Abstract

The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species (P. proteolytica, P. gessardii, P. lactis, P. fluorescens, P. protegens, P. lundensis, and P. fragi) in raw milk, we developed two triplex qPCR assays taking into account species-dependent differences in AprX activity. Besides five species-specific hydrolysis probes, targeting the aprX gene, a universal rpoB probe was included in the assay to determine the total Pseudomonas counts. For all six probes, linear regression lines between C_q value and target DNA concentration were obtained in singleplex as well as in multiplex approaches, yielding R² values of > 0.975 and amplification efficiencies of 85–97%. Moreover, high specificity was determined using genomic DNA of 75 Pseudomonas strains, assigned to 57 species, and 40 other bacterial species as templates in the qPCR. Quantification of the target species and total Pseudomonas counts resulted in linear detection ranges of approx. 10³–10⁷ cfu/ml, which correspond well to common Pseudomonas counts in raw milk. Application of the assay using 60 raw milk samples from different dairies showed good agreement of total Pseudomonas counts calculated by qPCR with cell counts derived from cultivation. Furthermore, a remarkably high variability regarding the species composition was observed for each milk sample, whereby P. lundensis and P. proteolytica/P. gessardii were the predominant species detected.

Key points

• Multiplex qPCR for quantification of seven proteolytic Pseudomonas species and total Pseudomonas counts in raw milk

• High specificity and sensitivity via hydrolysis probes against aprX and rpoB

• Rapid method to determine Pseudomonas contamination in raw milk and predict spoilage potential

中文翻译：

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通过多重qPCR同时定量原料乳中最常见和蛋白水解的假单胞菌种类

摘要

由抗精神病的假单胞菌属种在原奶中分泌的热稳定肽酶AprX是超高温（UHT）奶和奶制品不稳定和过早变质的主要原因。为了能够快速检测和七个频繁和蛋白定量假单胞菌属种（P. proteolytica，P. gessardii，P.球菌，荧光假单胞菌，P. protegens，P. lundensis和P. fragi原料乳），我们开发了两个三重qPCR分析要考虑到ApX活性的物种依赖性差异。除了五种针对特定物种的水解探针外，还针对aprX基因中，通用rpoB探针包含在测定中，以确定总的假单胞菌计数。对于所有六个探针，在单重和多重方法中均获得了C _q值与目标DNA浓度之间的线性回归线，得出的R ²值> 0.975，扩增效率为85-97％。此外，在qPCR中，使用75个假单胞菌菌株的基因组DNA作为模板，确定了高特异性，其中75个假单胞菌菌株属于57种，其他40种细菌为模板。靶标物种和假单胞菌总数的量化导致线性检测范围约为。10 ³ –10 ⁷cfu / ml，与生奶中常见的假单胞菌计数非常吻合。使用来自不同奶牛场的60个原始牛奶样品进行的测定表明，通过qPCR计算的假单胞菌总计数与培养细胞数非常吻合。此外，对于每个牛奶样品，在物种组成方面都观察到非常高的变异性，其中检测到的主要有隆德氏疟原虫和蛋白水解疟原虫/格氏疟原虫。

关键点

•多重定量PCR定量生乳中的7种蛋白水解假单胞菌种类和假单胞菌总数

•通过水解探针对aprX和rpoB的高特异性和敏感性

•快速测定生乳中假单胞菌污染并预测变质潜势的方法