实时 RT-PCR 检测和定量分析马鼻炎病毒。

Real-time RT-PCR for the detection and quantitative analysis of equine rhinitis viruses.

机构信息

Virology Unit, The Irish Equine Centre, Johnstown, Naas, Co. Kildare, Ireland.

出版信息

Equine Vet J. 2010 Mar;42(2):98-104. doi: 10.2746/042516409X479559.

DOI:10.2746/042516409X479559

PMID:20156243

Abstract

REASONS FOR PERFORMING STUDY

Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection.

OBJECTIVES

To develop a sensitive, rapid, real-time RT-PCR (rRT-PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV.

METHODS

TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5' UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses were employed for validation of the assays. ERAV samples positive by rRT-PCR were verified by virus isolation and ERBV positive samples were verified by rRT-PCR using a different set of primers.

RESULTS

The detection limit of the rRT-PCR for both viruses was 10-100 genome copies. Of 250 archival nasal swabs submitted for diagnostic testing over a 7 year period, 29 were ERAV positive and 3 were ERBV positive with an average incidence rate per year of 10 and 1.5%, respectively. There was evidence of co-circulation of ERAV and ERBV with equine influenza virus (EIV). Of 100 post race urine samples tested, 29 were ERAV positive by rRT-PCR. Partial sequencing of 2 ERBV positive samples demonstrated that one was 100% identical to ERBV1 from a 270 bp sequence and the other was more closely related to ERBV2 than ERBV1 (95% compared to 90% nucleotide identity in 178 bp).

CONCLUSIONS

The rRT-PCR assays described here are specific and more sensitive than virus isolation. They have good reproducibility and are suitable for the routine diagnosis of ERAV and ERBV.

POTENTIAL RELEVANCE

These assays should be useful for investigating the temporal association between clinical signs and rhinitis virus shedding.

摘要

研究目的

马传染性鼻炎病毒（ERV）可引起马的呼吸道疾病和生产性能下降。由于检测方法不敏感，这些病毒的经济意义可能被低估。

目的

开发一种敏感、快速的实时 RT-PCR（rRT-PCR）检测方法，适用于 ERV 的 A 和 B 变异株的常规诊断和流行病学监测。

方法

根据 ERV 基因组 5'UTR 的保守区设计了 ERAV 和 ERBV 的 TaqMan 引物探针。使用 400 多个来自临床感染和无症状马的样本验证了该方法。rRT-PCR 阳性的 ERAV 样本通过病毒分离验证，rRT-PCR 阳性的 ERBV 样本通过使用不同引物的 rRT-PCR 验证。

结果

两种病毒的 rRT-PCR 检测限均为 10-100 基因组拷贝。在 7 年期间提交进行诊断检测的 250 份存档鼻拭子中，29 份 ERAV 阳性，3 份 ERBV 阳性，每年的平均发病率分别为 10%和 1.5%。ERAV 和 ERBV 与马流感病毒（EIV）有共同循环。在 100 份赛后尿液样本中，29 份 rRT-PCR 检测 ERAV 阳性。对 2 份 ERBV 阳性样本的部分测序表明，其中 1 份与 270bp 序列的 ERBV1 完全相同，另 1 份与 ERBV2 的亲缘关系比 ERBV1 更密切（178bp 中有 95%与 90%的核苷酸相同）。