Development of a qPCR method targeting torA gene and application for the freshness monitoring of modified atmosphere-packed chilled whiting (Merlangius merlangus)
Résumé
Introduction:
Tracking the early decline of freshness is an important concern for the fishery industry to insure the best quality for foodstuffs highly liable to spoil. A lot of techniques have been developed like sensory, chemical and more slightly microbial analysis. Unfortunately, most of them have drawbacks of being subjective, or having the disadvantage of being either reliable once the freshness is lost or for the analysis of whole fish. The study presents the development of a qPCR method targeting a gene harboured by specific spoilage organisms (SSOs) of fish: torA. This gene encodes an enzyme responsible of the production of trimethylamine whose odour is characteristic of the spoiled fish.
Material and methods:
The study aimed to develop a degenerate primer pair able to amplify torA gene in a wide range of SSOs. For that, numerous software and algorithms were used for a maximal reliability of the in silico design process. A first selection of pairs where tested in vitro to further characterization. Finally a primer pair was conserved for efficiency and selectivity tests. The selected primer pair was tested for analysing MAP-chilled whiting along a 15 days storage study. Methods such as total volatile basic nitrogen (TVB-N) and trimethylamine (TMA) analysis or total viable count (TVC) analysis were performed simultaneously to evaluate fish overall quality.
Results and discussion:
A degenerate primer pair was selected after six steps of in silico design and selection. It amplified torA gene of both Vibrio and Photobacterium with good efficiencies, ranging from 91.6 to 93 % regarding species, on 7-log DNA dilutions. The best conditions of annealing temperature and primer concentration were found to be 62°C and 1 µM. As regards with selectivity, the degenerate primer pair allows to selectively amplify torA gene of Vibrio and Photobacterium compared with other tested species. TVC study led to inconclusive results, probably because ISO standard used was not suitable for these kinds of food and storage. However, throughout the storage of fillets, the qPCR approach allows detecting an increase of torA copies. Additionally, good correlation between qPCR results and the evolution of known spoilage markers were established, such as -0.86 (TVB-N) and -0.81 (TMA).
Conclusion:
This study, thanks to careful steps of primers characterization, allowed designing a primer pair able to amplify torA gene of both Vibrio and Photobacterium. A very promising, sensitive and time-effective qPCR technique is thus proposed to characterize the freshness of processed whiting.