Development of a PCR-based assay for rapid detection of class IIa bacteriocin genes

Abstract

Aims: We have developed a PCR‐based assay using custom designed panel of primers which allows rapid detection of class IIa bacteriocin‐coding genes. To demonstrate the applicability of the developed assay, the method was applied on 40 metagenomic DNA preparations isolated from native microbiota of Polish artisanal cheeses produced in the Tatra Mountains. Methods and Results: The developed assay was designed on the basis of a large scale alignment of class IIa bacteriocin‐coding genes. A panel of seven primer pairs with confirmed ability to detect class IIa bacteriocin‐coding sequences was obtained. The following study has revealed a superb bacteriocinogenic potential of all forty analysed cheese samples. Conclusions: The majority of obtained sequences were lactic acid bacteria (LAB) related, although some sequences showed significant similarity to bacteriocin‐coding sequences present in non‐LAB bacteriocin producers. The results suggest that several potentially new bacteriocin‐coding sequences were found. Significance and Impact of the Study: The developed assay can be extremely helpful in establishing whether isolates from the environment of interest have a potential of synthesizing antilisterial class IIa bacteriocins. Application of the approach may represent a useful tool contributing to ecological studies looking for valuable probiotic, bacteriocinogenic microbiota developing in foods.

Class IIa Bacteriocins: Diversity and New Developments

“…The screened bacteriocins are then purified and characterized. These classic screening strategies are time-consuming and labor-intensive, so researchers need to explore and develop more rapid and higher-throughput approaches for identification of bacteriocins potential [149–152]. The PCR assays that target bacteriocin-coding genes or bacteriocin regulation-related genes for rapid detection of bacteriocins have been developed [152–156].…”

“…These classic screening strategies are time-consuming and labor-intensive, so researchers need to explore and develop more rapid and higher-throughput approaches for identification of bacteriocins potential [149–152]. The PCR assays that target bacteriocin-coding genes or bacteriocin regulation-related genes for rapid detection of bacteriocins have been developed [152–156]. Most PCR assays can only detect known bacteriocins because they use specific primers which were designed according to previously characterized bacteriocins [154,155,157].…”

Sonorensin: an Antimicrobial Peptide, Belonging to the Heterocycloanthracin Subfamily of Bacteriocins, from a New Marine Isolate, Bacillus sonorensis MT93

“…They used degenerate primers based on the conserved N-terminal regions found in many class IIa bacteriocins and specific downstream primers to design PCRs, resulting in large amplimers specific for pediocin, enterocin, and plantari-cin. Wieckowicz et al used a PCR assay with a custom panel of bacteriocin-related primers to screen metagenomic DNA preparations obtained from the microflora of Polish artisanal cheeses and were able to identify class IIa bacteriocin sequences (22).…”

Isolation and characterization of bacteriocinogenic lactic bacteria from M‐Tuba and Tepache, two traditional fermented beverages in México

“…2007; Wieckowicz et al. 2011). Amplification was performed in a C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA) for 30 cycles as follows: 95°C for 30 sec; 45–55°C for 45 sec, 68°C for 45 sec, followed by a 4-min termination cycle at 68°C.…”