高分辨率熔解曲线分析法检测食源性副溶血性弧菌

Rapid Detection of Vibrio parahaemolyticusby Real-time PCR using High Resolution Melting Curve Analysis

摘要:

为建立一种快速鉴定副溶血性弧菌的HRM（高分辨率熔解曲线）real-time PCR法，以tox R为靶基因，结合特异性引物，通过优化反应体系及条件，进行特异性、敏感性及重复性评价，并初步应用于90份送检的鲜活海产品样本的检测。特异性试验表明，该方法能选择性检测副溶血弧菌，Tm值为76.64±0.57 ℃；而与创伤弧菌、霍乱弧菌、金黄色葡萄球菌等多种海产品中常见的食源性病原菌没有交叉反应。灵敏度试验表明，该方法最少可检测tox R重组质粒的浓度为3.50×102 copies/mL。重复性试验表明，同一样品于试验内及试验间的平均Tm值分别为76.53±0.35 ℃和76.74±0.52 ℃，变异系数分别为0.56±0.42%和1.11±0.73%。对90份鲜活海产品样本的检测证实该法可使阳性检出率从国标法的14.44%提高至18.89%。本研究所建立的副溶血性弧菌HRM real-time PCR法具有特异性好、灵敏度高、重复性好的特点，能应用于食品样本的检测，具有很好的研究价值和应用前景。

Abstract:

A rapid real-time PCR method for detecting Vibrio parahaemolyticus (VP) by high resolution melting curve analysis (HRM) was developed. A pair of specific primers targeting the toxR gene of Vibrio parahaemolyticus was used. After optimizing the reaction system and conditions,the specificity of the method was validated with 10 target strains and 10 non-target strains,Serial dilutions from 3.50×101 to 3.50×107 copies/mL purified constructed plasmids were analyzed to evaluation its sensitivity. Reproducibility assay was also conducted on purified constructed plasmids between 1.50×101 and 1.50×107 copies/mL. The method was applied to detect 90 live submitted seafood samples. The results showed that the developed HRM real-time PCR assay had a good specificity by detecting only VP with the Tm value of 76.64±0.57 ℃ and was not affected by other seafood pathogens such as Vibrio vulnificus, Vibrio cholerae,Staphylococcus aureus et al. The sensitivity of detection of the constructed plasmids were 3.50×102 copies/mL. In addition, the assay had a high reproducibility and the average Tm values were 76.53±0.35 ℃ and 76.74± 0.52 ℃. The sample’s variations (CVs) were 0.56±0.42% and 1.11±0.73%. A better detection rate of 18.89% than that of the traditional culture method (14.44%) was found in the detection of 90 live seafood samples. The established real-time PCR assay showed sufficient specificity, high sensitivity and good reproducibility. And it was rapid, simple and cheap for detection of VP.