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用于临床样本中猪支原体定量检测的首个LightCycler实时荧光定量PCR检测方法。

First LightCycler real-time PCR assay for the quantitative detection of Mycoplasma suis in clinical samples.

作者信息

Hoelzle Ludwig E, Helbling Marianne, Hoelzle Katharina, Ritzmann Mathias, Heinritzi Karl, Wittenbrink Max M

机构信息

Institute of Veterinary Bacteriology, Vetsuisse Faculty, University Zurich, Winterthurerstr. 270, 8057 Zürich, Switzerland.

出版信息

J Microbiol Methods. 2007 Aug;70(2):346-54. doi: 10.1016/j.mimet.2007.05.009. Epub 2007 May 29.

Abstract

Mycoplasma suis cannot be cultivated in vitro. Therefore, PCR-based methods are irreplaceable for the diagnosis of M. suis infections especially when clinical symptoms are not evident. Currently, no easy and reliable method allowing the quantitative detection of M. suis is available. This report describes the development of a quantitative LightCycler PCR assay based on the msg1 gene of M. suis (LC MSG1 PCR). No PCR signals were obtained with closely related haemotrophic and non-haemotrophic mycoplasmas, with other bacteria, and with M. suis-free blood and tissue arguing for a high analytical specificity. Test sensitivity was found to be 100%, and test specificity 96.7%. To test the diagnostic suitability of the LC MSG1 PCR, 25 pigs with clinical porcine eperythrozoonosis and 25 healthy pigs were investigated. All ill pigs revealed a positive real-time PCR result whereas only one healthy pig was detected to be M. suis-infected. M. suis was quantitatively detected in 19 blood specimens of 100 sows from Switzerland and in 17 of 160 post-weaning piglets from Germany. In conclusion, this new LC MSG1 PCR assay represents a powerful tool for the improvement of the current M. suis diagnosis and for prevalence and pathogenesis studies.

摘要

猪支原体不能在体外培养。因此,基于聚合酶链反应(PCR)的方法在诊断猪支原体感染方面具有不可替代的作用,尤其是在临床症状不明显时。目前,尚无简便可靠的方法可用于猪支原体的定量检测。本报告描述了一种基于猪支原体msg1基因的定量LightCycler PCR检测方法(LC MSG1 PCR)的开发。与密切相关的血营养型和非血营养型支原体、其他细菌以及不含猪支原体的血液和组织进行检测时,均未获得PCR信号,这表明该方法具有较高的分析特异性。检测灵敏度为100%,检测特异性为96.7%。为测试LC MSG1 PCR的诊断适用性,对25头患有临床猪附红细胞体病的猪和25头健康猪进行了调查。所有患病猪的实时PCR结果均为阳性,而仅检测到1头健康猪感染了猪支原体。在来自瑞士的100头母猪的19份血液样本以及来自德国的160头断奶后仔猪的17份样本中定量检测到了猪支原体。总之,这种新的LC MSG1 PCR检测方法是改进当前猪支原体诊断以及进行流行率和发病机制研究的有力工具。

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