Dingman Douglas W
Department of Biochemistry and Genetics, Connecticut Agricultural Experiment Station, New Haven, CT 06504, USA.
J Invertebr Pathol. 2009 Jan;100(1):16-21. doi: 10.1016/j.jip.2008.09.006. Epub 2008 Sep 25.
Failure to identify correctly the milky disease bacteria, Paenibacillus popilliae and Paenibacillus lentimorbus, has resulted in published research errors and commercial production problems. A DNA fingerprinting procedure, using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS) regions, has been shown to easily and accurately identify isolates of milky disease bacteria. Using 34 P. popilliae and 15 P. lentimorbus strains, PCR amplification of different ITS regions produced three DNA fingerprints. For P. lentimorbus phylogenic group 2 strains and for all P. popilliae strains tested, electrophoresis of amplified DNA produced a migratory pattern (i.e., ITS-PCR fingerprint) exhibiting three DNA bands. P. lentimorbus group 1 strains also produced this ITS-PCR fingerprint. However, the fingerprint was phase-shifted toward larger DNA sizes. Alignment of the respective P. popilliae and P. lentimorbus group 1 ITS DNA sequences showed extensive homology, except for a 108bp insert in all P. lentimorbus ITS regions. This insert occurred at the same location relative to the 23S rDNA and accounted for the phase-shift difference in P. lentimorbus group 1 DNA fingerprints. At present, there is no explanation for this 108bp insert. The third ITS-PCR fingerprint, produced by P. lentimorbus group 3 strains, exhibited approximately eight DNA bands. Comparison of the three fingerprints of milky disease bacteria to the ITS-PCR fingerprints of other Paenibacillus species demonstrated uniqueness. ITS-PCR fingerprinting successfully identified eight unknown isolates as milky disease bacteria. Therefore, this procedure can serve as a standard protocol to identify P. popilliae and P. lentimorbus.
未能正确识别乳状病细菌——日本金龟子芽孢杆菌和缓病芽孢杆菌，已导致已发表的研究出现错误以及商业生产出现问题。一种利用聚合酶链式反应（PCR）扩增16S - 23S核糖体DNA（rDNA）基因间隔转录区（ITS）的DNA指纹图谱技术，已被证明能够轻松、准确地识别乳状病细菌的分离株。使用34株日本金龟子芽孢杆菌和15株缓病芽孢杆菌菌株，对不同的ITS区域进行PCR扩增产生了三种DNA指纹图谱。对于缓病芽孢杆菌系统发育组2的菌株以及所有测试的日本金龟子芽孢杆菌菌株，扩增后的DNA电泳产生了一种迁移模式（即ITS - PCR指纹图谱），呈现出三条DNA条带。缓病芽孢杆菌组1的菌株也产生了这种ITS - PCR指纹图谱。然而，该指纹图谱向更大的DNA大小发生了相位偏移。日本金龟子芽孢杆菌和缓病芽孢杆菌组1各自的ITS DNA序列比对显示，除了缓病芽孢杆菌所有ITS区域中存在一个108bp的插入片段外，它们具有广泛的同源性。这个插入片段相对于23S rDNA出现在相同位置，并导致了缓病芽孢杆菌组1 DNA指纹图谱中的相位偏移差异。目前，对于这个108bp的插入片段尚无解释。缓病芽孢杆菌组3的菌株产生的第三种ITS - PCR指纹图谱显示出大约八条DNA条带。将乳状病细菌的三种指纹图谱与其他芽孢杆菌属物种的ITS - PCR指纹图谱进行比较，显示出其独特性。ITS - PCR指纹图谱技术成功地将八个未知分离株鉴定为乳状病细菌。因此，该方法可作为鉴定日本金龟子芽孢杆菌和缓病芽孢杆菌的标准方案。
