Zmudzki J, Szczotka A, Podgórska K, Nowak A, Grzesiak A, Dors A, Pejsak Z
Department of Swine Diseases, National Veterinary Research Institute, Partyzantow 57, 24-100 Pulawy, Poland.
Pol J Vet Sci. 2012;15(2):267-73. doi: 10.2478/v10181-011-0144-2.
The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis and Brachyspira hyodysenteriae in porcine feces. Before the optimization process was performed two different extraction methods were compared to select the more efficient one. Based on the results achieved at this stage the boiling procedure was rejected and a commercially available silica-membrane based method was chosen for further analysis. The primers and the Taqman probe for B. hyodysenteriae and L. intracellularis were based on the sequence of NADH oxidase gene and 16S rDNA gene, respectively. The detection limit of the real-time PCR for suspension of feces inoculated with B. hyodysenteriae and L. intracellularis was determined to be 1.5x10(3) CFU/ml and 6.5x10(1) CFU/ml, respectively. The results of this study demonstrate that our real-time PCR is able to detect low number of B. hyodysenteriae and L. intracellularis cells which is satisfying in routine diagnosis of swine dysentery and proliferative enteropathy. Therefore, it is possible to identify both subclinically infected pigs and those representing an acute form of mentioned diseases. In summary, the quantitative real-time PCR is useful for routine diagnosis of L. intracellularis and B. hyodysenteriae. Compared to conventional PCR, the new validated quantification method based on real-time PCR is fast and with reduced risk of laboratory contamination. The novel technique is specific and even more sensitive than the previously used one. Furthermore, the new real-time PCR enables quick detection and quantification of both pathogens in fecal samples, which helps to estimate the health status of a pig herd.
本研究的目的是开发并验证用于定量猪粪便中胞内劳森菌和猪痢疾短螺旋体的实时荧光定量PCR方法。在进行优化过程之前，比较了两种不同的提取方法以选择更有效的方法。基于此阶段获得的结果，摒弃了煮沸法，选择了市售的基于硅胶膜的方法进行进一步分析。用于猪痢疾短螺旋体和胞内劳森菌的引物和Taqman探针分别基于烟酰胺腺嘌呤二核苷酸氧化酶基因和16S rDNA基因的序列。接种猪痢疾短螺旋体和胞内劳森菌的粪便悬液的实时荧光定量PCR检测限分别确定为1.5×10³CFU/ml和6.5×10¹CFU/ml。本研究结果表明，我们的实时荧光定量PCR能够检测到少量的猪痢疾短螺旋体和胞内劳森菌细胞，这对于猪痢疾和增生性肠炎的常规诊断是令人满意的。因此，有可能识别亚临床感染的猪和那些表现为上述疾病急性形式的猪。总之，实时荧光定量PCR可用于胞内劳森菌和猪痢疾短螺旋体的常规诊断。与传统PCR相比，基于实时荧光定量PCR的新验证定量方法快速且实验室污染风险降低。该新技术具有特异性，甚至比以前使用的方法更灵敏。此外，新的实时荧光定量PCR能够快速检测和定量粪便样本中的两种病原体，这有助于评估猪群的健康状况。
