一种基于PCR技术混合样本高效定性筛查转基因阳性样本的方法

An Efficient Method of Qualitative Screening Positive Transgenic Samples Based on PCR Technique and Pooling Strategy

【目的】基于PCR技术与DNA样本混合策略建立一种高效定性筛查大量待检样本中转基因阳性样本的方法。【方法】以480个样本为研究案例,对待检样本DNA模板随机编号后构建虚拟三维混合池,置于5×96孔板中,将各板、各行和各列分别混合,构建所有板各行的X维行池(A,B,C,……G,H)和所有板各列的Y维混合池(1,2,3,……11,12),各板所有样本混合的Z维混合池(I,II,III,IV,V)。利用三维交叉唯一性原理与PCR检测的灵敏性与特异性,高效定性筛查样本中的阳性个体。将浓度为500 ng.μL-1的阳性对照分别用ddH2O和同浓度阴性DNA模板进行96倍(96×)稀释,检测该方法的灵敏性与特异性。5×96孔板480个样本中,双盲法随机掺入13个Bt176转化事件标准样本(Cry1Ab阳性与Bt176特异引物阳性),通过对25个混合池检测,得到候选阳性样本,并进行二次检测,筛查出真正的阳性样本。【结果】96×混合样本未影响检测的灵敏性与特异性。通过该种混合池得到可能的33个候选样本,再对33个候选样本进行二次检测,成功检测出所有与设置相符的盲样,即掺入的13个阳性样本。【结论】该方法适用于从大量的样本中用PCR方法定性筛查少量阳性样本的检测,工作量比普通单样本逐一检测的方法降低了80%左右。

【Objective】 The objective of this study is to establish an efficient method of qualitative screening a few of transgenic-positive individuals from a large number of samples based on PCR technique and DNA pooling strategy.【Method】 In a case study,480 samples were randomly numbered and then a virtual 3 dimensional pooling bulks of template DNA were constructed.Each of 480 samples was put into 5×96-well plates well by well.The row pools,X dimension pools,were the samples from same row number of all plates and therefore 8（A,B,C,…… G,H） pools were made.The line pools,Y dimension pools,were the samples from same line number of all plates and therefore 12（1,2,3,……11,12） pools were made.The plate pools,Z dimension pools,were the samples from the same plate and therefore 5（I,II,III,IV,V） pools were made.The rationals of the design was used in order to archive high efficiency because the cross among 3 dimensions is unique along with high sensitivity and specificity be provided with PCR technique.The 500 ng·μL-1 positive samples were 96× diluted by using ddH2O or negative DNA,respectively.At the same time,13 double blind samples of Bt176 events,Bt176 and Cry1Ab positive samples,were mixed in 480 samples.By screening the 25 pools,the candidate samples were detected and then screened them again.【Result】 The results showed that the sensitivity and specificity of the detection were not affected.What＇s more,this way exactly screened out 13 positive samples after screening of 25 pools and they were the positive 13 samples.【Conclusion】 This method can be used for screening a few numbers of positive individuals from a large number of accessions.The work load will be reduced around 80% compared with conventional sample by sample method.