Schaad N W, Berthier-Schaad Y, Sechler A, Knorr D
ARS-USDA Foreign Disease-Weed Science Research Unit, Frederick, MD 21702.
Perkin Elmer, Foster City, CA.
Plant Dis. 1999 Dec;83(12):1095-1100. doi: 10.1094/PDIS.1999.83.12.1095.
Ring rot of potato, caused by Clavibacter michiganensis subsp. sepedonicus, is one of the most regulated diseases of potatoes world wide. The organism is often difficult to detect in symptomless tubers because of low populations and slow competitive growth on available media. Polymerase chain reaction (PCR) primers and a fluorescent probe for use in the Perkin Elmer 7700 automated real time PCR detection system (TaqMan) were designed from a C. michiganensis subsp. sepedonicus-specific genomic DNA fragment for development of a BIO-PCR assay for C. michiganensis subsp. sepedonicus in potato tubers. Results of screening the primers with strains of C. michiganensis subsp. sepedonicus and other bacteria showed the primers to be specific. A total of 30 naturally infected ring rot suspect tubers were sampled by the core extract, shaker incubation procedure and assayed by (i) plating aliquots onto agar media, (ii) classical PCR, and (iii) BIO-PCR. In all, 4 tubers were positive by agar plating and pathogenicity tests, 8 by classical TaqMan PCR, and 26 by TaqMan BIO-PCR. We conclude that BIO-PCR combined with the TaqMan automated closed detection system is a rapid, reliable method of assaying large numbers of potato tuber extracts for C. michiganensis subsp. sepedonicus. Furthermore, for a large central laboratory running large numbers of PCR assays, the high-throughput TaqMan system can reduce costs per sample over the more labor-intensive classical PCR.
由密执安棒杆菌马铃薯环腐亚种（Clavibacter michiganensis subsp. sepedonicus）引起的马铃薯环腐病，是全球范围内受管控最为严格的马铃薯病害之一。由于该病菌数量较少且在现有培养基上竞争生长缓慢，所以在无症状块茎中往往难以检测到。从密执安棒杆菌马铃薯环腐亚种特异性基因组DNA片段设计了聚合酶链反应（PCR）引物及用于珀金埃尔默7700型自动实时PCR检测系统（TaqMan）的荧光探针，以开发用于检测马铃薯块茎中密执安棒杆菌马铃薯环腐亚种的生物PCR检测方法。用密执安棒杆菌马铃薯环腐亚种菌株和其他细菌对引物进行筛选的结果表明这些引物具有特异性。通过取核心提取物、振荡培养程序对30个自然感染环腐病的疑似块茎进行采样，并通过以下方法进行检测：（i）将等分试样接种到琼脂培养基上，（ii）进行经典PCR，（iii）进行生物PCR。通过琼脂平板培养和致病性测试，共有4个块茎呈阳性；通过经典TaqMan PCR检测，8个呈阳性；通过TaqMan生物PCR检测，26个呈阳性。我们得出结论，生物PCR与TaqMan自动封闭检测系统相结合，是一种快速、可靠的检测大量马铃薯块茎提取物中密执安棒杆菌马铃薯环腐亚种的方法。此外，对于进行大量PCR检测的大型中心实验室而言，高通量TaqMan系统相对于劳动强度更大的经典PCR能够降低每个样本的成本。
