Crop Diseases, Pests and Genetics Research Unit, San Joaquin Valley Agricultural Science Center, USDA ARS PWA, Parlier, CA 93648, USA.
J Microbiol Methods. 2010 Apr;81(1):17-25. doi: 10.1016/j.mimet.2010.01.014. Epub 2010 Jan 22.
An ultra-sensitive and quantitative diagnostic system by combining nested PCR and TaqMan PCR in a single tube was developed for detection of "Candidatus Liberibacter asiaticus". The procedure involves two PCR steps using the species-specific outer and inner primer pairs. Different annealing temperatures allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The first PCR with outer primers was performed at a higher annealing temperature and with limited amount of primers to prevent interference with the inner primers during the second round of PCR. The specificity of the dual primer TaqMan is high because the fluorescent signal can only be generated from the TaqMan probes that are homologous to the product amplified by the outer and inner primers. This new detection system can reliably detect as few as single copies of target DNA. The sensitivity of the dual primer system is comparable to the conventional two-tube nested PCR, but it eliminates the potential risk of cross contamination commonly associated with conventional nested PCR. This one-tube dual primer TaqMan PCR method is gel-free with reduced handling time and is cost effective. At the same time, this system provides significantly increased sensitivity, improved reliability and high through-put capability suitable for routine, large scale diagnoses of clinical plant tissue and insect samples. The technique described here is generic and can be applied to the detection of other plant pathogenic bacteria.
建立了一种通过在单个管中组合巢式 PCR 和 TaqMan PCR 来进行检测“亚洲韧皮杆菌”的超灵敏和定量诊断系统。该程序涉及两步 PCR，使用种特异性外引物和内引物对。不同的退火温度允许第一轮和第二轮 PCR 分别在同一封闭管中顺序进行。第一轮使用外引物的 PCR 在较高的退火温度下进行，并且使用有限量的引物，以防止在第二轮 PCR 期间干扰内引物。双重引物 TaqMan 的特异性很高，因为荧光信号只能从与外引物和内引物扩增的产物同源的 TaqMan 探针中产生。这种新的检测系统可以可靠地检测到仅单个拷贝的靶 DNA。双引物系统的灵敏度与传统的双管巢式 PCR 相当，但它消除了传统巢式 PCR 常见的潜在交叉污染风险。这种单管双引物 TaqMan PCR 方法是无凝胶的，处理时间缩短，并且具有成本效益。同时，该系统提供了显著提高的灵敏度、改进的可靠性和高通量能力，适用于临床植物组织和昆虫样本的常规大规模诊断。这里描述的技术是通用的，可以应用于其他植物病原菌的检测。
