Simultaneous Identification of Clinically Common Vibrio parahaemolyticus Serotypes Using Probe Melting Curve Analysis
Simultaneous Identification of Clinically Common Vibrio parahaemolyticus Serotypes Using Probe Melting Curve Analysis
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利用探针熔解曲线分析同时鉴定临床常见副溶血性弧菌血清型
DOI：
10.3389/fcimb.2019.00385
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发表时间：
2019-11
影响因子：
5.7
通讯作者：
Hu Qinghua
中科院分区：
文献类型：
--
作者：
Li Minxu;Jiang Yixiang;Shi Xiaolu;Li Yinghui;Jiang Min;Lin Yiman;Qiu Yaqun;Zuo Le;Deng Yinhua;Lin Zeren;Liao Yiqun;Li Qingge;Hu Qinghua
副溶血性弧菌流行病学的动态特性对疾病干预策略提出了独特的挑战。尽管疾病发病率和弧菌病暴发持续上升，以及全球出现了毒力增强的大流行克隆和血清变异体，但用于副溶血性弧菌菌株血清学分型的分子方法很少，以改善疾病监测和暴发调查。我们描述了一种基于探针熔化曲线分析(MLMA)的多重连接反应的发展，用于同时鉴定跨越10年的11个临床上最常见的副溶血性弧菌血清型。通过使用418个基因组进行广泛的序列分析，设计了针对O-和K-血清群的总共22个抗原基因靶点的特异性引物和探针。此外，还将toxR基因引入到检测中，用于副溶血性弧菌的确认。所有的基因靶标都被检测到，并给出了预期的Tm值，11个临床常见的血清型之间没有任何交叉反应，或者与其他38个血清型没有任何交叉反应。所有基因靶标的检出限为0.1~1 ng/μL，批内、批间标准偏差和变异系数分别不超过1℃和1%，具有较高的重复性。使用传统的副溶血性弧菌鉴定流程和MLMA检测流程并行进行了一项多中心双盲临床研究。从10家哨兵医院一年来收集的连续腹泻粪便标本(n=6118)中，两种工作流程共鉴定出153株副溶血性弧菌(2.5%)。用MLMA方法鉴定的血清型与传统的血清学方法完全一致(kappa=1.0)。这是第一个同时识别多个临床重要副溶血性弧菌血清型的分子检测方法，满足了对副溶血性弧菌血清型进行实用、快速和可靠鉴定的迫切需要，以便于及时检测弧菌病暴发和监测。
The dynamic nature of Vibrio parahaemolyticus epidemiology has presented a unique challenge for disease intervention strategies. Despite the continued rise of disease incidence and outbreaks of vibriosis, as well as the global emergence of pandemic clones and serovariants with enhanced virulence, there is a paucity of molecular methods for the serotyping of V. parahaemolyticus strains to improve disease surveillance and outbreak investigations. We describe the development of a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the simultaneous identification of 11 clinically most common V. parahaemolyticus serotypes spanning a 10-year period. Through extensive sequence analyses using 418 genomes, specific primers and probes were designed for a total of 22 antigen gene targets for the O- and K- serogroups. Additionally, the toxR gene was incorporated into the assay for the confirmation of V. parahaemolyticus. All gene targets were detected by the assay and gave expected Tm values, without any cross reactions between the 11 clinically common serotypes or with 38 other serotypes. The limit of identification for all gene targets ranged from 0.1 to 1 ng/μL. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 1°C and <1% respectively, indicating a highly reproducible assay. A multicenter double-blind clinical study was conducted using the traditional V. parahaemolyticus identification workflow and the MLMA assay workflow in parallel. From consecutive diarrheal stool specimens (n = 6118) collected over a year at 10 sentinel hospitals, a total of 153 V. parahaemolyticus isolates (2.5%) were identified by both workflows. A total agreement (kappa = 1.0) between the serotypes identified by the MLMA assay and conventional serological method was demonstrated. This is the first molecular assay to simultaneously identify multiple clinically important V. parahaemolyticus serotypes, which satisfies the acute need for a practical, rapid and robust identification of V. parahaemolyticus serotypes to facilitate the timely detection of vibriosis outbreaks and surveillance.
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