一种针对不同标本类型中16S - 23S rDNA内部转录间隔区的属特异性实时荧光定量PCR检测方法的开发。
Development of a Genus-Specific Real-Time PCR Assay Targeting the 16S-23S rDNA Internal Transcribed Spacer from Different Specimen Types.
作者信息
Nyarku Rejoice, Hassim Ayesha, Jonker Annelize, Quan Melvyn
机构信息
Vectors and Vector-borne Diseases Research Programme, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
出版信息
Vet Sci. 2020 Nov 11;7(4):175. doi: 10.3390/vetsci7040175.
The aim of this study was to develop a 16S-23S ribosomal deoxyribonucleic acid internal transcribed spacer (ITS) quantitative polymerase chain reaction (qPCR) assay for the early diagnosis and rapid screening of brucellosis. Blood, milk, and tissue samples were spiked with biovar 1 (B01988-18 strain) to determine the analytical sensitivity and specificity of the assay. The 95% limit of detection of the ITS qPCR assay was highest in tissue, followed by blood, then milk, i.e., 0.48, 4.43, and 15.18 bacteria/PCR reaction, respectively. The diagnostic performance of the assay was compared to the cell surface protein (BCSP) 31 qPCR assay and bacterial culture. Out of 56 aborted foetal tissue samples from bovine, ovine, and caprine, 33% (19/56) were positive for spp. The sensitivity and specificity of the ITS qPCR assay was 87% and 95% respectively, compared to 92% and 89% for the BCSP31 qPCR assay and 47% and 55% for bacterial culture, respectively. The assay was efficient, sensitive, and specific, making it a valuable tool in the early detection of the pathogen.
本研究的目的是开发一种用于布鲁氏菌病早期诊断和快速筛查的16S-23S核糖体脱氧核糖核酸内转录间隔区（ITS）定量聚合酶链反应（qPCR）检测方法。向血液、牛奶和组织样本中添加生物变种1（B01988-18菌株），以确定该检测方法的分析灵敏度和特异性。ITS qPCR检测方法的95%检测限在组织中最高，其次是血液，然后是牛奶，即分别为0.48、4.43和15.18个细菌/PCR反应。将该检测方法的诊断性能与细胞表面蛋白（BCSP）31 qPCR检测方法和细菌培养进行比较。在56份来自牛、羊和山羊的流产胎儿组织样本中，33%（19/56）的样本对布鲁氏菌呈阳性。ITS qPCR检测方法的灵敏度和特异性分别为87%和95%，而BCSP31 qPCR检测方法的灵敏度和特异性分别为92%和89%，细菌培养的灵敏度和特异性分别为47%和55%。该检测方法高效、灵敏且特异，使其成为早期检测布鲁氏菌病原体的有价值工具。
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