转基因番木瓜基因组DNA的快速制备和PCR检测方法

Genomic DNA Rapid Preparation and PCR Detection Methods for Genetically Modified Papaya

转基因番木瓜检测中,模板DNA的制备是关键一步。为了提高检测效率,建立快速制备番木瓜样品基因组DNA和PCR检测方法十分重要。建立了番木瓜基因组DNA的快速制备方法,包括样品准备、制备缓冲液匀浆和稀释上清。在完成不同番木瓜样品的基因组DNA快速制备后,对获得的DNA进行PCR检测验证。普通PCR验证结果显示,本方法制备的叶片和果肉基因组DNA溶液稀释5倍时,PCR扩增条带清晰,更高稀释倍数时,叶片基因组DNA扩增条带强于果肉基因组DNA;实时荧光PCR验证结果显示,叶片基因组DNA的PCR扩增效率位于合理区间。灵敏度测试结果表明,含量低至0.1%的叶片和果肉样品,内标准基因和外源基因特异性片段普通PCR和实时荧光PCR均能得到预期扩增,且重复性较好。本研究建立的番木瓜基因组DNA快速制备与PCR方法效果良好,体系稳定。

The preparation of genomic DNA template is the key step in the detection of genetically modified papaya.In order to improve detection efficiency,it is very important to establish rapid genomic DNA preparation and PCR detection methods for papaya samples.In this study,a rapid preparation method of papaya genomic DNA was developed,including sample preparation,homogenisation with preparation buffer and supernatant dilution.After the rapid preparation of genomic DNA solutions from different papaya samples,the obtained genomic DNA solutions were verified by PCR method.The results of qualitative PCR showed that the amplified bands were clear when the genomic DNA solutions of the leaves and fruits were diluted for 5 times.The PCR amplification results of leaf genomic DNA were better than that of the fruits when the DNA solution were diluted for 25 times.The PCR amplification efficiencies of leaf genomic DNA were reasonable in the realtime fluorescence PCR reaction.Sensitivity testing results indicated the specific fragments of endogenous reference and exogenous genes were amplified as expected by both qualitative and real-time fluorescence PCR methods when the contents in the leaves and fruits samples was 0.1%.The rapid preparation and PCR detection methods of papaya genomic DNA established here is efficient and stable.