Quantitative detection of Vibrio parahaemolyticus in aquatic products by duplex droplet digital PCR combined with propidium monoazide
Quantitative detection of Vibrio parahaemolyticus in aquatic products by duplex droplet digital PCR combined with propidium monoazide
复制标题
DOI：
10.1016/j.foodcont.2022.109353
复制
发表时间：
2022-09
期刊：
影响因子：
6
通讯作者：
H. Zhou;Xinmei Liu;Zhaoxin Lu;A. Hu;W. Ma;C. Shi;X. Bie;Yiyu Cheng;Haijing Wu;Jun Yang
中科院分区：
文献类型：
--
作者：
H. Zhou;Xinmei Liu;Zhaoxin Lu;A. Hu;W. Ma;C. Shi;X. Bie;Yiyu Cheng;Haijing Wu;Jun Yang
将微滴数字PCR（ddPCR）与叠氮丙啶（PMA）结合，建立了一种检测水产品中副溶血性弧菌活菌的PMA-双重ddPCR方法。针对本实验室自行筛选的编码DNA聚合酶III β链的新基因vp 0488和传统的靶基因热不稳定溶血素（tlh），设计并验证了特异性引物和探针。优化了双ddPCR体系和条件以及PMA处理条件。对PMA-双链ddPCR的检测限（LOD）进行了评价，并应用于检测V。人工污染样品和实际样品中的副溶血性毒素。结果表明，双链ddPCR的最佳退火温度为60 °C，最佳扩增循环数为40，最佳引物与探针的比例为1：0. 5。PMA处理的最佳条件为：孵育时间5 min，PMA浓度30 μmol/L，暴露时间15 min。PMA-双链ddPCR的检出限（7.32 × 101 CFU/mL）低于双链定量PCR（7.32 × 102 CFU/mL）。牡蛎中副溶血性弧菌的最低检出量为8.15 × 101 CFU/g。本研究建立的方法特异性好、灵敏度高、准确度高，为水产品中副溶血性弧菌活菌的检测提供了一种有效的方法。
Based on the combination of droplet digital PCR (ddPCR) and propidium monoazide (PMA), a new method called PMA-duplex ddPCR was developed for the quantitative determination of viable Vibrio parahaemolyticus in aquatic products. Specific primers and probes targeting a new gene,vp0488, coding for putative DNA polymerase III beta chain, which was self-screened by this laboratory, and a traditional target gene, thermolabile hemolysin (tlh), were designed and verified. The duplex ddPCR system and conditions and PMA treatment conditions were optimized. The limit of detection (LOD) for PMA-duplex ddPCR was evaluated and applied to detectV. parahaemolyticusin artificially contaminated samples and actual samples. The results indicate that the optimal duplex ddPCR annealing temperature was 60 °C, the optimal number of amplification cycles was 40, and the optimal ratio of primer to probe was 1:0.5. The optimal PMA treatment conditions were as follows: incubation time 5 min, concentration 30 μmol/L, and exposure time 15 min. The LOD of PMA-duplex ddPCR (7.32 × 101CFU/mL) was lower than that of duplex quantitative PCR (qPCR) (7.32 × 102CFU/mL). The lowest detectable V. parahaemolyticus in oysters was 8.15 × 101CFU/g without any pre-enrichment. The method established in this study has the advantages of good specificity, high sensitivity and accuracy, which provides an effective method for the detection of viableVibrioparahaemolyticusin aquatic foods.