甜瓜种子细菌性果斑病菌PCR检测方法的研究

PCR detection of Acidovorax avenae subsp.citrulli in melon seeds

以携带细菌性果斑病菌(Acidovoras avenae subsp.citrulli)的甜瓜种子为材料,比较了不同引物、不同种子处理方式、不同浸提液及不同提取时间对细菌性果斑病菌PCR及实时荧光定量PCR检测结果的影响.结果表明:以完整带菌种子浸提液为模板进行PCR扩增,操作最简单且特异条带亮度最高,“种壳+种仁”处理次之,而以磨碎种子浸提液为模板,未能扩增出特异条带.以无菌水作为浸提液的PCR扩增效果明显优于磷酸缓冲液和液体KB培养基,浸提时间以3-12 h为宜.实时荧光定量PCR检测结果与普通PCR一致.在3对特异引物中,引物BX-L1/BX-S-R2的普通PCR扩增效果最佳,引物SEQID4m/SEQID5更适用于实时荧光定量PCR检测.

Taking the melon seeds with Acidovoras avenae subsp. citrulli as materials,the effects of different primers,different seed treatments,different extracts and different extraction time on the detection results of PCR and quantitative real-time PCR of Acidovoras avenae subsp. citrulli were compared. The results showed that the PCR amplification with complete seed extract as template was the simplest and the brightness of the specific bands was maximum,followed by the treatment of "seed shell + seed kernel",but the specific bands could not be amplified by using the extract of ground seeds as the template. Compared to PBS buffer and liquid KB medium,sterile water was more suitable for extracting Ac from melon seeds,and the optimum extraction time was3 to 12 hours. The results of quantitative real-time PCR were consistent with those of ordinary PCR. Among the 3 pairs of specific primers,primers BX-L1/BX-S-R2 had the best PCR amplification results,and primers SEQID4m/SEQID5 were more suitable for quantitative real-time PCR detection.