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Abstract：
In this study, three generations of polymerase chain reaction (PCR) assays: (i) conventional PCR, (ii) qPCR and (iii) droplet digital PCR (ddPCR), were systematically tested for their abilities to detect non-pathogenic and pathogenic populations of Vibrio parahaemolyticus. The limit of detection (LOD) for the ddPCR was 1.1 pg/µL of purified DNA, followed by the qPCR (5.6 pg/µL) and the conventional PCR (8.8 pg/µL). Regarding the LOD for V. parahaemolyticus cells, the ddPCR assay was able to detect 29 cells, followed by the conventional PCR assay (58 cells) and the qPCR assay (115 cells). Regarding the sensitivities to detect this pathogen from PCR inhibition prone samples (naturally contaminated mussels), the ddPCR assay significantly outperformed the conventional PCR and qPCR. The ddPCR assay was able to consistently detect non-pathogenic and pathogenic populations of V. parahaemolyticus from naturally contaminated mussels, indicating its tolerance to various PCR inhibitors. This study also revealed the significant difference between conventional PCR and qPCR. The conventional PCR assay showed significantly greater sensitivity than that of the qPCR assay in detecting V. parahaemolyticus in crude samples, whereas the qPCR assay showed better sensitivity in detecting the presence of V. parahaemolyticus in purified DNA samples.
摘要：
在这项研究中,三代聚合酶链反应（PCR）检测：（i）常规PCR，（ii）qPCR和（iii）液滴数字PCR（ddPCR），系统测试了其检测副溶血性弧菌非致病性和致病性种群的能力。ddPCR的检测限（LOD）为1.1pg/µL纯化的DNA，然后进行qPCR（5.6pg/µL）和常规PCR（8.8pg/µL）。关于副溶血性弧菌细胞的LOD，ddPCR检测能够检测到29个细胞，然后是常规PCR测定(58个细胞)和qPCR测定(115个细胞)。关于从PCR抑制倾向样品（自然污染的贻贝）中检测这种病原体的敏感性，ddPCR测定显著优于常规PCR和qPCR。ddPCR分析能够从自然污染的贻贝中持续检测出副溶血弧菌的非致病性和致病性种群，表明其对各种PCR抑制剂的耐受性。这项研究还揭示了常规PCR和qPCR之间的显着差异。在检测粗样品中的副溶血性弧菌时，常规PCR法显示出明显高于qPCR法的灵敏度。而qPCR分析在检测纯化的DNA样品中副溶血性弧菌的存在方面显示出更好的灵敏度。
