Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milk

ABSTRACT The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.

摘要 本研究旨在建立一套用于牛与水牛奶源鉴定，以及沙门氏菌属（Salmonella spp.）和单核细胞增生李斯特菌（Listeria monocytogenes）检测的标准化聚合酶链式反应（Polymerase Chain Reaction，PCR）方案。为此，我们提取靶DNA并进行混合，随后开展PCR检测实验。实验中，我们对牛奶样品进行掺假处理并人工接种微生物，以此评估不同培养时长、细菌滴度条件下的靶DNA检测效果，以及遗传物质浓度。此外，我们还在未经过预富集步骤的情况下，使用直接从食品中提取的DNA对该方案进行了测试。所提出的四重PCR在鉴定靶DNA序列方面表现出良好的准确性：当样品以2 CFU/250mL的剂量被污染时，可在接种即刻（0h）同时检出所有DNA序列；当初始接种量为1 CFU/250mL时，可在培养6小时后完成检出。当食品以3 CFU/mL的剂量接种细菌时，也可直接从其中检出DNA序列。综上，所提出的方法性能良好，优化了分析时长，且具备检出低滴度微生物的潜力，可用于奶源掺假及食品污染的检测。