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周可欣, 潘晓艺, 蔺凌云, 黄雷, 穆雪姣, 王丛旭, 姚嘉赟, 劳顺健, 沈锦玉. 基于SecA基因的鱼诺卡氏菌qPCR检测方法的建立及进化分析[J]. 水生生物学报, 2023, 47(10): 1553-1560. DOI: 10.7541/2023.2022.0380
引用本文: 周可欣, 潘晓艺, 蔺凌云, 黄雷, 穆雪姣, 王丛旭, 姚嘉赟, 劳顺健, 沈锦玉. 基于SecA基因的鱼诺卡氏菌qPCR检测方法的建立及进化分析[J]. 水生生物学报, 2023, 47(10): 1553-1560. DOI: 10.7541/2023.2022.0380
ZHOU Ke-Xin, PAN Xiao-Yi, LIN Ling-Yun, HUANG Lei, MU Xue-Jiao, WANG Cong-Xu, YAO Jia-Yun, LAO Shun-Jian, SHEN Jin-Yu. ESTABLISHMENT OF QPCR DETECTION ASSAY AND PHYLOGENETIC ANALYSIS BASED ON NOCARDIA SERIOLAE SECA GENE[J]. ACTA HYDROBIOLOGICA SINICA, 2023, 47(10): 1553-1560. DOI: 10.7541/2023.2022.0380
Citation: ZHOU Ke-Xin, PAN Xiao-Yi, LIN Ling-Yun, HUANG Lei, MU Xue-Jiao, WANG Cong-Xu, YAO Jia-Yun, LAO Shun-Jian, SHEN Jin-Yu. ESTABLISHMENT OF QPCR DETECTION ASSAY AND PHYLOGENETIC ANALYSIS BASED ON NOCARDIA SERIOLAE SECA GENE[J]. ACTA HYDROBIOLOGICA SINICA, 2023, 47(10): 1553-1560. DOI: 10.7541/2023.2022.0380

基于SecA基因的鱼诺卡氏菌qPCR检测方法的建立及进化分析

ESTABLISHMENT OF QPCR DETECTION ASSAY AND PHYLOGENETIC ANALYSIS BASED ON NOCARDIA SERIOLAE SECA GENE

  • 摘要: 为对诺卡氏菌病进行快速早期诊断, 建立了鰤鱼诺卡氏菌(Nocardia seriolae)TaqMan qPCR检测方法。以鰤鱼诺卡氏菌的管家基因SecA为靶标设计引物和探针, 对反应条件进行优化, 制作标准曲线, 进行灵敏性、重复性和特异性测试, 并将建立的方法应用于临床样品的检测。结果表明, 优化后, 引物和探针终浓度分别为0.3和0.1 μmol/L, 退火延伸温度60℃时, qPCR在9.85×1010—9.85×100 copies内呈良好的线性关系, 灵敏度最高达9.85 copies; 重复性实验结果显示, 组间和组内变异系数均小于1%; 特异性结果表明, 对无乳链球菌、弗氏柠檬酸杆菌和维氏气单胞菌等13种病菌均无扩增曲线; 临床对42份大口黑鲈样品进行检测, 结果表明, qPCR检出率比普通PCR提高14.24%; 对发病大口黑鲈的组织及结节部位检测显示, 结节部位菌载量大幅高于非结节部位, 最高相差1000倍; SecA基因分析结果显示, SecA基因在传代中和种内高度保守, 种间具有一定的离散性, 是个很好的用于诺卡氏菌种鉴定的靶基因。研究建立的鰤鱼诺卡氏菌qPCR检测方法灵敏性高、特异性强、重复性好, 可用于对鰤鱼诺卡氏菌病的早期诊断和定量检测, 为诺卡氏菌病的早诊断早治疗提供了有效手段。

     

    Abstract: Nocardia seriolae is one of the most common pathogenic bacteria in aquaculture animals, and the nocardiosis caused by it has brought huge economic losses to the global aquaculture industry. In order to perform diagnosis of N. seriolae infection quickly and early, a TaqMan qPCR detection method for N. seriolae was established. The primers and probes were designed with the housekeeping gene SecA of N. seriolae, and the reaction conditions were optimized, and standard curves was developed. Then the established qPCR method was tested for sensitivity, reproducibility and specificity, and applied for the detection of clinical diseased fish samples. The assay of qPCR showed high linearity in the range of 9.85×1010—9.85×100 copies with a sensitivity of up to 9.85 copies under the optimal primers concentration (0.3 μmol/L), the optimal probe concentration (0.1 μmol/L), and the optimal annealing extension temperature (60℃). The results of the repeatability experiment showed that the coefficient of variation between groups and within groups was less than 1%. The specificity showed that there was no amplification curve for 13 pathogens such as S. agalactiae, C. freundii and A. veronii. The test results of the 42 diseased M. salmoides samples showed that the positive rate of qPCR was 14.24% higher than that of normal PCR, and the content of nocardia in the nodules was significantly higher than that in the non-nodal site of tissues, with a maximum difference of 1000-fold. The results of SecA gene analysis showed that SecA gene was highly conserved in passage and within species, and has certain discreteness among species, so it was a good target gene for Nocardia species identification. The qPCR method established in this study exhibits remarkable sensitivity, specificity and repeatability, and can be used in early diagnosis and quantitative detection of N. seriolae, and provides an effective tool for early diagnosis and treatment of nocardiosis.

     

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