Detection of Canine Distemper Virus Nucleoprotein RNA by Reverse Transcription-PCR Using Serum, Whole Blood, and Cerebrospinal Fluid from Dogs with Distemper

Reverse transcription-PCR (RT-PCR) was used to detect canine distemper virus (CDV) nucleoprotein (NP) RNA in serum, whole blood, and cerebrospinal fluid (CSF) samples from 38 dogs with clinically suspected distemper. Results were correlated to clinical findings, anti-CDV neutralizing antibody titers, postmortem findings, and demonstration of CDV NP antigen by immunohistochemistry. The specificity of the RT-PCR was ensured by amplification of RNA from various laboratory CDV strains, restriction enzyme digestion, and Southern blot hybridization. In 29 of 38 dogs, CDV infection was confirmed by postmortem examination and immunohistochemistry. The animals displayed the catarrhal, systemic, and nervous forms of distemper. Seventeen samples (serum, whole blood, or CSF) from dogs with distemper were tested with three sets of primers targeted to different regions of the NP gene of the CDV Onderstepoort strain. Expected amplicons were observed in 82, 53, and 41% of the 17 samples, depending upon the primer pair used. With the most sensitive primer pair (primer pair I), CDV NP RNA was detected in 25 of 29 (86%) serum samples and 14 of 16 (88%) whole blood and CSF samples from dogs with distemper but not in body fluids from immunohistochemically negative dogs. Nucleotide sequence analysis of five RT-PCR amplicons from isolates from the field revealed few silent point mutations. These isolates exhibited greater homology to the Rockborn (97 to 99%) than to the Onderstepoort (95 to 96%) CDV strain. In summary, although the sensitivity of the RT-PCR for detection of CDV is strongly influenced by the location of the selected primers, this nucleic acid detection system represents a highly specific and sensitive method for the antemortem diagnosis of distemper in dogs, regardless of the form of distemper, humoral immune response, and viral antigen distribution.

逆转录聚合酶链反应（Reverse transcription-PCR, RT-PCR）被用于检测38只临床疑似犬瘟热的犬只的血清、全血及脑脊液（cerebrospinal fluid, CSF）样本中的犬瘟热病毒（canine distemper virus, CDV）核蛋白（nucleoprotein, NP）RNA。将上述检测结果与临床症状、抗CDV中和抗体效价、死后剖检结果以及免疫组化法（immunohistochemistry）检测到的CDV NP抗原进行关联分析。本RT-PCR方法的特异性通过对多种实验室CDV毒株的RNA进行扩增、限制性酶切（restriction enzyme digestion）以及Southern印迹杂交（Southern blot hybridization）得以验证。38只受试犬中，有29只经死后剖检和免疫组化确认感染CDV，这些受试犬表现出卡他性、全身性与神经性三种犬瘟热感染类型。针对CDV Onderstepoort株NP基因的不同区域设计了三对引物，对17份来自犬瘟热患犬的样本（血清、全血或脑脊液）进行检测，结果显示：17份样本中，根据所用引物对的不同，可检出预期扩增子（amplicon）的比例分别为82%、53%与41%。使用灵敏度最高的引物对（引物对I）时，在29只确诊CDV感染的患犬中，25份（86%）血清样本、16份全血与脑脊液样本中的14份（88%）可检测到CDV NP RNA，而免疫组化结果为阴性的犬只的体液样本则未检出该RNA。对5份来自野外分离株的RT-PCR扩增产物进行核苷酸序列分析后发现，仅存在少量沉默点突变；这些野外分离株与Rockborn株的同源性为97%~99%，高于与Onderstepoort株的同源性（95%~96%）。综上所述，尽管RT-PCR检测CDV的敏感性受所选引物的位置显著影响，但该核酸检测系统仍是一种高特异性、高灵敏度的犬瘟热生前诊断方法，且不受犬瘟热感染类型、体液免疫应答以及病毒抗原分布的限制。