| 阪崎克罗诺杆菌中转座子拷贝数实时定量PCR检测方法的建立 |
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| 引用本文： | 王菲,杜欣军,张荣,徐桂香,王硕.阪崎克罗诺杆菌中转座子拷贝数实时定量PCR检测方法的建立[J].安徽农业科学,2012,40(10):5800-5802,5807. |
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| 作者姓名： | 王菲 杜欣军 张荣 徐桂香 王硕 |
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| 作者单位： | 天津科技大学食品工程与生物技术学院,食品营养与安全省部共建教育部重点实验室,天津300457;天津科技大学食品工程与生物技术学院,食品营养与安全省部共建教育部重点实验室,天津300457;天津科技大学食品工程与生物技术学院,食品营养与安全省部共建教育部重点实验室,天津300457;天津科技大学食品工程与生物技术学院,食品营养与安全省部共建教育部重点实验室,天津300457;天津科技大学食品工程与生物技术学院,食品营养与安全省部共建教育部重点实验室,天津300457 |
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| 基金项目： | 天津市教委重点项目(2010ZD01) |
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| 摘 要： | 目的]建立检测阪崎克罗诺杆菌中转座子拷贝数的实时定量PCR方法。方法]以单拷贝持家基因atpD作为内参基因,建立同时含有单拷贝持家基因atpD和EZ-TN5转座子的重组质粒;建立atpD基因与EZ-TN5转座子实时定量检测的标准曲线,并利用所建立的标准曲线,对3株阪崎肠杆菌突变株中atpD基因和EZ-TN5转座子的拷贝数进行检测,并计算其比值。结果]atpD基因与EZ-TN5转座子实时定量检测标准曲线的相关系数分别为0.999和0.998;3株突变株中atpD基因和EZ-TN5转座子拷贝数比值分别为0.98、1.17与0.91,证明突变株中转座子为单拷贝。结论]该研究所建立的实时定量PCR方法实用性强,可替代Southern杂交用于不同细菌中EZ-TN5转座子拷贝数的检测。
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| 关 键 词： | 转座子 拷贝数 实时定量PCR 阪崎克罗诺杆菌 |
Establishment of Real-Time Quantitative PCR Method for Determination of Transposon Copy Number in Cronobacter sakazakii |
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| Institution: | WANG Fei et al(Key Laboratory of Food Nutrition and Safety,Ministry of Education,Tianjin University of Science & Technology,Tianjin 300457) |
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| Abstract: | Objective] This study aimed to establish the Real-Time quantitative PCR method for determination of transposon copy number in C.sakazakii.Method] With single-copy housekeeping gene atpD as the reference gene,recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed;based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon,copy number of atpD gene and EZ-TN5 transposon in three C.sakazakii mutants was detected and the ratio was calculated.Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0.999 and 0.998,respectively;the ratios of copy number of atpD gene and EZ-TN5 transposon in three C.sakazakii mutants were 0.98,1.17 and 0.91,respectively,which indicated that EZ-TN5 transposon in C.sakazakii mutants was single-copy.Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy number of EZ-TN5 transposon in different bacteria. |
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| Keywords: | Transposon Copy number Real-time quantitative PCR Cronobacter sakazakii |
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