Stulberg Michael J, Rascoe John, Li Wenbin, Yan Zonghe, Nakhla Mark K, Huang Qi
Floral and Nursery Plants Research Unit, Agricultural Research Service, United States Department of Agriculture, United States National Arboretum, Beltsville, MD, USA.
Oak Ridge Institute for Science and Education, Oak Ridge, TN, USA.
Curr Microbiol. 2016 Oct;73(4):542-9. doi: 10.1007/s00284-016-1091-z. Epub 2016 Jul 11.
Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperate climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is commonly used in federal and state diagnostic laboratories over conventional PCR due to its speed and sensitivity. We developed the Rs16S primers and probe set and compared it with a widely used set (RS) for detecting R. solanacearum species complex strains. We also developed the RsSA3 primers and probe set and compared it with the previously published B2 and RsSA2 sets for specific detection of r3b2 strains. Both comparisons were done under standardized qPCR master mix and cycling conditions. The Rs16S and RS assays detected all 90 R. solanacearum species complex strains and none of the five outgroups, but the former was more sensitive than the latter. For r3b2 strain detection, the RsSA2 and RsSA3 sets specifically detected the 34 r3b2 strains and none of the 56 R. solanacearum non-r3b2 strains or out-group strains. The B2 set, however, detected five non-r3b2 R. solanacearum strains and was less sensitive than the other two sets under the same testing conditions. We conclude that the Rs16S, RsSA2, and RsSA3 sets are best suited under the standardized conditions for the detection of R. solanacearum species complex and r3b2 strains by TaqMan-based qPCR assays.
由青枯雷尔氏菌引起的青枯病对全球许多植物物种具有毁灭性。青枯雷尔氏菌的3号生理小种2型(r3b2)菌株在温带气候下感染马铃薯,被美国政府列为特定生物制剂。基于TaqMan的实时定量PCR(qPCR)由于其速度和灵敏度,在联邦和州诊断实验室中比传统PCR更常用。我们开发了Rs16S引物和探针组,并将其与一种广泛使用的引物和探针组(RS)进行比较,以检测青枯雷尔氏菌复合种菌株。我们还开发了RsSA3引物和探针组,并将其与先前发表的用于特异性检测r3b2菌株的B2和RsSA2组进行比较。这两个比较均在标准化的qPCR预混液和循环条件下进行。Rs16S和RS检测法检测了所有90株青枯雷尔氏菌复合种菌株,而5个外群菌株均未检测到,但前者比后者更灵敏。对于r3b2菌株检测,RsSA2和RsSA3组特异性检测了34株r3b2菌株,而56株非r3b2青枯雷尔氏菌菌株或外群菌株均未检测到。然而,B2组检测到5株非r3b2青枯雷尔氏菌菌株,并且在相同测试条件下比其他两组灵敏度更低。我们得出结论,在标准化条件下,Rs16S、RsSA2和RsSA3组最适合通过基于TaqMan的qPCR检测法来检测青枯雷尔氏菌复合种和r3b2菌株。