双重PCR检测黄秋葵储藏期间青霉和链格孢菌条件的优化

Optimum conditions for detecting Penicillium and Alternaria spp. during the storage period of okra by Dual-PCR

以黄秋葵为材料,分离纯化在贮藏过程中引起腐烂的主要病原真菌青霉和链格孢菌。根据基因库上的序列,针对青霉和链格孢菌在18SRNA的序列设计2对引物,建立了用于检测黄秋葵青霉和链格孢菌双重PCR检测方法,并对PCR扩增条件进行了优化,优化条件如下:引物Alt4、Alt5和Pen F'、Pen R的比例为2∶1、退火温度为52℃、Mg^(2+)浓度为2.5 mmol/L、d NTP浓度为0.08 mmol/L、循环数为35、延伸时间为50 s、退火时间为30 s。结果表明,该方法灵敏度较高、特异性强。该试验可以为黄秋葵在储藏期间的病害防治提供重要指导依据。

Using okra as test materials, pathogenic fungi of okra caused by rotting Penicillium and Alternaria during storage process were isolated and purified. According to the sequences of Penicillium and Alternaria gene in Gene Bank, a pair of specific primers in 18SRNA sequence were designed. Then a double polymerase chain reaction (PCR) was developed for the simultaneous detection of Penicillium and Alternaria of okra during storage. Otherwise, the conditions for Dual-PCR amplification were optimized. The optimal conditions of PCR were determined as following: primer proportion was 2 ∶ 1, annealing temperature was 52 ℃, Mg^2+ concentration was 2.5 mmol/L, dNTP concentration was 0.08 mmol/L, the number of amplification cycles was 35, extended time was 50 s, the annealing time was 30 s. The results showed that a fast, accurate method with high sensitivity and specificity were founded, which provided important guidance for disease prevention and control of okra during storage.