Annis Seanna L, Dastoor Farahad P, Ziel Heather, Daszak Peter, Longcore Joyce E
Department of Biological Sciences, University of Maine, Orono, Maine 04469, USA.
J Wildl Dis. 2004 Jul;40(3):420-8. doi: 10.7589/0090-3558-40.3.420.
Chytridiomycosis caused by Batrachochytrium dendrobatidis (Chytridiomycota) has been implicated in declines of amphibian populations on four continents. We have developed a sensitive and specific polymerase chain reaction-based assay to detect this pathogen. We isolated B. dendrobatidis from captive and wild amphibians collected across North America and sequenced the internal transcribed spacer regions of the rDNA cassette of multiple isolates. We identified two primers (Bd1a and Bd2a) that are specific to B. dendrobatidis under amplification conditions described in this study. DNA amplification with Bd1a/Bd2a primers produced a fragment of approximately 300 bp from B. dendrobatidis DNA but not from DNA of other species of chytrids or common soil fungi. The assay detected 10 zoospores or 10 pg of DNA from B. dendrobatidis and detected infections in skin samples from a tiger salamander (Ambystoma tigrinum), boreal toads (Bufo boreas), Wyoming toads (Bufo baxteri), and smooth-sided toads (Bufo guttatus). This assay required only small samples of skin and can be used to process a large number of samples.
由蛙壶菌（壶菌门）引起的蛙壶菌病与四大洲两栖动物种群数量下降有关。我们开发了一种基于聚合酶链反应的灵敏且特异的检测方法来检测这种病原体。我们从北美各地收集的圈养和野生两栖动物中分离出蛙壶菌，并对多个分离株的核糖体DNA盒的内部转录间隔区进行了测序。我们鉴定出了两种引物（Bd1a和Bd2a），在本研究描述的扩增条件下，它们对蛙壶菌具有特异性。用Bd1a/Bd2a引物进行DNA扩增时，从蛙壶菌DNA中产生了一个约300 bp的片段，但其他壶菌物种或常见土壤真菌的DNA未产生该片段。该检测方法能检测到10个游动孢子或10 pg的蛙壶菌DNA，并能检测到虎螈（Ambystoma tigrinum）、北方蟾蜍（Bufo boreas）、怀俄明蟾蜍（Bufo baxteri）和平侧蟾蜍（Bufo guttatus）皮肤样本中的感染情况。该检测方法仅需少量皮肤样本，可用于处理大量样本。
