National Dairy Development Board Research and Development Laboratory, IIL Campus, Hyderabad 500032, Telangana, India.
National Dairy Development Board, Anand 388001, Gujarat, India.
J Virol Methods. 2018 Jul;257:1-6. doi: 10.1016/j.jviromet.2018.03.009. Epub 2018 Mar 26.
The extended frozen semen (EFS) batches produced from infectious bovine rhinotracheitis (IBR) sero-positive cattle and buffalo bulls housed in various semen stations in India are transported to the testing laboratory in liquid nitrogen (LN) for screening bovine herpesvirus-1 (BoHV-1). This procedure is laborious and poses LN related hazards. An alternative logistics for transportation of samples was investigated. Use of Flinders Technology Associates (FTA) elute card was evaluated for transportation of extended bovine semen to screen BoHV-1 DNA by real-time PCR targeting gB gene and the method was compared with the OIE approved Chelex resin based method. A protocol for extraction of BoHV-1 DNA from FTA card spotted with extended semen was optimized. The viral DNA was found to be stable on FTA card for at least 28 days when the cards are stored at 4°-37 °C. The analytical sensitivity for the assay was determined using variable dilutions of BoHV-1 spiked semen and positive plasmid harbouring gB gene (97bp) spotted onto FTA card and it was found to be 10 TCID/ml or 100 copies respectively in real-time PCR. The test could detect as low as 10 TCID/ml or 1 copy of positive plasmid when more number of replicates (n = 6) of the same sample were tested. This sensitivity was found to be comparable to Chelex method and both the methods demonstrated a very strong correlation (r = 0.9774; 95% CI: 0.9620-0.9860) in terms of Ct value (p < 0.0001). The diagnostic sensitivity and specificity of the FTA method in comparison to the Chelex method was 83.08% (95% CI: 71.73%-91.24%) and 93.23% (95% CI: 89.38%-96.01%) respectively when 316 samples were screened by both the methods. The degree of agreement between these two tests was good (Kappa value: 0.738; 95% CI: 0.646-0.829). The method was found to be robust and highly repeatable in inter-assay and intra-assay precision testing. The result suggests that the FTA card holds promise as an alternative system for transportation of EFS for downstream screening of BoHV-1 DNA.
从印度各精液站中饲养的感染传染性牛鼻气管炎（IBR）血清阳性的牛和水牛公牛中生产的延长冷冻精液（EFS）批次，被运送到液态氮（LN）检测实验室进行牛疱疹病毒 1（BoHV-1）的筛选。该过程繁琐，且存在 LN 相关的危险。因此，研究了一种替代的样本运输方式。本研究评估了 Flinders 技术协会（FTA）洗脱卡是否可用于通过实时 PCR 针对 gB 基因筛选 BoHV-1 DNA，该方法与 OIE 批准的 Chelex 树脂法进行了比较。优化了从 FTA 卡点取的延长牛精液中提取 BoHV-1 DNA 的方案。研究发现，当 FTA 卡存放在 4°C-37°C 时，卡上的病毒 DNA 至少稳定 28 天。使用不同稀释度的 BoHV-1 接种精液和点样到 FTA 卡上的携带 gB 基因的阳性质粒（97bp）进行了分析灵敏度测定，结果表明实时 PCR 的检测下限分别为 10 TCID/ml 或 100 个拷贝。当同一样本的重复（n=6）数量增加时，该试验可以检测到低至 10 TCID/ml 或 1 个阳性质粒拷贝。该灵敏度与 Chelex 法相当，两种方法的 Ct 值相关性非常强（r=0.9774；95%CI：0.9620-0.9860；p<0.0001）。与 Chelex 法相比，FTA 法在 316 个样本中的诊断灵敏度和特异性分别为 83.08%（95%CI：71.73%-91.24%）和 93.23%（95%CI：89.38%-96.01%）。这两种方法的一致性较好（Kappa 值：0.738；95%CI：0.646-0.829）。该方法在室内和室内精密度测试中表现出稳健性和高度可重复性。结果表明，FTA 卡是一种有前途的替代系统，可用于运输 EFS，用于下游 BoHV-1 DNA 的筛选。
