鉴别布鲁氏菌疫苗株与野毒株多重TaqMan qPCR检测方法的建立

Establishment of a multiplex TaqMan qPCR assay for distinguishing between vaccine strains and wild-type strains of Brucella

摘要:

背景 布鲁氏菌病是一种由布鲁氏菌(Brucella)引起的重要人兽共患病，该病可导致家畜流产、不孕等严重生殖障碍。疫苗接种是防控布鲁氏菌病的有效手段，实现疫苗免疫株与野毒株的有效区分是准确诊断及防控布鲁氏菌病的关键。目的 建立一种区分布鲁氏菌疫苗(A19, S2)免疫与自然感染的灵敏、特异的多重TaqMan qPCR检测方法。方法 通过Clustal Omega软件对比相同物种来源的布鲁氏菌强毒株与疫苗株的基因组序列，分别确定A19和S2特异缺失序列；根据布鲁氏菌通用的IS711基因序列、A19和S2疫苗株特异性缺失序列设计引物与探针，优化检测方法的反应体系与程序；构建重组质粒标准品，确定标准曲线；进一步对检测方法的敏感性、特异性、重复性及临床应用效果进行评价。结果 分别筛选出A19疫苗株(68个碱基)和S2疫苗株(26个碱基)特有缺失序列作为靶标。最佳退火温度为60 ℃，IS711、A19、S2引物与探针浓度分别为0.25和0.15、0.10和0.25、0.20和0.20 μmol/L；标准曲线线性关系较好，R²分别为0.999、0.999、0.996，扩增效率(E)分别为100.547%、102.489%、98.895%。敏感性高，最小检测量为10 copies/μL；特异性强，可区分布鲁氏菌疫苗株与野毒株，并且与大肠埃希氏菌(Escherichia coli) O₁₅₇、小肠结肠炎耶尔森氏菌(Yersinia enterocolitica) O₉、都柏林沙门氏菌(Salmonella Dublin)等6种病原菌均无交叉反应；重复性好，批间与批内试验变异系数均小于2%。临床随机采集的247份样本(鼻/肛拭子、牛奶、血液，病变组织)的检出率高于《动物布鲁氏菌病诊断技术》的检测。结论 成功建立敏感性高、特异性强、能快速区分疫苗免疫和野毒株感染的多重TaqMan qPCR检测方法，适合背景清楚的动物布病临床样本风险监测。

Abstract:

Background Brucellosis is a major zoonotic disease caused by Brucella, which can lead to severe reproductive disorders such as abortion and infertility in livestock. Vaccination is an effective strategy for the prevention and control of brucellosis, and the accurate distinguishing between vaccine strains and wild-type strains is crucial for reliable diagnosis and effective disease management.Objective To establish a sensitive and specific multiplex TaqMan qPCR method for distinguishing Brucella vaccination (strains A19 and S2) from natural infections.Methods By using Clustal Omega to compare the genome sequences of Brucella virulent strains and vaccine strains from the same species, we identified the specific deletion sequences of A19 and S2. Primers and probes were designed based on the conserved IS711 sequence of Brucella and the specific deletion sequences of the vaccine strains A19 and S2. The reaction system and program of the detection method were optimized. Recombinant plasmid standards were constructed to establish standard curves. The sensitivity, specificity, repeatability, and clinical application performance of the method were evaluated.Results The unique deletion sequences of A19 (68 bp) and S2 (26 bp) were identified as targets. The optimal reaction conditions were as follows: annealing temperature of 60 ℃ final primer and probe concentrations of 0.25 μmol/L and 0.15 μmol/L for IS711, 0.10 μmol/L and 0.25 μmol/L for A19, and 0.20 μmol/L and 0.20 μmol/L for S2, respectively. The standard curves showed good linearity, with R² values of 0.999, 0.999, and 0.996 and amplification efficiencies of 100.547%,102.489%, and 98.895%, respectively. The method demonstrated high sensitivity, with a minimum limit of detection being 10 copies/μL, and strong specificity, effectively distinguishing Brucella vaccine strains from wild-type strains without cross-reactivity with six pathogens including Escherichia coli O₁₅₇, Yersinia enterocolitica O₉, and Salmonella Dublin. It exhibited excellent repeatability, with inter- and intra-assay coefficients of variation (CV) both below 2%. The detection rate of the established method for 247 randomly collected clinical samples (nasal/anal swabs, milk, blood, and lesion tissue) was higher than that of the method specified in the Diagnostic Techniques for Animal Brucellosis.Conclusion A highly sensitive, specific, and rapid multiplex TaqMan qPCR assay was successfully established for distinguishing between vaccination and wild-type Brucella infection. This method is suitable for risk monitoring of brucellosis in animals with well-documented backgrounds.