056021, China;College of Landscape and Ecological Engineering,Hebei University of Engineering,Handan 056021,China;Gaoyi County A gricultural and Pastoral Bureau,Gaoyi 051330,China;Wuyi County A gricultural and Pastoral Bureau, Wuyi 053400,China;Handan City Animal Husbandry Technology Promotion Station,Handan 056000,China) |
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| Abstract: | LIU Jian-chai;LIU Huan-zhang;LIU Yan-wei;ZHANG He-ping;SONG Kuo-kuo;LI Ju-qin;QIAO Tie-ku;WU Zhi-yong(The Efficient BreedingPostfor LayingHens ,Hebei Province Animal-derived Food Safety Testing Technology and Engineering Laboratory ,College of Life Science and Food Engineering,Hebei University of Engineering,Handan 056021, China;College of Landscape and Ecological Engineering,Hebei University of Engineering,Handan 056021,China;Gaoyi County A gricultural and Pastoral Bureau,Gaoyi 051330,China;Wuyi County A gricultural and Pastoral Bureau, Wuyi 053400,China;Handan City Animal Husbandry Technology Promotion Station,Handan 056000,China) |
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| Keywords: | specific primer Candida real-time qPCR melting curve rapid test |
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