4种中国常见致病性基因种钩端螺旋体标准菌株的建立与评价

邱海燕; 张翠彩

doi:10.3784/jbjc.202311250636

4种中国常见致病性基因种钩端螺旋体标准菌株的建立与评价

Establishment and evaluation of standard strains of four major pathogenic Leptospira species in China

摘要

摘要:
目的依据《病原微生物菌（毒）种国家标准株评价技术标准》（WS/T 812—2022），建立Leptospira interrogans、L. borgpetersenii、L. alexanderi和L. weilii 4种中国常见致病性基因种钩端螺旋体标准菌株。
方法以56601、56604、56615、56655 4株致病性代表菌株为研究对象，利用暗视野显微镜开展形态学观察。以16S rRNA基因为靶基因进行聚合酶链式反应扩增、测序确定所属基因种。通过多位点序列分型（MLST）方法，对4株代表株进行分子分型分析，确定每株菌株7个位点等位基因号和序列型别（ST）。综合暗视野显微镜形态学观察、16S rRNA基因测序和MLST分型结果系统评价4株代表菌株经不同传代次数后的分子遗传学稳定性。同时通过Illumina NovaSeq测序平台对56604、56615和56655开展全基因组测序分析，了解菌株基因组特征。
结果暗视野显微镜下，4株代表菌株均具有典型的钩端螺旋体形态。16S rRNA基因序列分析显示，56601、56604、56615和56655分别隶属于L. interrogans、L. borgpetersenii、L. alexanderi和L. weilii 4种不同致病性基因种。 MLST分型结果显示4株代表株隶属于4种不同ST型。 4株代表株在亲代株及在体外连续传代一定代次后，暗视野显微镜下形态不变，所属基因种和MLST基因型结果一致，证明该4株代表株具有良好的分子遗传学稳定性。基因组测序分析显示，4株代表株基因组大小为3.93～4.69 Mb之间，G+C含量为36.00%～40.87%。
结论初步建立并评价了4种中国常见致病性基因种钩端螺旋体标准菌株，为钩端螺旋体病的疾病控制、临床诊断、教学科研、疫苗研发提供菌株资源。

Abstract:
Objective To establish Chinese standard strains of four major pathogenic Leptospira species, i.e. L. interrogans, L. borgpetersenii, L. alexanderi and L. weilii, according to the Technical Requirements for Evaluation of National Standard Strains of Pathogenic Microorganisms (WS/T 812—2022).
Methods Four reference strains of Leptospira, i.e. 56601, 56604, 56615 and 56655, belonging to four pathogenic species of Leptospira were selected. The morphological characteristics of the 4 strains were observed under the dark-field microscope. Based on 16S rRNA gene sequencing, species identification of the 4 strains was performed. Multilocus sequence typing (MLST) was conducted, and the alleles and sequence types (STs) of the 4 strains were obtained. The morphological and genetic stability of the 4 strains among different generations was evaluated by using dark-field microscope, 16S rRNA gene sequencing and MLST analysis.
Results All the 4 strains had typical morphological characteristics of Leptospira. Species identification revealed that the 4 strains, i.e. 56601, 56604, 56615 and 56655, belonged to four pathogenic Leptospira species, L. interrogans, L. borgpetersenii, L. alexanderi and L. weilii, respectively. A total of four different STs were identified from the 4 pathogenic Leptospira strains. The morphological characteristics of the 4 strains were stable after successive generations. The stability experiments results showed that the sequences of 16S rRNA gene among different generations were 100.00% homologous. In addition, the ST types among different generations were completely consistent. The genetic stability of the 4 strains was good. The whole genome sequencing analysis revealed that the genome size was 3.93–4.69 Mb and the G+C content was 36.00%–40.87%.
Conclusion The 4 strains, i.e. 56601, 56604, 56615 and 56655, have typical morphological and biological characteristics of pathogenic Leptospira, and their molecular genetic characteristics were stable, indicating that they can be used as four standard strains of pathogenic Leptospira species in disease control, clinical diagnosis, teaching and scientific research and vaccine development in China.

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