Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars
Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars
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DOI：
10.1186/s12941-019-0338-5
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发表时间：
2019-12-05
影响因子：
5.7
通讯作者：
Hu, Qinghua
中科院分区：
文献类型：
--
作者：
Zuo, Le;Jiang, Min;Hu, Qinghua
背景资料：虽然沙门氏菌血清分型对于沙门氏菌病的疾病干预至关重要，但快速且易于操作的分子血清分型解决方案仍然难以捉摸。我们建立了一种基于探针熔解曲线分析（MLMA）的多重连接反应，用于30种常见沙门氏菌血清型的鉴定。方法：根据5868株沙门氏菌基因组中编码体细胞抗原的基因（wzx和wzy）和鞭毛抗原的基因（fliC和fljB），设计血清型特异性引物和探针。ssaR基因，一个III型分泌系统的组成部分，被列入沙门氏菌的确认。在30个血清型（n = 211）之间或与另外120个血清型（n = 120）和其他肠杆菌科（n = 3）之间未证实交叉反应。所有靶标的鉴定限范围为1.2 ng/μ L至1.56 ng/μ L DNA。批内和批间标准偏差和变异系数分别不超过0.5 ℃和小于1%，表明重现性高。我们从中国深圳11家哨点医院10个月内收集的连续门诊粪便样本（n = 3590）中，并行使用传统沙门氏菌鉴定工作流程和MLMA检测工作流程进行了一项多中心研究。沙门氏菌分离株（n = 496，13.8%）由两种工作流程得出，完全一致MLMA检测与常规血清学分型之间的差异（kappa = 1.0）得到证实。测定时间为2.5小时，这种简单的测定法显示出提供快速和高-为临床和公共卫生实验室提供沙门氏菌血清型的通量鉴定，以促进沙门氏菌病的及时监测。
Background: While Salmonella serotyping is of paramount importance for the disease intervention of salmonellosis, a fast and easy-to-operate molecular serotyping solution remains elusive. We have developed a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the identification of 30 common Salmonella serovars.Methods: Serovar-specific primers and probes were designed based on a comparison of gene targets (wzx and wzy encoding for somatic antigen biosynthesis; fliC and fljB for flagellar antigens) from 5868 Salmonella genomes. The ssaR gene, a type III secretion system component, was included for the confirmation of Salmonella.Results: All gene targets were detected and gave expected Tm values during assay evaluation. Cross reactions were not demonstrated between the 30 serovars (n = 211), or with an additional 120 serovars (n = 120) and other Enterobacteriaceae (n = 3). The limit of identification for all targets ranged from using 1.2 ng/mu L to 1.56 ng/mu L of DNA. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 0.5 degrees C and less than 1% respectively, indicating high reproducibility. From consecutive outpatient stool samples (n = 3590) collected over a 10-month period at 11 sentinel hospitals in Shenzhen, China, we conducted a multicenter study using the traditional Salmonella identification workflow and the MLMA assay workflow in parallel. From Salmonella isolates (n = 496, 13.8%) derived by both workflows, total agreement (kappa = 1.0) between the MLMA assay and conventional serotyping was demonstrated.Conclusions: With an assay time of 2.5 h, this simple assay has shown promising potential to provide rapid and high-throughput identification of Salmonella serovars for clinical and public health laboratories to facilitate timely surveillance of salmonellosis.