Location: Floral and Nursery Plants ResearchTitle: Development and comparison of TaqMan-based real-time PCR assays for detection and differentiation of Ralstonia solanacearum strains Author
|Stulberg, Michael - Orise Fellow|
|Rascoe, John - Animal And Plant Health Inspection Service (APHIS)|
|Yan, Zonghe - Animal And Plant Health Inspection Service (APHIS)|
|Nakhla, Mark - Animal And Plant Health Inspection Service (APHIS)|
Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/21/2016
Publication Date: 7/13/2016
Citation: Stulberg, M.J., Rascoe, J., Li, W.N., Yan, Z., Nakhla, M.K., Huang, Q. 2016. Development and comparison of TaqMan-based real-time PCR assays for detection and differentiation of Ralstonia solanacearum strains. Current Microbiology. doi:10.1007/s00284-016109-z.
Interpretive Summary: A strain of the bacterium Ralstonia solanacearum causes destructive diseases in potato and geranium and is not allowed to enter the United States, Canada and Europe. To make sure that plants imported from other countries do not carry this particular bacterium, a fast, accurate and reliable method to detect it is needed. We developed assays that allow detection of the bacterium in quantity, real time and with confidence under standardized conditions, and compared them to previously published methods. Our newly developed assays and a previously published method performed the best under our standardized testing conditions. The methods developed here will benefit federal regulators and diagnostic labs by providing assays for detection of the regulated bacterium with accuracy, confidence and ease.
Technical Abstract: 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 temperature 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 labs over conventional PCR due to its speed and sensitivity. We developed a Rs16S primer and probe set and compared it with a widely-used published set for detecting R. solanacearum species complex strains, RS. 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 using 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 tested, 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 outgroup strains tested. 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 primer sets are best suited under standardized conditions for the detection of the R. solanacearum species complex and r3b2 strains by TaqMan-based qPCR assays.