多重富集定量PCR(ME-qPCR)同时检测4种食源性病原弧菌

【目的】溶藻弧菌、副溶血弧菌、创伤弧菌、霍乱弧菌是4种重要的食源性病原弧菌,能够造成人类多种疾病,建立同时检测这4种食源性病原弧菌的检测方法是保障食品安全的基础。本研究旨在建立同时检测溶藻弧菌、副溶血弧菌、创伤弧菌、霍乱弧菌的多重富集定量PCR(multiplex enrichment quantitative PCR,ME-q PCR)方法,并含扩增内标用于指示PCR反应的假阴性,创新一种高通量、高灵敏度、具备定量能力的检测方法,为这4种食源性病原弧菌的检测提供新方法。【方法】以细菌16S r RNA为扩增内标靶序列设计引物,并针对副溶血弧菌的collagenase、溶藻弧菌的gyr B、霍乱弧菌的omp W、创伤弧菌的vvh A,分别设计内、外2对特异性引物,首先将所有内、外引物混合,进行一个循环数较少(10—20 cycles)的高通量多重富集PCR将靶基因富集出来,由于循环数较少,各个基因得到均匀的扩增,每个基因均有4种可能的产物,每1种产物均能作为第二轮巢氏荧光定量PCR的模板,这增加了靶基因从模板中被富集出来的概率,然后将产物稀释后作为模板,利用内引物分别进行巢式荧光定量PCR检测各个基因,最后根据扩增曲线和熔融曲线分析结果。通过设置不同的第一轮多重富集PCR循环数(10、15和20个循环),优化ME-q PCR第一轮循环数。采用14株标准菌株的基因组DNA作为模板,评价ME-q PCR的特异性。并以4种弧菌基因组DNA混合物梯度稀释的样品(100、10、1、0.1、0.01和0.001 ng·μL-1)作为模板,对建立的ME-q PCR进行灵敏度和定量能力的评价。并将该方法应用于已分离到的69株疑似弧菌菌落的鉴定中,与传统生理生化鉴定结果进行对比。【结果】优化后,ME-q PCR的第一轮循环数确定为15,特异性评价结果显示该方法特异性强,灵敏度达0.001 ng,高于普通荧光定量PCR约1个数量级,并能有效指示PCR反应的假阴性,且拥有与普通荧光定量PCR相同的定量能力,扩增效率和R2符合定量的要求;将建立的ME-q PCR方法应用于69株疑似弧菌菌株的鉴定,24个绿色菌落为副溶血弧菌,22个黄色菌落为溶藻弧菌,1个黄色菌落为创伤弧菌,没有检出霍乱弧菌,其结果与生理生化鉴定结果一致。【结论】该方法能够定量、快速准确地检测副溶血弧菌、溶藻弧菌、霍乱弧菌和创伤弧菌这4种食源性病原弧菌,灵敏度高,并能有效指示PCR反应的假阴性,结果无需凝胶电泳,适用于食品中4种常见病原弧菌的快速筛检。

Multiplex Enrichment Quantitative PCR Assays for the Detection of Vibrio alginolyticus,Vibrio parahaemolyticus,Vibrio vulnificus and Vibrio cholerae

【Objective】Vibrio parahaemolyticus, V. alginolyticus, V. cholerae and V. vulnificus have been recognized as the important foodborne pathogens causing human disease. It is important to establish a detection method to identify these 4 foodborne pathogenic Vibrio species in order to ensure food safety. To develop a multiplex enrichment quantitative PCR (ME-qPCR) assay that can simultaneously detect Vibrio parahaemolyticus, V. alginolyticus, V. cholerae and V. vulnificus in the presence of an internal amplification control (IAC), to make it possible for researchers and technical staff achieve simple detection of these 4 foodborne pathogens. 【Method】 Inner and outer species-specific PCR primers were designed based on gyrB gene for V. alginolyticus, collagenase gene for V. parahaemolyticus, vvhA gene for V. vulnificus and ompW gene for V. cholerae, 16S rRNA gene of bacteria as IAC primers was used to indicate false-negative results. All inner and outer primers were used in first round multiplex enrichment PCR with a small number of cycles so as to avoid competition between amplicons. Each gene has 4 types of probable products which can be the templates for the next nested real-time PCR. This can enrich the target genes from the genomic DNA template successfully. The reaction product was then diluted and analyzed individual real-time PCRs using inner primers. The results were analysed by amplification curve and melting curve. ME-qPCR method was developed after optimization of the first round multiplex enrichment PCR cycle numbers (10, 15 and 20 cycles). The specificity of ME-qPCR was validated by 14 bacteria standard strains. The sensitivity and quantitative capability of ME-qPCR were tested by using 10-fold serially diluted genomic DNA of 4 Vibrio strains. Strains as templates, and then, the ME-qPCR was used to detect 69 suspicious Vibrio strains and the results were compared with physiological and biochemical experiments. 【Result】 Fifteen cycles were determined to use in first round multiplex enrichment PCR in this study finally. The results showed that the ME-qPCR assay was rapid, high-throughput, sensitive and specificand the existence of IAC could successfully eliminate false-negative results. The sensitivity of ME-qPCR was 0.001ng per reaction, about 10 times higher than real-time PCR. The ME-qPCR was validated with 69 suspicious Vibrio strains. The result showed that 27 green bacteria colonies were V. parahaemolyticus, 22 yellow bacteria colonies were V. alginolyticus, 1 yellow bacteria colony was V. vulnificus, and V. cholerae was not detected. The results are consistent with physiological and biochemical experiments. 【Conclusion】 The ME-qPCR assay is specific, stable and reliable for the detection of V. parahaemolyticus, V. alginolyticus, V. cholerae and V. vulnificus. It’s sensitivity is high, and can effectively indicate the false negative of PCR reaction, the results without gel electrophoresis, and is suitable for the rapid screening of 4 common pathogenic vibrio. in food