**8.3. Sensitive methods (including ELISA and molecular DNA techniques) at the level of academic or innovation labs**

Plaque assays and acidification tests are microbiological methods that are economically accessible and sensitive enough for detection of phages in the dairy industry. These techniques are time consuming, but provide many practical data for both dairy plants and starter producers. The polymerase chain reaction (PCR), ELISA and flow cytometry-based methods have been designed for detecting phages and are often used to complement microbiology tests. However, they have still many drawbacks to be applied for routine analyses in the dairy industry [104].

PCR-based methods detect virulent and non-virulent phages; thus, microbial methods should be used in parallel to precisely distinguish the virulent phages. PCR-based methods can also be too expensive and too specific (only phages targeted by specifically-designed primers are detected) for routine experiments. However, PCR is a fast method able to confirm the presence of bacteriophages within 30 minutes and can be applied to determine the potential utility and quality of big batches of milk. At the same time, the method could be handy in finding niches of phage accumulation, in order to reduce their impact in dairy fermentations [105-108].

ELISA techniques use for phage detection antibodies which are highly specific against structural proteins of phage capsids. Due to the wide phage diversity in the dairy environment, development of several antibodies detecting various groups of phages was required. ELISA is regarded as a highly useful method for monitoring specific phages in the dairy environment, but a single assay cannot be used to detect phages with different structural proteins. For this reason, the sensitivity of an ELISA method to detect phages in dairy a sample is rather low.

Flow-cytometry can also be used for detection of phages in dairy samples by discriminating the phage-infected cells from non-infected based on cell morphological changes leading to lysis. Running on the flow-cytometry of samples containing phages gives a broad distribution of cell mass (wide peak), which demonstrates the presence of both lysed and live cells, while non-infected samples give narrow peaks. Flow-cytometry allows detection of phages in real time, but expensive equipment and well-trained staff needed to perform the assays limits application of this technique in the dairy industry [104].
