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Development of a real-time PCR assay for detection and quantification of Streptococcus iniae using the lactate permease gene.
Journal of Fish Diseases ( IF 2.5 ) Pub Date : 2020-09-21 , DOI: 10.1111/jfd.13267
Yolanda Torres-Corral 1 , Ysabel Santos 1
Affiliation  

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 101 amplicon copies per assay (equivalent to 2 × 10–9 ng/µl) using bacterial DNA and of 1.44 × 101 gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.

中文翻译:

使用乳酸通透酶基因开发实时荧光定量PCR检测和定量检测链球菌的方法。

这项研究的目的是开发和评估一种快速,准确的基于定量PCR(qPCR)的方案,用于检测病鱼细菌培养和组织中的人畜共患病原体链球菌。为此,基于比较基因组分析,使用从NCBI基因组数据库检索到的45个序列,选择乳酸通透酶编码(lldY)基因作为S. iniae特异性引物设计的目标。使用115种感染链球菌的细菌菌株和鱼组织测试了这些引物的特异性和适用性。还确定了qPCR测定的灵敏度,可重复性和效率。发达的qPCR检测结果表明,其对纯细菌培养物或从海豚链球菌或感染该细菌的鱼组织中提取的DNA具有100%的特异性。该方法灵敏度高, 使用细菌DNA的每次检测检出限为1.12×10 1个扩增子拷贝(相当于2×10 –9 ng /μl),感染海豚链球菌的鱼组织的检出限为1.44×10 1个基因拷贝。总之,此qPCR协议为准确鉴定海豚链球菌及其在鱼组织上的检测提供了一种准确而敏感的替代方法,可以将其作为微生物实验室中的常规工具来实施。
更新日期:2020-09-21
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