Oidtmann Birgit, Geiger Sheila, Steinbauer Peter, Culas Annabelle, Hoffmann Rudolf W
Cefas Weymouth Laboratory, Barrack Road, Weymouth, Dorset, UK.
Dis Aquat Organ. 2006 Sep 14;72(1):53-64. doi: 10.3354/dao072053.
We present a PCR based method to detect Aphanomyces astaci in North American crayfish. Primers were designed to specifically amplify parts of the internal transcribed spacer (ITS) regions and the 5.8 rRNA gene of A. astaci. A single round and a semi-nested assay were tested for their sensitivity and specificity. Specificity of the PCR assays was tested against several closely related Aphanomyces species, other Oomycetes and some non-A. astaci DNA that might be found in or on crayfish. The single round assay was fully specific against all DNA tested. In the semi-nested assay, cross-reaction was seen when the equivalent of 40,000 or more genomic units of A. invadans or A. frigidophilus were entered into the PCR reaction. The lower detection limit of both assays lies around 1 genomic unit of A. astaci. Investigation of various parts of the exoskeleton of 3 North American crayfish species revealed that for O. limosus and P. leniusculus the telson and soft abdominal cuticle yielded a positive PCR reaction most frequently. For the third species, Procambarus clarkii, only 1 individual tested positive, so no conclusion as to preferred infestation site(s) could be drawn.
我们提出了一种基于聚合酶链式反应（PCR）的方法来检测北美小龙虾中的螯虾瘟病菌（Aphanomyces astaci）。设计了引物以特异性扩增螯虾瘟病菌的内部转录间隔区（ITS）部分和5.8 rRNA基因。对单轮PCR和半巢式PCR检测方法的灵敏度和特异性进行了测试。针对几种密切相关的丝囊霉属物种、其他卵菌纲物种以及一些可能在小龙虾体内或体表发现的非螯虾瘟病菌DNA，测试了PCR检测方法的特异性。单轮PCR检测方法对所有测试DNA均具有完全特异性。在半巢式PCR检测中，当将相当于40,000个或更多基因组单位的入侵丝囊霉（A. invadans）或嗜冷丝囊霉（A. frigidophilus）加入PCR反应时，出现了交叉反应。两种检测方法的最低检测限约为1个螯虾瘟病菌基因组单位。对三种北美小龙虾物种的外骨骼不同部位进行的调查显示，对于光滑河螯虾（O. limosus）和细足螯虾（P. leniusculus），尾节和柔软的腹部表皮最常产生阳性PCR反应。对于第三种物种，克氏原螯虾（Procambarus clarkii），只有1只个体检测呈阳性，因此无法得出关于偏好侵染部位的结论。
