基于锁核苷酸(LNA)增敏的植烟土壤中烟草黑胫病菌定量PCR检测方法

摘要

为了快速有效地检测植烟土壤中的烟草黑胫病菌的定殖数量,以一种锁核苷酸(LNA)引物为基础,进行了植烟土壤中烟草黑胫病菌数量的定量PCR检测方法研究.结果表明:①与普通DNA引物相比,LNA引物提高了PCR反应的退火温度,减少了引物二聚体和非特异性产物的形成,提高了烟草黑胫病菌分子检测的灵敏度与特异性;②定量分析检测出14个烟草-大蒜轮作土样中烟草黑胫病菌的定殖数量为2.76×103～5.20×104个/g,且在4～5 h内完成检测,具有较高的灵敏度和检测效率.%In order to efficiently detect the colonization of Phytophthora parasitica var. nicotianae in tobacco planting soil, a quantitative PCR method was researched based on locked nucleic acid (LNA) primer. The results showed that: 1) Comparing with conventional DNA primers, the LNA primer raised PCR reaction annealing temperature, reduced the formation of primer dimers and non-specific products, and improved the sensitivity and specificity of detection. 2) LNA quantitative PCR analysis revealed that the concentration of Phytophthora parasitica var. nicotianae in 14 soil samples of tobacco / garlic rotation fields ranged from 2.76 × 103 to 5.20 × 104 per gram. This method is sensitive and efficient, the detection can be completed within 4-5 hours.