Mycology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208, USA.
Mycopathologia. 2011 Oct;172(4):247-56. doi: 10.1007/s11046-011-9435-5. Epub 2011 Jun 26.
Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS-PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10(-1)-5 × 10(7)), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)-PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.
嗜角质真菌（Geomyces destructans）是蝙蝠地霉病（俗称“白鼻综合征”）的病原体。这种感染已导致小褐蝙蝠（Myotis lucifugus）严重发病和死亡，也已传播到其他蝙蝠物种，导致其数量显著下降。目前，通过培养、ITS-PCR 和组织病理学来鉴定嗜角质真菌（Geomyces destructans）的感染。我们假设实时 PCR 检测可以大大提高蝙蝠中嗜角质真菌（Geomyces destructans）的检测率。α-L-鼠李糖苷酶基因的 100bp 序列被验证为实时 PCR 的靶序列。通过从嗜角质真菌（Geomyces destructans）分生孢子提取的 DNA 进行连续稀释，确定了该检测方法的灵敏度（5×10(-1)-5×10(7)），并用 30 种密切相关和远缘相关的真菌和 5 种常见细菌病原体的 DNA 进行了特异性测试。该实时 PCR 检测方法具有高度的敏感性，在 40 个 PCR 循环时，每个反应的检测限为 2 个嗜角质真菌（Geomyces destructans）分生孢子。该检测方法也具有高度的特异性，因为在实时 PCR 检测中，没有其他真菌或细菌 DNA 发生交叉反应。用 147 份疑似感染嗜角质真菌（Geomyces destructans）的蝙蝠组织样本对实时 PCR 检测与用于检测嗜角质真菌（Geomyces destructans）的其他方法进行了比较。实时 PCR 检测对 80 份（55%）样本的嗜角质真菌（Geomyces destructans）DNA 检测呈阳性，具有高度的敏感性。相比之下，组织病理学检查显示 64/147（44%）阳性样本。ITS-PCR 从 37 份组织样本中获得了嗜角质真菌（Geomyces destructans）的阳性扩增子（25%）。最不敏感的检测方法是真菌培养，只有 17 份（12%）组织样本在培养中产生了嗜角质真菌（Geomyces destructans）。数据表明，实时 PCR 检测非常有希望用于快速、敏感、特异性地鉴定嗜角质真菌（Geomyces destructans）。建议进一步进行该新型检测方法的试验和实验室间比较，以提高蝙蝠地霉病的诊断水平。
