应用实时 PCR 靶向 21 个可变基因组座对马耳他布鲁氏菌和流产布鲁氏菌进行基因分型。
Brucella melitensis and Brucella abortus genotyping via real-time PCR targeting 21 variable genome loci.
机构信息
Microbiology Reference Laboratories, General Directorate of Public Health, Minister of Health, Ankara, Turkey; University of Health Sciences, Institute of Defence Health Sciences, Department of Medical CBRN, Ankara, Turkey.
Department of Zonotic and Vector Borne Diseases, General Directorate of Public Health, Minister of Health, Ankara, Turkey.
出版信息
J Microbiol Methods. 2021 Jan;180:106125. doi: 10.1016/j.mimet.2020.106125. Epub 2020 Dec 14.
Brucella melitensis and Brucella abortus account for almost all cases of brucellosis in Turkish population. We developed a fourplex quantitative real-time PCR (qPCR) assay for the electrophoresis-free, rapid and cost-effective differentiation of B. abortus and B. melitensis from the other Brucella spp. The 4-plex species differentiation assay was combined with a qPCR assay targeting 17 different single nucleotide polymorphism (SNP) loci in Brucella genomes. This combination resulted in a 21 Variable Genome Loci (21-VGL) qPCR assay for high resolution genotyping of B. abortus and B. melitensis. A total of 486 Brucella was analyzed using the qPCR assay to create a 21-VGL profile database. The database contained the profiles of 55 B. abortus, 352 B. melitensis, 3 B. ceti, 6 B. neotomae, 7 B. ovis, 6 B. pinnipedialis, 44 B. suis and 13 B. canis strains. The 21-VGL Brucella genotyping clearly distinguished B. abortus, B. melitensis, B. neotomae and B. ovis. The 21-VGL approach could not distinguish B. pinnipedialis from B. ceti and some B. suis genotypes from B. canis. The results revealed that more than 99% of the Brucella isolates in Turkey were B. melitensis and 21-VGL genotyping can be reduced to 8-VGL B. melitensis genotyping without any loss of genotyping resolution. To our knowledge, we introduced the fastest and the lowest-cost B. abortus and B. melitensis genotyping and species differentiation methodology in the literature.
布鲁氏菌属 melitensis 和布鲁氏菌属 abortus 几乎占土耳其人群中布鲁氏菌病的所有病例。我们开发了一种四重实时荧光定量 PCR (qPCR) 无电泳检测方法，用于快速、经济有效地区分布鲁氏菌属 abortus 和布鲁氏菌属 melitensis 与其他布鲁氏菌属物种。该四重种间区分检测方法与针对布鲁氏菌属基因组中 17 个不同单核苷酸多态性 (SNP) 位点的 qPCR 检测方法相结合，形成了布鲁氏菌属 abortus 和布鲁氏菌属 melitensis 的高分辨率基因分型 21 可变基因组座 (21-VGL) qPCR 检测方法。使用 qPCR 检测方法对总共 486 株布鲁氏菌进行了分析，以建立 21-VGL 图谱数据库。该数据库包含 55 株布鲁氏菌属 abortus、352 株布鲁氏菌属 melitensis、3 株布鲁氏菌属 ceti、6 株布鲁氏菌属 neotomae、7 株布鲁氏菌属 ovis、6 株布鲁氏菌属 pinnipedialis、44 株布鲁氏菌属 suis 和 13 株布鲁氏菌属 canis 菌株的图谱。21-VGL 布鲁氏菌基因分型清楚地区分了布鲁氏菌属 abortus、布鲁氏菌属 melitensis、布鲁氏菌属 neotomae 和布鲁氏菌属 ovis。21-VGL 方法无法区分布鲁氏菌属 pinnipedialis 和布鲁氏菌属 ceti，也无法区分某些布鲁氏菌属 suis 基因型和布鲁氏菌属 canis。结果表明，土耳其超过 99%的布鲁氏菌分离株为布鲁氏菌属 melitensis，21-VGL 基因分型可简化为 8-VGL 布鲁氏菌属 melitensis 基因分型，而不会降低基因分型分辨率。据我们所知，我们在文献中引入了最快和最低成本的布鲁氏菌属 abortus 和布鲁氏菌属 melitensis 基因分型和种间区分方法。
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