Vydelingum S, Tao T, Balazsi K, Hecker R
Pasteur Mérieux Connaught, North York, Ontario, Canada.
Rev Sci Tech. 1998 Dec;17(3):674-81. doi: 10.20506/rst.17.3.1122.
The authors evaluated the ability of a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect classical swine fever virus (CSFV) in comparison with virus isolation and detection by an indirect immunoperoxidase assay (VI-IPA). To determine the specificity of the assay, samples from 60 spleens, 45 tonsils, ten submandibular lymph nodes, eight mesenteric lymph nodes and four kidneys, collected from pigs of various ages which had been slaughtered in abattoirs in Canada (a population free from CSFV), were tested. All the samples tested gave negative results by both VI-IPA and RT-PCR. A total of 20 samples were passaged in porcine kidney (PK) 15 cells and retested by both assays. All were found to be negative, giving a specificity of 100%. To determine the analytical sensitivity of the assay, a similar comparative study was conducted, using CSFV grown in tissue culture and tonsil tissues from a CSFV-infected pig. For both infected tissues and tissue culture fluids, RT-PCR was ten times more sensitive than VI-IPA. Amounts as small as 0.6 infectious units per 100 mg of tissue were detected by RT-PCR, compared to 6 infectious units by VI-IPA. Similarly, RT-PCR could detect as little as 0.1 infectious unit per ml in tissue culture fluids, compared to one infectious unit per ml by VI-IPA. To determine diagnostic sensitivity, three coded panels (two internal and one external), comprising 45 samples from 14 pigs, were tested. The diagnostic sensitivity of both RT-PCR and VI-IPA was found to be 100% for both internal panels. The results of the external panel, apart from two samples that were missed by both RT-PCR and VI-IPA, were found to be in total agreement. These two samples remained negative after amplification in PK15 cells. All the RT-PCR results were based on a single test whereas, for the VI-IPA results, positive results were obtained for five samples only after an amplification round in PK15 cells. Application of the RT-PCR assay for the diagnosis of CSFV would enable improved detection of the virus in a shorter time period.
作者评估了逆转录-聚合酶链反应（RT-PCR）检测经典猪瘟病毒（CSFV）的能力，并与病毒分离及间接免疫过氧化物酶检测法（VI-IPA）进行了比较。为确定该检测方法的特异性，对从加拿大屠宰场（无CSFV的猪群）不同年龄段猪采集的60份脾脏、45份扁桃体、10份下颌下淋巴结、8份肠系膜淋巴结和4份肾脏样本进行了检测。所有检测样本经VI-IPA和RT-PCR检测均呈阴性。总共20份样本在猪肾（PK）15细胞中传代，并再次用两种检测方法进行检测。所有样本均为阴性，特异性为100%。为确定该检测方法的分析灵敏度，使用在组织培养中培养的CSFV和来自感染CSFV猪的扁桃体组织进行了类似的比较研究。对于感染组织和组织培养液，RT-PCR的灵敏度比VI-IPA高10倍。RT-PCR可检测到每100毫克组织中低至0.6个感染单位，而VI-IPA为6个感染单位。同样，RT-PCR在组织培养液中可检测到低至每毫升0.1个感染单位，而VI-IPA为每毫升1个感染单位。为确定诊断灵敏度，对三个编码样本组（两个内部样本组和一个外部样本组）进行了检测，这些样本组包括来自14头猪的45份样本。发现RT-PCR和VI-IPA对两个内部样本组的诊断灵敏度均为100%。除了RT-PCR和VI-IPA均遗漏的两个样本外，外部样本组的结果完全一致。这两个样本在PK15细胞中扩增后仍为阴性。所有RT-PCR结果均基于单次检测，而对于VI-IPA结果，仅在PK15细胞中进行一轮扩增后，五个样本才获得阳性结果。应用RT-PCR检测方法诊断CSFV能够在更短时间内实现对病毒的更好检测。
