Garrido-Maestu Alejandro, Azinheiro Sarah, Roumani Foteini, Carvalho Joana, Prado Marta
Food Quality and Safety Research Group, International Iberian Nanotechnology Laboratory, Braga, Portugal.
Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain.
Front Microbiol. 2020 Oct 9;11:591041. doi: 10.3389/fmicb.2020.591041. eCollection 2020.
Molecular methods, particularly those based on real-time PCR (qPCR), have become a popular approach to detect pathogens in food samples. This technique may take advantage of hydrolysis fluorescent probes for increased specificity. Even though suitable, this approach loses the capacity of performing result confirmation by melt curve analysis. In the current study, we developed an alternative approach, combining fluorescent probes along with an intercalating dye (SYBR Green) in order to simultaneously detect, and confirm the result, of two foodborne pathogens ( spp. and O157). This new approach named double chemistry qPCR was combined with a short pre-enrichment in order to obtain a multiplex "same-day" detection method for the selected pathogens. The evaluation of the novel method in spiked food samples (ground beef and chicken breast) obtained values of relative sensitivity, specificity, and accuracy higher than 95%, and Cohen's kappa of 0.92, with a Limit of Detection below 5 cfu/25 g, demonstrating its reliability. In addition to this, the method was challenged by inoculating heat-stressed bacteria as well as dead ones. It was observed that it was also possible to detect stressed bacteria with an initial inoculation level below 10 cfu/25 g. Also, it was noticed that high initial concentration of either pathogen (higher than 10 cfu/25 g) was needed in order to generate false positive results due to the presence of dead bacteria, thus the method presents potential for its application in the specific detection of live microorganisms.
分子方法，尤其是基于实时荧光定量PCR（qPCR）的方法，已成为检测食品样本中病原体的常用方法。该技术可利用水解荧光探针提高特异性。尽管这种方法适用，但它失去了通过熔解曲线分析进行结果确认的能力。在本研究中，我们开发了一种替代方法，将荧光探针与嵌入染料（SYBR Green）结合，以便同时检测和确认两种食源性病原体（[具体病原体1]和O157）的结果。这种名为双化学qPCR的新方法与短时间预富集相结合，以获得针对所选病原体的多重“同日”检测方法。在加标食品样本（绞碎牛肉和鸡胸肉）中对该新方法进行评估，得到的相对灵敏度、特异性和准确度值均高于95%，科恩kappa系数为0.92，检测限低于5 cfu/25 g，证明了其可靠性。除此之外，该方法还对接种热应激细菌和死细菌进行了验证。结果发现，对于初始接种水平低于10 cfu/25 g的应激细菌也能够进行检测。此外，还注意到由于死细菌的存在，需要较高的任一病原体初始浓度（高于10 cfu/25 g）才能产生假阳性结果，因此该方法在活微生物的特异性检测中具有应用潜力。
