基于微滴式和芯片式数字PCR技术的转基因克螟稻成分的定量检测

以转基因克螟稻品系为研究对象,通过大米内源蔗糖磷酸合成酶基因和转基因克螟稻品系特异性序列的绝对拷贝数分析,建立转基因克螟稻成分的双重数字聚合酶链式反应(polymerase chain reaction,PCR)定量分析方法。本研究中转基因克螟稻定量体系中最适DNA添加量在10 pg～13 ng之间,模板断裂程度、PCR扩增退火温度等因素对定量结果的影响不大。其定量的绝对灵敏度达1 copies/μL,可在微滴式和芯片式数字PCR平台上准确检测质量分数在0.1%～100%之间的转基因克螟稻成分,尤其是对低于1%的样品,其定量准确性高于传统的实时荧光PCR方法。本研究建立的转基因克螟稻成分定量分析方法适用性较好,可用于转基因水稻规范化与标准化的定量分析。

Quantification of Genetically Modified Rice (Oryza sativa L.) Event KMD by Digital PCR Based on Droplet and Microfluidic Chip

In this study, a quantitative duplex digital PCR method was established for insect-resistant genetically modified rice event KMD by using the sucrose phosphate synthase gene of rice and the KMD event-specific gene as the endogenous and exogenous genes, respectively. The optimal concentration of rice genomic DNA in the quantitative system was in the range of 10 pg to 13 ng, and the degree of template fracture and the annealing temperature of PCR amplification had little effect on the quantitative results. The absolute sensitivity and relative sensitivity of digital PCR were 1 copies/μL and 0.1%, respectively, while the relative sensitivity of the real-time PCR method established with the same pairs of primers and probes was only 1%. For standardization of the application of this method, an intra-laboratory repeatability validation was performed on two chip-based digital PCR platforms 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 requirements for quantitative analysis. The method was proved to highly applicable the standardized quantitative analysis of GM rice KMD.