Polymerase chain reaction for verification of fluorescent colonies of Erwinia chrysanthemi and Pseudomonas putida WCS358 in immunofluorescence colony staining.

J M van der Wolf, J R van Beckhoven, P M de Vries, J M Raaijmakers, P A Bakker, Y Bertheau, J W van Vuurde
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引用次数: 13

Abstract

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.

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聚合酶链反应验证菊花Erwinia和恶臭假单胞菌WCS358免疫荧光菌落染色。
研究了聚合酶链反应(PCR)用于验证免疫荧光菌落染色(IFC)程序染色的菌落身份的潜力。作者利用引物直接指向编码菊花欧文氏菌同工酶PLa、PLd和PLe的果胶裂解酶基因的保守序列,从纯培养的ifc染色样品中获得了20个荧光靶菌落的96%。在倒入混合了Erw的盘子里。从马铃薯皮提取的113个目标菌落中,鉴定了90%的目标菌落的同源性。利用引物直接指向恶臭假单胞菌WCS358的铁-假bactin受体基因pupA序列,在ifc染色的纯培养样品中,22个目标菌落的96%得到了鉴定。在混合了恶臭p.s . putida WCS358和来自堆肥提取物的非目标菌的培养皿中,108个荧光菌落中有59%通过PCR鉴定。结果表明,来自非目标菌的组分使恶臭p.s . putida WCS358的PCR阈值降低100倍。
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