Departamento de Microbiología, Instituto de Análisis Químico y Biológico (IAQBUS), Universidad de Santiago de Compostela, 15782, Santiago de Compostela, Spain.
Appl Microbiol Biotechnol. 2020 Jul;104(14):6211-6222. doi: 10.1007/s00253-020-10683-z. Epub 2020 May 21.
This paper describes the predicted structure for the cps loci involved in capsule biosynthesis for Streptococcus parauberis serotypes III, IV, and V. Based on the specific serotype regions I, II, and III, a multiplex PCR protocol (mPCR) was designed to differentiate the main serotypes causing fish diseases. A real-time PCR method (qPCR) is also described to identify S. parauberis of serotype III in bacterial cultures and fish tissues. In silico and in vitro analyses revealed that both methods have a 100% specificity. The mPCR assay was optimized for the detection of S. parauberis strains of subtypes Ia (amplicon size 213 bp), subtypes Ib and Ic (both amplicon size 303 bp), serotype II (amplicon size 403 bp), and serotype III (amplicon size 130 bp) from bacterial cultures. The qPCR assay was optimized for the identification and quantification of S. parauberis serotype III strains in bacterial cultures and fish tissues. This assay achieved a sensitivity of 2.67 × 10 gene copies (equivalent to 3.8 × 10 ng/μl) using pure bacterial cultures of S. parauberis serotype III and 1.76 × 10 gene copies in fish tissues experimentally and naturally infected with S. parauberis of the serotype III. The specificity and sensitivity of the protocols described in this study suggest that these methods could be used for diagnostic and/or epidemiological purposes in clinical diagnostic laboratories. KEY POINTS: • Structure of loci cps for S. parauberis of serotypes III, IV and V was described. • mPCR to differentiate S. parauberis serotypes causing disease in fish was optimized. • qPCR assay to quantify strains of S. parauberis serotype III in fish tissues.
本文描述了与猪链球菌血清型 III、IV 和 V 相关荚膜生物合成的 cps 基因座的预测结构。基于特定的血清型区域 I、II 和 III，设计了一种多重 PCR 协议（mPCR）来区分引起鱼类疾病的主要血清型。还描述了一种实时 PCR 方法（qPCR）来鉴定细菌培养物和鱼类组织中的血清型 III 的 S. parauberis。计算机分析和体外分析表明，这两种方法均具有 100%的特异性。mPCR 检测优化用于检测细菌培养物中的 S. parauberis 亚型 Ia（扩增子大小 213 bp）、亚型 Ib 和 Ic（均扩增子大小 303 bp）、血清型 II（扩增子大小 403 bp）和血清型 III（扩增子大小 130 bp）菌株。qPCR 检测优化用于鉴定和定量细菌培养物和鱼类组织中的 S. parauberis 血清型 III 菌株。该检测法在使用纯细菌培养物的情况下，对 S. parauberis 血清型 III 的灵敏度达到 2.67×10 基因拷贝（相当于 3.8×10 ng/μl），在经实验和自然感染 S. parauberis 血清型 III 的鱼类组织中灵敏度达到 1.76×10 基因拷贝。本研究中描述的协议的特异性和灵敏度表明，这些方法可用于临床诊断实验室的诊断和/或流行病学目的。关键点：• 描述了 S. parauberis 血清型 III、IV 和 V 的 cps 基因座结构。• 优化了用于区分引起鱼类疾病的 S. parauberis 血清型的 mPCR。• qPCR 检测法用于定量鱼类组织中 S. parauberis 血清型 III 的菌株。
