Vidovic Sinisa, Taylor Roland, Hedderley Duncan, Fletcher Graham C, Wei Nicola
The New Zealand Institute for Plant and Food Research Limited, 120 Mount Albert Road, Sandringham, 1025, Auckland, New Zealand.
The New Zealand Institute for Plant and Food Research Limited, Palmerston North, New Zealand.
Sci Rep. 2024 Feb 19;14(1):4137. doi: 10.1038/s41598-024-54753-y.
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.
在本研究中，系统测试了三代聚合酶链反应（PCR）检测方法：（i）传统PCR，（ii）定量PCR（qPCR）和（iii）数字液滴PCR（ddPCR）检测副溶血性弧菌非致病群体和致病群体的能力。ddPCR的检测限（LOD）为1.1 pg/µL纯化DNA，其次是qPCR（5.6 pg/µL）和传统PCR（8.8 pg/µL）。关于副溶血性弧菌细胞的检测限，ddPCR检测方法能够检测到29个细胞，其次是传统PCR检测方法（58个细胞）和qPCR检测方法（115个细胞）。关于从易于PCR抑制的样本（天然污染的贻贝）中检测这种病原体的灵敏度，ddPCR检测方法明显优于传统PCR和qPCR。ddPCR检测方法能够始终如一地从天然污染的贻贝中检测到副溶血性弧菌的非致病群体和致病群体，表明其对各种PCR抑制剂具有耐受性。本研究还揭示了传统PCR和qPCR之间的显著差异。传统PCR检测方法在检测粗样本中的副溶血性弧菌时显示出比qPCR检测方法更高的灵敏度，而qPCR检测方法在检测纯化DNA样本中的副溶血性弧菌存在时显示出更好的灵敏度。
