Abstract：
The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe hybridization in the presence of carrier DNA. It is based on the transfer of a known amount of DNA onto an inert solid support, such as a nylon membrane, utilizing the dot-blot apparatus and without electrophoretic separation. Nylon membranes have the advantage of high nucleic acid binding capacity (400 µg/cm2), high strength, and are positively or neutrally charged. The probe used is a highly specific ssDNA fragment of 18 to 20 bases long labeled with digoxigenin (DIG). The probe will conjugate with the Leptospira DNA. Once the probe has hybridized with the target DNA, it is detected by an anti-digoxigenin antibody, allowing its easy detection through its emissions revealed in an X-ray film. The dots with an emission will correspond to the DNA fragments of interest. This method employs the non-isotopic labeling of the probe, which may have a very long half-life. The drawback of this standard immuno-label is a lower sensitivity than isotopic probes. Nevertheless, it is mitigated by coupling polymerase chain reaction (PCR) and dot-blot assays. This approach enables the enrichment of the target sequence and its detection. Additionally, it may be used as a quantitative application when compared against a serial dilution of a well-known standard. A dot-blot application to detect Leptospira from the three main clades in water samples is presented here. This methodology can be applied to large amounts of water once they have been concentrated by centrifugation to provide evidence of the presence of Leptospiral DNA. This is a valuable and satisfactory tool for general screening purposes, and may be used for other non-culturable bacteria that may be present in water, enhancing the comprehension of the ecosystem.
摘要：
斑点印迹是一个简单的，快,敏感,和多功能技术，能够在载体DNA存在的情况下鉴定最小量的探针杂交特异性靶向的DNA。它基于将已知量的DNA转移到惰性固体支持物上，比如尼龙膜，利用斑点印迹装置，无需电泳分离。尼龙膜具有高核酸结合能力（400µg/cm2）的优势，高强度,并且带正电荷或中性电荷。使用的探针是用地高辛(DIG)标记的18至20个碱基长的高度特异性ssDNA片段。探针将与钩端螺旋体DNA缀合。一旦探针与靶DNA杂交，它通过抗洋地黄毒苷抗体检测到，通过X射线胶片中显示的发射，可以很容易地检测到它。具有发射的点将对应于感兴趣的DNA片段。该方法采用探针的非同位素标记，可能有很长的半衰期。这种标准免疫标记的缺点是灵敏度低于同位素探针。然而，它通过偶联聚合酶链反应(PCR)和斑点印迹分析得到缓解.该方法使得能够富集靶序列及其检测。此外,当与已知标准的连续稀释进行比较时,其可用作定量应用。此处介绍了用于检测水样中三个主要进化枝的钩端螺旋体的斑点印迹应用。一旦通过离心浓缩了大量的水，就可以将该方法应用于大量的水，以提供存在钩端螺旋体DNA的证据。这是一个有价值的和令人满意的工具，用于一般的筛选目的，并可用于其他可能存在于水中的不可培养细菌，增强对生态系统的理解。
