van der Wolf J M, van Beckhoven J R, de Vries P M, Raaijmakers J M, Bakker P A, Bertheau Y, van Vuurde J W
DLO Research Institute for Plant Protection (IPO-DLO), Wageningen, The Netherlands.
J Appl Bacteriol. 1995 Nov;79(5):569-77. doi: 10.1111/j.1365-2672.1995.tb03178.x.
The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi, the authors confirmed the identity of 96% of 20 fluorescent target colonies, punched from IFC-stained samples with pure cultures. In pour plates with mixtures of Erw. chrysanthemi and non-target colonies from potato peel extracts, the identity of 90% of 113 target colonies was confirmed. Using primers directed against sequences of the ferric-pseudobactin receptor gene pupA of Pseudomonas putida WCS358, the identity of 96% of 22 target colonies was confirmed in IFC-stained samples with pure cultures. In pour plates with mixtures of Ps. putida WCS358 and non-target bacteria from compost extracts, the identity of 59% of 108 fluorescent colonies was confirmed by PCR. It was shown that components from non-target bacteria lowered the threshold level of PCR for Ps. putida WCS358 100-fold.
研究了聚合酶链反应（PCR）用于验证经免疫荧光菌落染色（IFC）程序染色的菌落身份的潜力。作者使用针对编码菊欧文氏菌果胶酸裂解酶同工酶PLa、PLd和PLe的果胶酸裂解酶基因保守序列的引物，确认了从IFC染色样品中用纯培养物打出的20个荧光目标菌落中96%的菌落身份。在含有菊欧文氏菌与来自马铃薯皮提取物的非目标菌落混合物的倾注平板中，113个目标菌落中90%的菌落身份得到了确认。使用针对恶臭假单胞菌WCS358铁假菌素受体基因pupA序列的引物，在IFC染色的纯培养样品中，22个目标菌落中96%的菌落身份得到了确认。在含有恶臭假单胞菌WCS358与来自堆肥提取物的非目标细菌混合物的倾注平板中，通过PCR确认了108个荧光菌落中59%的菌落身份。结果表明，来自非目标细菌的成分将恶臭假单胞菌WCS358的PCR阈值水平降低了100倍。
