应用多重PCR技术快速检测抗虫转基因水稻

Application of multiplex polymerase chain reaction to rapidly detect insect-resistant transgenic rice

通过优化PCR扩增体系中缓冲液的浓度、退火温度、引物的组合及其浓度等参数,建立了转基因水稻及其加工品中几种外源基因成分的多重PCR检测体系。利用该体系检测CaMV35S、NOS、Cry1Ac或Cry1Ab的三重PCR条件为:缓冲液工作浓度2.0x、引物各为0.2μM、退火温度为58℃,循环数为35个。缓冲液工作浓度为1.5x,其他条件不变,可进行CaMV35S、NOS、Bt和CpTi的四重PCR检测。缓冲液工作浓度为1.0x,其他条件不变,可进行CaMV35S、NOS、Bt和hpt的四重PCR检测。该多重PCR检测体系具有特异性好、简便、快速、准确等优点,能够有效地检测出转基因水稻质量百分比含量(w/w)0.1%的转基因成分。

Through optimizing the concentration of mediate liquid, annealing temperature, primers, etc., multiplex polymerase chain reaction （PCR） method was designed for simultaneously detecting CaMV35S promoter, NOS terminator, hpt （hygromycin B phosphotransferase gene）, insect-resistante gene CrylAc, CrylAb, CpTi which is frequently used in transgenic rice. The result show that the high efficiency PCR system was obtained when the primer is 0.2 μM for each, and annealing temperature is 58℃. The final concentration of PCR buffer is 2.0x for detection of CaMV35S, NOS, Bt; the final concentration of PCR buffer is 1.5x for detection of CaMV35S, NOS, Bt and CpTi; the final concentration of PCR buffer is 1.0x for detection of CaMV35S, NOS, Bt and hpt. And the limits of detection are 0.1%. This method was stable, reliable, rapid and accurate for testing different transgenic rice.