转基因大米TT51-1品系精准定量检测方法的比较研究

基于大米蔗糖磷酸合成酶(SPS)基因和TT51-1品系特异性基因序列筛选适用于数字PCR的内、外源基因特异性引物探针并建立转基因大米TT51-1品系的双重数字PCR定量方法。其定量的绝对灵敏度和相对灵敏度分别达2 copies/μL和0.1%。当样品中TT51-1转基因大米成分含量低至0.1%时,6次定量值的相对标准偏差在7.30%~18.63%之间,偏差在-8.77%~9.62%之间,精密度和稳定性均较为理想,而同样的引物探针所建立的实时荧光PCR方法定量的相对灵敏度仅达到1%。为促进该方法的标准化应用,将微滴式数字PCR平台上建立的定量方法在芯片式数字PCR平台上进行室内验证,结果表明该方法的定量精密度和准确性符合要求。该方法可应用于大米、稻谷及其初加工产品中TT51-1转基因大米成分的精准定量检测。

Comparative Analysis on the Quantitative Detection of Genetically Modified Rice(Oryza sativa L.)TT51-1 Event

This study designed two pairs of primers and probes and established a duplex digital PCR method for the quantitative detection of GM rice TT51-1 event based on the sequence of sucrose phosphate synthase gene of rice and TT51-1 event-specific gene,respectively.The absolute sensitivity and relative sensitivity were 2 copies/μL and 0.1%respectively.The relative standard deviation(RSD)was among 7.30%to 18.63%while the deviation was among-8.77%to 9.62%in 6 replicates,even the content of GM rice TT51-1 event was as low as 0.1%,met the requirements of standardized detection of GMO.The relative sensitivity of real-time PCR method established with the same pairs of primers and probes could reach only 1%.In order to promote the standardization the application of this method,an intra-laboratory repeatability validation was performed on the chip-based digital PCR platform by different operators to verify the quantitative detection method established on the droplet digital PCR platform,and the results showed that the precision and accuracy of the assay still met the quantitative requirements.It was proved that this method could be applied to the quantitative detection of TT51-1 GM rice ingredients in rice and its preliminary processing products.