可视化牛传染性鼻气管炎病毒快速检测

A visual rapid detection method for bovine infectious rhinotracheitis virus

摘要:

【背景】牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus, IBRV)是牛常见的易感病原体，给畜牧业带来了重大经济损失，亟须建立一种快速检测IBRV的方法。【目的】建立一种重组酶聚合酶扩增(recombinase polymerase amplification, RPA)和微流控芯片技术相结合的IBRV可视化快速检测方法。【方法】实验选择IBRV的ul52基因作为分子检测的目标片段。选择伪狂犬病毒、火鸡疱疹病毒、鸭瘟疱疹病毒作为对照组。通过不同稀释倍数IBRV的DNA提取液确定灵敏度。通过对34份疑似感染IBRV急性期病牛的血清和鼻拭子样本病原分离株的检测来分析本方法的适用性。【结果】ul52基因扩增子大小为403 bp，扩增子测序结果显示，得到的目标片段核酸序列与IBRV的ul52基因(NC_063268.1)相似性达100%，对照组未见扩增子，检测限为6.9×10³ copies/µL。对34份疑似感染IBRV急性期病牛血清和鼻拭子样本病原分离株的检测结果分析发现，编号分别为SD0213、SD0518和SD0701的牛感染了IBRV，血清和鼻拭子样本病原分离株检测结果一致。另外，从样本DNA提取到获得检测结果的全流程时长为110 min。【结论】本研究将RPA、磁性探针捕获和微流控技术相结合，建立了一种IBRV病毒的快速可视化分子检测方法，该方法具有良好的特异性和灵敏度。

Abstract:

[Background] Bovine infectious rhinotracheitis virus (IBRV) represents a common pathogenic threat to cattle, causing substantial economic losses in the livestock industry. Therefore, it is urgent to establish a rapid detection method for IBRV. [Objective] To establish a visual rapid detection method for IBRV by leveraging recombinase polymerase amplification (RPA) in conjunction with microfluidic chip. [Methods] The ul52gene of IBRV was selected as the target fragment for molecular detection. Pseudorabies virus, herpesvirus of turkey, and duck plague herpesvirus were selected as the controls. The DNA extract dilutions of IBRV were used to evaluate the sensitivity of the established method. Thirty-four serum and nasal swab specimens from the cattle exhibiting symptoms of acute IBRV infection were used to evaluate the applicability of the established method. [Results] The size of ul52 gene amplicon was 403 bp. Sequencing results confirmed the amplicon as the ul52 gene (NC_063268.1) of IBRV, with the homology of 100%, and no amplification was observed in the control groups. The limit of detection of the established method was 6.9×10³ copies/µL. Furthermore, the established method detected IBRV in cattle with the identification number of SD0213, SD0518, and SD0701, which were consistent with the strain isolation results from both serum and nasal swab specimens. The entire process, from sample preparation to result acquisition, required 110 min. [Conclusion] We successfully developed a visual rapid molecular detection method for IBRV by combining RPA, magnetic probe capture, and microfluidic chip. The established method demonstrates good specificity and sensitivity.