Cui Z, Ojaghian M R, Tao Z, Kakar K U, Zeng J, Zhao W, Duan Y, Vera Cruz C M, Li B, Zhu B, Xie G
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China.
Chinese Academy of Inspection and Quarantine, Beijing, China.
J Appl Microbiol. 2016 May;120(5):1357-67. doi: 10.1111/jam.13094.
The aim of this study was to develop a multiplex PCR (mPCR) assay for rapid, sensitive and simultaneous detection of six important rice pathogens: Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Pseudomonas fuscovaginae, Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae subsp. avenae.
Specific primers were designed through a bioinformatics pipeline. Sensitivity of detection was established using both traditional PCR and quantitative real-time PCR on isolated DNA and on bacterial cells both in vitro and in simulated diseased seeds and the parameters were optimized for an mPCR assay. A total of 150 bacterial strains were tested for specificity. The mPCR assay accurately predicted the presence of pathogens among 44 symptomatic and asymptomatic rice seed, sheath and leaf samples.
This study confirmed that this mPCR assay is a rapid, reliable and simple tool for the simultaneous detection of six important rice bacterial pathogens.
This study is the first report of a method allowing simultaneous detection of six major rice pathogens. The ability to use crude extracts from plants without bacterial isolation or DNA extraction enhances the value of this mPCR technology for rapid detection and aetiological/epidemiological studies.
本研究旨在开发一种多重聚合酶链反应（mPCR）检测方法，用于快速、灵敏且同时检测六种重要的水稻病原体：水稻白叶枯病菌、水稻条斑病菌、水稻褐鞘假单胞菌、水稻细菌性谷枯病菌、唐菖蒲伯克霍尔德菌和燕麦嗜酸菌燕麦亚种。
通过生物信息学流程设计了特异性引物。利用传统PCR和定量实时PCR对分离的DNA以及体外和模拟染病种子中的细菌细胞进行检测，确定了检测灵敏度，并对mPCR检测方法的参数进行了优化。共对150株细菌菌株进行了特异性测试。mPCR检测方法准确预测了44份有症状和无症状的水稻种子、叶鞘和叶片样本中病原体的存在情况。
本研究证实，这种mPCR检测方法是一种用于同时检测六种重要水稻细菌病原体的快速、可靠且简单的工具。
本研究是关于一种能够同时检测六种主要水稻病原体的方法的首次报道。无需细菌分离或DNA提取即可使用植物粗提物的能力提高了这种mPCR技术在快速检测以及病因学/流行病学研究中的价值。
