**Conflict of interest**

*Synthetic Biology - New Interdisciplinary Science*

**Water and vegetables**

**Table 2.**

*different food matrices.*

causing loss of genomic DNA during extraction [35] and reducing the efficiency of PCR. However, PMA has been shown to be highly selective in penetrating only bacterial cells with compromised membrane integrity, but not in cells with intact cell membranes. After the DNA intercalation of nonviable cells, the azide group, present in the dye molecule, forms a covalent grid and when exposed to halogen light makes the DNA insoluble, which results in its loss during the extraction process of the genomic DNA. Thus, exposing a bacterial population composed of living and dead cells to PMA treatment results in the selective removal of DNA from dead cells [32]. Nevertheless, the dose of PMA must be carefully adjusted because this reagent becomes increasingly toxic to cells at higher concentrations. It is important to note that the cost of method may become prohibitive in the case of increasing concentra-

*Summary of the studies using PMA or EMA prior to PCR methods for microbiological analysis applied in* 

Lettuce *Salmonella typhimurium* PMA-qPCR [66] Lettuce and soya sprouts *E. coli* O157:H7 PMA-qPCR [67] Fresh spinach *Salmonella* spp. PMA-qPCR [43]

**Food matrix Microorganisms Cell viability** 

Water *Campylobacter jejuni; Campylobacter coli*

*\*Bacterial culture in physiological status of "viable but nonculturable" (VBNC).*

**dye-PCR method**

EMA-qPCR [65]

**References**

tion of PMA for its use in different food matrix, or its use in large scale [36].

intended testing situation prior to implementation.

granted to Amanda Teixeira Sampaio Lopes.

The qPCR came with the intention of reducing the time of analysis and laborious work of the microbiological culture method. The analysis of a food sample performed by qPCR allows the monitoring of amplification while it runs; therefore, it does not need to perform any postreaction processing, such as the electrophoresis gel, allowing results available in around 2 h. Nevertheless, the difficulty of distinguishing living cells from dead cells is the great obstacle when using this methodology as routine food analysis laboratories. In this way, the pretreatment of food samples using PMA (or EMA) aims at eliminating false-positive results, as it only allows the quantification of viable cells. Thus, the PMA/EMA-qPCR promises to be a valuable tool in food safety management and microbiological quality control, especially as a method for quantitative microbial risk assessment. It is critical, therefore, that assays are comparatively evaluated in different food matrices for the detection and quantification of different pathogens and their reproducibility must be validated with intralaboratory experiments to ensure their effectiveness in the

The authors thank Universidade Estadual de Santa Cruz for the financial support. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES), Finance Code 001, for the scholarship

**188**

**4. Conclusion**

**Acknowledgements**

The authors declare no conflict of interest.
