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一种用于鉴定牛支原体亚种和其他支原体物种的微生物诊断微阵列检测方法。

One test microbial diagnostic microarray for identification of Mycoplasma mycoides subsp. mycoides and other Mycoplasma species.

机构信息

Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", (Istituto G. Caporale), Teramo, Italy.

出版信息

Mol Biotechnol. 2012 Nov;52(3):285-99. doi: 10.1007/s12033-012-9497-8.

Abstract

The present study describes the use of microarray technology for rapid identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. A microarray containing genetic sequences of 55 different bacterial species from Acholeplasma, Mycoplasma, Spiroplasma and Ureaplasma genera was constructed. Sequences to genes of interest were collected in FASTA format from NCBI. The collected sequences were processed with OligoPicker software. Oligonucleotides were then checked for their selectivity with BLAST searches in GenBank. The microarray was tested with ATCC/NCTC strains of Mycoplasma spp. of veterinary importance in ruminants including Mycoplasma belonging to the mycoides cluster as well as Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri field strains. The results showed that but one ATCC/NCTC reference strains hybridized with their species-specific sequences showed a profile/signature different and distinct from each other. The heat-map of the hybridization results for the nine genes interrogated for Mycoplasma mycoides subsp. mycoides demonstrated that the reference strain Mycoplasma mycoides subsp mycoides PG1 was positive for all of the gene sequences spotted on the microarray. CBPP field, vaccine and reference strains were all typed to be M. mycoides subsp. mycoides, and seven of the nine strains gave positive hybridization results for all of the nine genes. Two Italian strains were negative for some of the genes. Comparison with non-Mycoplasma mycoides subsp. mycoides reference strains showed some positive signals or considerable homology to Mycoplasma mycoides subsp. mycoides genes. As expected, some correlations were observed between the strictly genetically and antigenically correlated Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri strains. Specifically, we observed that some Italian Mycoplasma mycoides subsp. mycoides strains were positive for two out of the three Mycoplasma mycoides subsp. capri genes, differently from what has been observed for other European or African Mycoplasma mycoides subsp. mycoides strains. This study highlighted the use of microarray technology as a simple and effective method for a single-step identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. The opportunity to discriminate several mycoplasmas in a single analysis enhances diagnostic rapidity and may represent a useful tool to screen occasionally mycoplasmas affecting animal farming in territories where diagnostic laboratory support is limited. The heat-map of the hybridization results of the comparative genomic hybridizations DNA-designed chip clearly indicates that the microarray performs well for the identification of the tested Mycoplasma mycoides subsp. mycoides reference and field strains, discriminating them from other mycoplasmas.

摘要

本研究描述了使用微阵列技术快速鉴定和区分绵羊肺炎支原体亚种与可能对反刍动物致病的其他支原体,包括与绵羊肺炎支原体亚种在遗传和抗原上严格相关的支原体支原体群。构建了一个包含来自 Acholeplasma、Mycoplasma、Spiroplasma 和 Ureaplasma 属的 55 种不同细菌的遗传序列的微阵列。从 NCBI 以 FASTA 格式收集感兴趣的基因序列。使用 OligoPicker 软件处理收集的序列。然后使用 BLAST 在 GenBank 中搜索来检查寡核苷酸的选择性。该微阵列经过了 ATCC/NCTC 兽医重要的绵羊支原体属菌株的测试,包括属于支原体群的支原体以及绵羊肺炎支原体亚种和绵羊肺炎支原体亚种。Capri 田间菌株。结果表明,只有一个 ATCC/NCTC 参考菌株与其种特异性序列杂交,其图谱/特征与其他菌株不同且明显不同。用于鉴定绵羊肺炎支原体亚种的九个基因的杂交结果热图表明,参考菌株绵羊肺炎支原体亚种 PG1 对微阵列上点样的所有基因序列均呈阳性。CBPP 田间、疫苗和参考菌株均被鉴定为 M. mycoides subsp. mycoides,其中 7 株对所有 9 个基因的杂交结果均呈阳性。意大利的两个菌株对某些基因呈阴性。与非绵羊肺炎支原体亚种的比较。mycoides 参考株显示与绵羊肺炎支原体亚种的某些基因有阳性信号或相当大的同源性。正如预期的那样,在遗传和抗原上严格相关的绵羊肺炎支原体亚种和绵羊肺炎支原体亚种之间观察到一些相关性。Capri 菌株。具体来说,我们观察到一些意大利绵羊肺炎支原体亚种的菌株对三个绵羊肺炎支原体亚种中的两个基因呈阳性。capri 基因,与其他欧洲或非洲绵羊肺炎支原体亚种的菌株不同。mycoides 菌株。这项研究强调了使用微阵列技术作为一种简单有效的方法,可对可能对反刍动物致病的绵羊肺炎支原体亚种进行一步鉴定和区分。mycoides 包括与绵羊肺炎支原体亚种在遗传和抗原上严格相关的支原体支原体群。能够在单次分析中区分多种支原体可提高诊断速度,可能是筛选偶发支原体的有用工具影响在诊断实验室支持有限的地区进行动物养殖。比较基因组杂交设计芯片的杂交结果热图清楚地表明,微阵列在鉴定测试的绵羊肺炎支原体亚种方面表现良好。mycoides 参考和田间菌株,将它们与其他支原体区分开来。

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