Brunnert S R, Dai Y, Kohn D F
Institute of Comparative Medicine, College of Physicians & Surgeons, Columbia University, New York, NY 10032.
Lab Anim Sci. 1994 Jun;44(3):257-60.
The polymerase chain reaction (PCR) is a reliable method of detecting specific DNA sequences. The purpose of this study was to develop a PCR method of detecting Mycoplasma pulmonis in paraffin-embedded tissue sections and to compare the sensitivity of this method with that of the avidin-biotin complex (ABC) immunohistochemistry technique. We infected mice intranasally with 10(7) M. pulmonis and sacrificed them 28 days later. Using a published oligonucleotide primer sequence for M. pulmonis, we examined 8-microns paraffin sections by PCR for a 710 base-pair segment of the genome. A sequential 8-microns paraffin section was stained for M. pulmonis, using standard ABC immunohistochemistry methods. By PCR and dot blot hybridization, 60 of 62 paraffin-embedded lungs were positive for M. pulmonis, whereas only 17 of 62 lungs were positive, using the ABC method. In this study, we demonstrated that PCR of paraffin-embedded tissues followed by dot blot hybridization of the PCR products was much more sensitive than the ABC method in identifying mouse lungs infected with M. pulmonis.
聚合酶链反应（PCR）是检测特定DNA序列的可靠方法。本研究的目的是开发一种在石蜡包埋组织切片中检测肺支原体的PCR方法，并将该方法的灵敏度与抗生物素蛋白-生物素复合物（ABC）免疫组织化学技术的灵敏度进行比较。我们经鼻给小鼠接种10⁷ 个肺支原体，28天后将其处死。使用已发表的肺支原体寡核苷酸引物序列，我们通过PCR检测8微米厚的石蜡切片中基因组的一个710碱基对片段。使用标准ABC免疫组织化学方法，对连续的8微米厚石蜡切片进行肺支原体染色。通过PCR和斑点印迹杂交，62个石蜡包埋肺中有60个肺支原体呈阳性，而使用ABC方法时，62个肺中只有17个呈阳性。在本研究中，我们证明，对石蜡包埋组织进行PCR，然后对PCR产物进行斑点印迹杂交，在鉴定感染肺支原体的小鼠肺时，比ABC方法灵敏得多。
