Elder R O, Duhamel G E, Mathiesen M R, Erickson E D, Gebhart C J, Oberst R D
Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln 68583-0905, USA.
J Vet Diagn Invest. 1997 Jul;9(3):281-6. doi: 10.1177/104063879700900309.
Proliferative enteritis, swine dysentery, and porcine salmonellosis are the most common enteric bacterial diseases affecting pigs in the growing and finishing stages of production. Currently, diagnoses of these diseases by standard cultural techniques of intestinal specimens can be laborious, time consuming, and expensive (swine dysentery, porcine salmonellosis) or impossible (proliferative enteritis). Amplification by polymerase chain reaction (PCR) of DNA sequences specific for each bacterial agent is a highly sensitive and specific method that overcomes the limitations associated with standard detection methods. A multiplex PCR (M-PCR) assay was developed for simultaneous detection and identification of the etiologic agents associated with proliferative enteritis, swine dysentery, and porcine salmonellosis in a single reaction using total DNA obtained directly from intestinal specimens. Purified DNA obtained from pure cultures of each bacterial agent alone or mixed in different combinations and concentrations and total DNA from intestinal specimens were amplified using the Lawsonia intracellularis-, Serpulina hyodysenteriae-, and salmonellae-specific M-PCR assay. Intestinal specimens consisted of feces and mucosal scrapings obtained from field cases of each disease alone or in combinations and feces obtained from pigs challenged with S. hyodysenteriae. The banding pattern of the amplified PCR products, after agarose gel electrophoresis and staining, indicated the presence of individual or combinations of etiologic agents in each specimen. Results from this study indicated that simultaneous amplification of L. intracellularis-, S. hyodysenteriae-, and salmonellae-specific DNA sequences by M-PCR can be used for specific detection and identification of three major enteric bacterial pathogens associated with proliferative enteritis, swine dysentery, and porcine salmonellosis occurring alone or in combinations. Also, the M-PCR assay can be done using DNA obtained directly from intestinal specimens submitted for diagnostic investigation.
增生性肠炎、猪痢疾和猪沙门氏菌病是影响生长育肥阶段猪群的最常见肠道细菌性疾病。目前，通过肠道标本的标准培养技术诊断这些疾病可能费力、耗时且昂贵（猪痢疾、猪沙门氏菌病），或者无法诊断（增生性肠炎）。通过聚合酶链反应（PCR）扩增每种细菌病原体特异的DNA序列是一种高度灵敏且特异的方法，克服了与标准检测方法相关的局限性。开发了一种多重PCR（M-PCR）检测方法，用于使用直接从肠道标本获得的总DNA在单个反应中同时检测和鉴定与增生性肠炎、猪痢疾和猪沙门氏菌病相关的病原体。使用仅由每种细菌病原体的纯培养物或以不同组合和浓度混合得到的纯化DNA以及来自肠道标本的总DNA，通过胞内劳森菌、猪痢疾蛇形螺旋体和沙门氏菌特异的M-PCR检测方法进行扩增。肠道标本包括单独或组合取自每种疾病现场病例的粪便和黏膜刮片，以及用猪痢疾蛇形螺旋体攻毒的猪的粪便。琼脂糖凝胶电泳和染色后，扩增的PCR产物的条带模式表明每个标本中存在单个或组合的病原体。本研究结果表明，通过M-PCR同时扩增胞内劳森菌、猪痢疾蛇形螺旋体和沙门氏菌特异的DNA序列可用于特异性检测和鉴定单独或组合发生的与增生性肠炎、猪痢疾和猪沙门氏菌病相关的三种主要肠道细菌病原体。此外，M-PCR检测可使用直接从提交用于诊断调查的肠道标本获得的DNA进行。
