Verification of Real-Time PCR Methods for Qualitative and Quantitative Testing of Genetically Modified Organisms
Verification of Real-Time PCR Methods for Qualitative and Quantitative Testing of Genetically Modified Organisms
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DOI：
10.1111/jfq.12001
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发表时间：
2012-12-01
影响因子：
3.3
通讯作者：
Cipollaro, M.
中科院分区：
文献类型：
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作者：
Del Gaudio, S.;Cirillo, A.;Cipollaro, M.
本文报道了某高校实验室按照国际标准化组织/国际电工委员会17025标准对转基因生物检测的分子生物学方法进行认证的经验。对一种筛选(35S启动子)、一种结构特异性(P35S/叶绿体转运肽-5-烯丙酮基莽草酸-3-磷酸合成酶[CTP-EPSPS])和一种事件特异性(BT11玉米)实时聚合酶链式反应方法进行了验证研究。除了通过DNA Engine Opticon 2 MJ研究仪进行的分析外，我们的程序按照2005年国际标准化组织21570标准中的描述进行。对扩增效率、线性、动态范围、检出限、定量限、精密度和真实性进行了评价。将定量方法获得的性能结果与由欧洲联盟转基因食品和饲料参考实验室协调的实验室间环试验的值进行比较，证实该实验室能够满足公布的标准。实际应用本文介绍了一所大学实验室获得国际标准化组织/国际电工委员会17025转基因生物检测认证的经验。已经对一种筛选(35S启动子)、一种结构特异性(P35S/叶绿体转运肽-5-烯丙酮基莽草酸-3-磷酸合成酶[CTP-EPSPS])和一种事件特异性(BT11玉米)实时聚合酶链式反应方法进行了内部验证。我们首次报告了通过DNA Engine Opticon 2 MJ Research仪器获得的验证数据。所描述的实验程序可以代表一种模式，供研究人员对验证转基因生物定量和定性测试的有效方法感兴趣。
This article reports the experience of a university laboratory in accreditation of molecular biology methods for genetically modified (GM) organisms detection according to the International Organization for Standardization (ISO)/International Electrotechnical Commission 17025 standard. Verification studies were performed for one screening (35S promoter), one construct-specific (p35S/Chloroplast Transit Peptides-5-enolpyruvylshikimate-3-phosphate synthase [CTP-EPSPS]) and one event-specific (Bt11 maize) real-time polymerase chain reaction methods. Our procedures were performed as described in the ISO 21570 2005, except for the analysis done through a DNA Engine Opticon 2 MJ research instrument. The following methodperformance characteristics were evaluated: amplification efficiency, linearity, dynamic range, limit of detection, limit of quantification, precision and trueness. Comparison of the performance results obtained for quantitative methods with the values of inter-laboratory ring trials, coordinated by the European Union Reference Laboratory for GM Food and Feed, confirmed that the laboratory is able to fulfill the published criteria. Practical Applications This article illustrates the experience of a university laboratory with the International Organization for Standardization/International Electrotechnical Commission 17025 accreditation for genetically modified organisms testing. In-house verification has been carried out for one screening (35S promoter), one construct-specific (p35S/Chloroplast Transit Peptides-5-enolpyruvylshikimate-3-phosphate synthase [CTP-EPSPS]) and one event-specific (Bt11 maize) real-time polymerase chain reaction methods. We reported for the first time validation data obtained through a DNA Engine Opticon 2 MJ Research instrument. The described experimental procedure could represent a pattern for researchers interested in setting up verification of validated methods for genetically modified organism quantitative and qualitative testing.