Ouyang Ping, Arif Mohammad, Fletcher Jacqueline, Melcher Ulrich, Ochoa Corona Francisco Manuel
National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Oklahoma State University, Stillwater, Oklahoma, United States of America ; Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, Oklahoma, United States of America.
PLoS One. 2013 Nov 29;8(11):e81647. doi: 10.1371/journal.pone.0081647. eCollection 2013.
A reliable, accurate and rapid multigene-based assay combining real time quantitative PCR (qPCR) and a Razor Ex BioDetection System (Razor Ex) was validated for detection of Xylella fastidiosa subsp. pauca (Xfp, a xylem-limited bacterium that causes citrus variegated chlorosis [CVC]). CVC, which is exotic to the United States, has spread through South and Central America and could significantly impact U.S. citrus if it arrives. A method for early, accurate and sensitive detection of Xfp in plant tissues is needed by plant health officials for inspection of products from quarantined locations, and by extension specialists for detection, identification and management of disease outbreaks and reservoir hosts. Two sets of specific PCR primers and probes, targeting Xfp genes for fimbrillin and the periplasmic iron-binding protein were designed. A third pair of primers targeting the conserved cobalamin synthesis protein gene was designed to detect all possible X. fastidiosa (Xf) strains. All three primer sets detected as little as 1 fg of plasmid DNA carrying X. fastidiosa target sequences and genomic DNA of Xfp at as little as 1 - 10 fg. The use of Razor Ex facilitates a rapid (about 30 min) in-field assay capability for detection of all Xf strains, and for specific detection of Xfp. Combined use of three primer sets targeting different genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a field-deployable rapid and reliable bioforensic detection and discrimination method for a bacterial phytopathogen based on multigene targets.
一种可靠、准确且快速的基于多基因的检测方法，结合了实时定量聚合酶链反应（qPCR）和Razor Ex生物检测系统（Razor Ex），已通过验证，用于检测桑氏木质部小菌（Xfp，一种导致柑橘杂色黄化病[CVC]的木质部受限细菌）。CVC对美国来说是外来病害，已在南美洲和中美洲传播，如果传入美国，可能会对美国柑橘造成重大影响。植物卫生官员在检查来自检疫地区的产品时，以及推广专家在检测、识别和管理疾病爆发及宿主时，都需要一种在植物组织中早期、准确且灵敏地检测Xfp的方法。设计了两组特异性PCR引物和探针，分别靶向Xfp的菌毛蛋白基因和周质铁结合蛋白基因。还设计了第三对引物，靶向保守的钴胺素合成蛋白基因，以检测所有可能的桑氏木质部小菌（Xf）菌株。所有这三组引物都能检测到携带桑氏木质部小菌靶序列的低至1 fg的质粒DNA以及低至1 - 10 fg的Xfp基因组DNA。Razor Ex的使用实现了一种快速（约30分钟）的现场检测能力，可用于检测所有Xf菌株以及特异性检测Xfp。联合使用靶向不同基因的三组引物提高了检测准确性并拓宽了检测范围。据我们所知，这是第一份基于多基因靶点的用于细菌植物病原体的可现场部署的快速可靠生物法医检测和鉴别方法的报告。
