当前位置: X-MOL 学术Microbiologyopen › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Novel multiplex TaqMan assay for differentiation of the four major pathogenic Brachyspira species in swine
MicrobiologyOpen ( IF 3.4 ) Pub Date : 2021-02-16 , DOI: 10.1002/mbo3.1169
Simone Scherrer 1 , Roger Stephan 1
Affiliation  

A novel TaqMan 5‐plex real‐time PCR using a combination of locked nucleic acid‐modified (LNA)‐ and minor groove binding (MGB)‐conjugated DNA probes was developed for identification and differentiation between the four main pathogenic Brachyspira species in swine. B. hyodysenteriae, B. pilosicoli, and B. suanatina are identified using three hydrolysis probes targeting cpn60, while B. hampsonii is recognized by another nox specific probe. The assay also includes an exogenous internal control simultaneously verifying the PCR competency of the DNA samples. Validation of the novel assay was performed using DNA samples from 18 Brachyspira reference strains and 477 clinical samples obtained from porcine rectal swabs by comparing them with different PCR‐based methods targeting nox, 16S rDNA, and 23S rDNA. The specificity of the assay was 100% without cross‐reactivity or detection of different pathogens. Depending on the Brachyspira species, the limit of detection was between 10 and 20 genome equivalents with a cut‐off threshold cycle (Ct) value of 37. The developed highly sensitive and specific 5‐plex real‐time PCR assay is easy to implement in routine veterinary diagnostic laboratories and enables rapid differentiation between the main four pathogenic Brachyspira species recognized in pigs using a single‐tube approach.

中文翻译:

用于区分猪四种主要致病性短螺旋体物种的新型多重 TaqMan 检测

使用锁核酸修饰 (LNA) 和小沟结合 (MGB) 结合 DNA 探针的组合开发了一种新型 TaqMan 5 重实时 PCR,用于鉴定和区分猪的四种主要致病性短螺旋体物种。 猪痢疾杆菌B .  pilosicoli,和 suanatina使用三种针对cpn60 的水解探针进行鉴定,而B .  hampsonii被另一个nox认出特异探针。该测定还包括同时验证 DNA 样品的 PCR 能力的外源内部对照。使用来自 18 个短螺旋体参考菌株的DNA 样本和从猪直肠拭子获得的 477 个临床样本,通过将它们与针对nox、16S rDNA 和 23S rDNA 的不同基于 PCR 的方法进行比较,对新测定进行了验证。该测定的特异性为 100%,没有交叉反应或检测到不同的病原体。取决于短螺旋体物种,检测限在 10 到 20 个基因组当量之间,截止阈值循环 (Ct) 值为 37。 开发的高度灵敏和特异的 5 重实时 PCR 检测易于在常规兽医诊断实验室中实施并能够使用单管方法在猪中识别的主要四种致病性短螺旋体物种之间进行快速区分。
更新日期:2021-02-17
down
wechat
bug