三重荧光定量聚合酶链反应法检测唐菖蒲伯克霍尔德氏菌及椰毒致病型菌株与洋葱伯克霍尔德氏菌

Determination of Burkholderia gladioli, Burkholderia gladioli pathovar cocovenenans and Burkholderia cepacia by triplex fluorescent quantitative polymerase chain reaction

中文关键词:  唐菖蒲伯克霍尔德菌椰毒致病型  洋葱伯克霍尔德氏菌  多重实时荧光定量PCR

英文关键词:Burkholderia gladioli pathovar cocovenenans  Burkholderia cepacia  triple fluorescent quantitative polymerase chain reaction

中文摘要:

      目的 建立可同时快速鉴别唐菖蒲伯克霍尔德氏菌及椰毒致病型菌株及洋葱伯克霍尔德氏菌的三重荧光定量聚合酶链式反应(triple fluorescent quantitative polymerase chain reaction, Triple-qPCR)方法。方法 针对唐菖蒲伯克霍尔德氏菌的16S-23S rRNA基因、其椰毒致病型的产毒基因bonM及洋葱伯克霍尔德氏菌的secY基因设计特异性引物与探针, 构建Triple-qPCR检测体系; 以16株唐菖蒲伯克霍尔德氏菌(含8株产毒株、8株非产毒株)、8株洋葱伯克霍尔德氏菌及6株近缘/环境干扰菌为试验菌株, 优化体系反应条件(退火温度、引物浓度), 并验证体系的特异性、灵敏度、重复性及抗干扰能力。结果 优化后体系的最佳退火温度为59 ℃, 引物终浓度为0.40 μmol/L, 探针终浓度为0.20 μmol/L; 特异性试验中, 3个靶基因对目标菌的阴阳性符合率均达100%, 无交叉反应; 靶基因16S-23S rRNA与bonM的最低检出限(limit of detection, LOD)为1.4×102 CFU/mL, secY基因的LOD为1.6×102 CFU/mL; 标准曲线相关系数r2>0.990, 扩增效率介于100.9%~106.3%; 重复性试验中, 组内变异系数(coefficient of variation, CV)<5%, 生物重复组间无显著性差异(P>0.05); 抗干扰试验显示, 复杂菌群混合体系中目标菌仍可被特异性检出。结论 建立的Triple-qPCR体系具有快速、特异、灵敏、稳定的优势, 可实现3种目标的同步鉴别, 适用于食品安全领域中唐菖蒲伯克霍尔德氏菌及椰毒致病型菌株与洋葱伯克霍尔德氏菌的高效筛查。

英文摘要:

      Objective To establish a method for the simultaneous and rapid identification of Burkholderia gladioli, Burkholderia gladioli pathovar cocovenenans, and Burkholderia cepacia by triple fluorescent quantitative polymerase chain reaction (Triple-qPCR). Methods Specific primers and probes were designed targeting the 16S-23S rRNA gene of Burkholderia gladioli, the toxin-encoding bonM gene of its toxigenic pathovar, and the secY gene of Burkholderia cepacia to construct an Triple-qPCR detection system. A total of 16 strains of Burkholderia gladioli (including 8 toxigenic strains and 8 non-toxigenic strains), 8 strains of Burkholderia cepacia, and 6 strains of phylogenetically related/environmental interfering bacteria were used as test strains. The reaction conditions of the system (annealing temperature, primer concentration) were optimized, and the specificity, sensitivity, repeatability and anti-interference ability of the system were verified. Results The optimized system had an optimal annealing temperature of 59 ℃, with a final primer concentration of 0.40 μmol/L and a final probe concentration of 0.20 μmol/L. In the specificity test, the positive and negative coincidence rates of the 3 target genes for the target bacteria all reached 100% without cross-reactivity. The limit of detection (LOD) for the target genes 16S-23S rRNA and bonM was 1.4×102 CFU/mL, and the LOD for the secY gene was 3.1×102 CFU/mL. The correlation coefficients (r2) of the standard curves were all>0.990, and the amplification efficiencies ranged from 100.9% to 106.3%. In the repeatability test, the intra-group coefficient of variation (CV) was all < 5%, and there was no significant difference among biological replicate groups (P>0.05). The anti-interference test showed that the target bacteria could still be specifically detected in the mixed system of complex flora. Conclusion The established Triple-qPCR system has the advantages of rapidity, specificity, sensitivity and stability, which can realize the simultaneous identification of 3 kinds of targets and is suitable for the efficient screening of toxigenic Burkholderia gladioli pathovar cocovenenans and Burkholderia cepacia in the field of food safety.