von Samson-Himmelstjerna G, Woidtke S, Epe C, Schnieder T
Institute of Parasitology, School of Veterinary Medicine, Hanover, Germany.
Vet Parasitol. 1997 Jan;68(1-2):119-26. doi: 10.1016/s0304-4017(96)01064-3.
Using substantial interspecific differences between the second internal transcribed spacer (ITS2) region within the rDNA gene of Dictyocaulus eckerti and Dictyocaulus viviparus a species-specific PCR was developed to distinguish between lungworm larvae of the two species from fallow deer and cattle. It was found that the method of DNA extraction was crucial for the sensitivity of the PCR. With serial dilutions of DNA extracted from 10,000 larvae the ITS2 fragment could be amplified from all dilutions down to a calculated amount of DNA equivalent to one larva. Using lower numbers of larvae, DNA from at least 100 larvae was necessary for a successful amplification. From this extraction a species-specific polymerase chain reaction (PCR) product was generated with a calculated amount of DNA equivalent to 33 larvae, whereas amplification of further diluted DNA was not successful. However, in a direct PCR single larvae could be detected after direct PCR amplification without preceding DNA extraction.
利用埃氏网尾线虫和胎生网尾线虫核糖体DNA基因内第二内部转录间隔区（ITS2）之间存在的显著种间差异，开发了一种种特异性PCR方法，以区分来自狍和牛的这两种肺线虫幼虫。结果发现，DNA提取方法对PCR的灵敏度至关重要。对从10000条幼虫中提取的DNA进行系列稀释，ITS2片段可从所有稀释度中扩增出来，直至计算得出相当于一条幼虫的DNA量。使用较少数量的幼虫时，成功扩增至少需要100条幼虫的DNA。从这种提取物中，用计算得出相当于33条幼虫的DNA量产生了种特异性聚合酶链反应（PCR）产物，而进一步稀释的DNA扩增未成功。然而，在直接PCR中，无需预先提取DNA，直接PCR扩增后即可检测到单个幼虫。
