Crawford T B, Li H, O'Toole D
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman 99164, USA.
J Vet Diagn Invest. 1999 Mar;11(2):111-6. doi: 10.1177/104063879901100201.
A previously described polymerase chain reaction (PCR) assay (amplification of a 238-bp fragment of ovine herpesvirus 2 [OHV-2] genomic DNA) for diagnosis of sheep-associated malignant catarrhal fever (MCF) was adapted for use on formalin-fixed, paraffin-embedded tissues. Variables affecting its use were examined. Archived tissues from cattle, white-tailed deer (Odocoileus virginianus), and bison (Bison bison) diagnosed with MCF by clinical signs or histologic lesions were obtained from 2 veterinary diagnostic laboratories. Tissues from healthy animals and from animals diagnosed with other common bovine viral diseases were examined as controls. A total of 86 blocks from 37 suspect MCF cases were examined. Forty-one blocks from healthy animals and animals with unrelated viral diseases were examined as controls. The assay was specific for sheep-associated MCF and did not yield false-positive signals from healthy animals or from cases of infectious bovine rhinotracheitis, bovine virus diarrhea, mucosal disease, or parainfluenza-3 virus infection. A wide variety of tissues were suitable substrates, including spleen, lymph node, intestine, brain, lung, and kidney. Extracted DNA provided a more suitable target than did unextracted tissue lysate. The highest levels of viral DNA were present in lymphoid organs and intestine, but the data indicate that in acute clinical cases, most organs contain sufficient viral DNA to serve as a suitable diagnostic specimen. Fixation of 0.5-cm3 blocks of tissue in 10% neutral buffered formalin was deleterious to the target DNA, and PCR signals progressively diminished after fixation for >45 days. Detection of genomic DNA of OHV-2 by PCR was successful for archived tissues that were 15 years old.
一种先前描述的用于诊断绵羊相关恶性卡他热（MCF）的聚合酶链反应（PCR）检测方法（扩增绵羊疱疹病毒2 [OHV-2]基因组DNA的238 bp片段）被改编用于福尔马林固定、石蜡包埋组织。研究了影响其使用的变量。从2个兽医诊断实验室获得了通过临床症状或组织学病变诊断为MCF的牛、白尾鹿（弗吉尼亚鹿）和野牛的存档组织。检查了来自健康动物和诊断为其他常见牛病毒性疾病的动物的组织作为对照。共检查了来自37例疑似MCF病例的86个组织块。检查了来自健康动物和患有无关病毒性疾病的动物的41个组织块作为对照。该检测方法对绵羊相关MCF具有特异性，未从健康动物或传染性牛鼻气管炎、牛病毒性腹泻、黏膜病或副流感3病毒感染病例中产生假阳性信号。多种组织都是合适的底物，包括脾脏、淋巴结、肠道、大脑、肺和肾脏。提取的DNA比未提取的组织裂解物提供了更合适的靶标。病毒DNA水平最高的是淋巴器官和肠道，但数据表明，在急性临床病例中，大多数器官都含有足够的病毒DNA作为合适的诊断标本。将0.5 cm³组织块固定在10%中性缓冲福尔马林中对靶标DNA有害，固定超过45天后PCR信号逐渐减弱。通过PCR检测OHV-2基因组DNA在15年的存档组织中是成功的。
