段立科, 侯学霞, 张琳, 郝琴. 基于不同基因型的中国莱姆病螺旋体菌株标准化研究[J]. 凯发娱乐加盟代理, 2023, 38(12): 1453-1459. DOI: 10.3784/jbjc.202309280515
引用本文: 段立科, 侯学霞, 张琳, 郝琴. 基于不同基因型的中国莱姆病螺旋体菌株标准化研究[J]. 凯发娱乐加盟代理, 2023, 38(12): 1453-1459. DOI: 10.3784/jbjc.202309280515
Duan Like, Hou Xuexia, Zhang Lin, Hao Qin. Standardized study of Chinese Borrelia strains causing Lyme disease based on different genotypes [J]. Disease Surveillance, 2023, 38(12): 1453-1459. DOI: 10.3784/jbjc.202309280515
Citation: Duan Like, Hou Xuexia, Zhang Lin, Hao Qin. Standardized study of Chinese Borrelia strains causing Lyme disease based on different genotypes [J]. Disease Surveillance, 2023, 38(12): 1453-1459. DOI: 10.3784/jbjc.202309280515

基于不同基因型的中国莱姆病螺旋体菌株标准化研究

Standardized study of Chinese Borrelia strains causing Lyme disease based on different genotypes

  • 摘要:
    目的 建立莱姆病螺旋体中国株5种基因型(巴伐利亚疏螺旋体 、 伽氏疏螺旋体、阿弗西尼疏螺旋体、狭义伯氏疏螺旋体和扬子疏螺旋体)的标准菌株,为菌株鉴定、诊断试剂和疫苗研发提供基础。
    方法 依据《病原微生物菌(毒)种国家标准株评价技术标准》(WS/T 812-2022),针对中国莱姆病螺旋体5种主要基因型,选取5株菌株(PD91、MD86、FP1、CS4和QX-S13)。 首先接种到BSKⅡ培养基中培养,暗视野显微镜下观察菌株的形态学特点。 提取全基因组DNA,聚合酶链式反应扩增莱姆病螺旋体5S~23S rRNA基因间隔区并测序,将每株菌株不同代次培养物的产物序列分别进行比对分析。 随后使用多位点序列分析(MLSA)方法鉴定菌株基因型,同时将不同代次菌株MLSA聚类分析结果进行比对,验证菌株遗传学稳定性。 通过Illumina NovaSeq平台进行全基因组测序分析菌株基因组特征。
    结果 5株菌株均具有莱姆病螺旋体典型形态学特征。 经特异性基因检测及MLSA分型鉴定,依次为巴伐利亚疏螺旋体(PD91), 伽氏疏螺旋体(MD86), 阿弗西尼疏螺旋体(FP1), 狭义伯氏疏螺旋体(CS4)和扬子疏螺旋体(QX-S13)基因型,聚类分析显示与相应基因型国际参考菌株位于同一分支。 稳定性实验结果显示5株菌株不同代次培养物5S~23S rRNA基因间隔区序列相似性达到100.00%,5株菌株10代次和20代次培养物MLSA聚类分析结果一致,遗传学稳定性良好。 全基因组测序结果显示,基因组大小为1.26Mbp~1.80 Mbp,G+C含量为27.63%~28.20%,基因组功能和生物代谢模块分布基本相同,主要为维持细菌生长、繁殖、运动和应激等相关成分。
    结论 PD91、FP1、MD86、CS4和QX-S13共5株菌株具有莱姆病螺旋体典型生物学、形态学特征,基因型及分子特征稳定,可作为中国莱姆病螺旋体5种基因型的标准菌株。 本研究可为建立中国莱姆病螺旋体标准化菌株体系提供基础,同时为菌株鉴定、莱姆病诊断试剂和疫苗研发提供相关资源。

     

    Abstract:
    Objective To establish the standard strains of Borrelia burgdorferi of five genotypes (B. bavariensis, B. garinii, B. afzelii, B. burgdorferi sensu stricto and B. yangtzensis) in China and provide evidence for the pathogen identification, diagnostic reagents and vaccine development for Lyme disease prevention and control.
    Methods According to Technical Requirements for Evaluation of National Standard Strains of Pathogenic Microorganisms (WS/T 812-2022), B. burgdorferi strains of five genotypes isolated in China (PD91, MD86, FP1, CS4 and QX-S13) were selected. First, the five strains were cultured in BSK II medium and the morphological characteristics of the strains were observed under darkfield microscope. The strain's whole genome DNA was extracted, the 5S–23S rRNA intergenic spacer region was amplified by polymerase chain reaction and sequenced, and the product sequences of different generations of each strain were compared. Multilocus sequence analysis (MLSA) was used to genotype the isolates, meantime the MLSA clustering analysis results of different generations of the strains were compared to verify the genetic stability of the strains. Whole genome sequencing was conducted to analyze genomic features of the 5 strains through the Illumina NovaSeq platform.
    Results All the 5 strains had typical morphological characteristics of B. burgdorferi. Through Specific genetic testing and MLSA, strain PD91, MD86, FP1, CS4 and QX-S13 were identified as B. bavariensis, B. garinii, B. afzelii, B. burgdorferi sensu stricto and B. yangtzensis, respectively, and phylogenetic analysis showed that the strains were the same branches genotypes of international reference strains. The stability experiments results showed that the sequence similarity of the 5S–23S rRNA intergenic spacer region of different generations of each strain reached 100.00%, the results of MLSA clustering of the 5 strains in the 10th and 20th cultures were consistent, indicating good genetic stability. The whole genome sequencing results showed that the genome size was 1.26–1.80 Mbp and the G+C content was 27.63%–28.20%. The functional annotation and distribution of metabolic modules of chromoso genome for the five strains were almost same: mainly for cell growth maintaining, reproduction, cell motility, defense and others for cell survival.
    Conclusion The five strains have typical biological and morphological characteristics of B. burgdorferi, and their genotypes and molecular characteristics are stable, indicating that they can be used as standard strains for B. burgdorferi of five genotypes in China. This study provided evidence for the establishment of a standardized strain system of B. burgdorferi in China, and relevant resources for the strain identification, diagnostic reagents and vaccine development in Lyme disease prevention and control.

     

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