采后蓝莓果实表面病原菌的分离鉴定及PCR检测

Study on the optimization of PCR detection conditions for pathogen isolated from the postharvest blueberry surface

以采后贮藏过程中自然腐败变质的蓝莓果实为材料,采用传统纯培养法和平板划线法对蓝莓果实表面致腐优势菌进行了分离与形态学鉴定,最终确定葡萄孢霉(Botrytis cinema)为主要病原菌。采用基因组DNA提取试剂盒法提取葡萄孢霉的DNA,并筛选具有特异性的引物,通过聚合酶链式反应(PCR)对葡萄孢霉特定的DNA片段进行扩增,得到具有特异性的目的基因,对其进行分子生物学的特异性鉴定,并对其检测条件进行优化。最终得到具有特异性的引物序列为Bc F-TGTAATTTCAATGTGCAGAATCC;BcR-TTGAAATGCGATTAATTGTTGC,最适的扩增引物浓度为0.25μmol/L,PCR扩增的最适退火温度为60℃。该方法可有效提高蓝莓病原菌的检测效率,可将其应用于实践中,为蓝莓的贮藏保鲜提供理论依据和技术指导。

The main pathogens were separated and identified from the rotting postharvest blueberry fruits using traditional pure culture method and plate streaking method.The results showed that the main pathogen was Botrytis cinerea.ln order to identify Botrytis cinerea with PCR method and found its best experimental conditions, the Genomic DNA was extracted and the target gene was screened with specific primers. The optimum PCR experimental condition was as following.the primer sequences were BcF-TGTAAT-FICAATGTGCAGAATCC and BcR-TTGAAATGCGATTAATT GTTGC,the best amplification primer concentration was 0.25 μmol/L,the optimal annealing temperature was 60 ℃.This PCR method could effectively improve the detection efficiency of blueberry pathogens.Moreover, it helped to set up a theoretical basis and technical guidance for the preservation of blueberries in production.