POLYMERASE CHAIN REACTION‐BASED ASSAYS FOR THE DETECTION AND DIFFERENTIATION OF POULTRY SIGNIFICANT PSEUDOMONADS

Abstract

Pseudomonas genus‐specific primers targeting the 16s rRNA gene were used in a real‐time polymerase chain reaction (PCR) assay for rapid analysis of Pseudomonas isolated from retail chicken carcasses. A multiplex PCR assay was also designed using specific primers targeting the gyrase B sub‐unit gene to rapidly distinguish between several species of poultry significant Pseudomonads. The assays were used to evaluate the species and level of spoilage Pseudomonads on raw chicken carcasses over an 8‐day storage period. No Pseudomonas were detected on the chicken carcasses until 4 days after storage using culturing and plating techniques, but the PCR‐based assays developed in this research were more sensitive and detected Pseudomonas in carcass rinses performed immediately after processing. With the multiplex PCR assay, it was determined that most of the Pseudomonas spoilage was because of Pseudomonas fluorescens and Pseudomonas fragi. PRACTICAL APPLICATIONS Economic losses as a result of spoilage are estimated between 5 and 17 billion dollars annually. In the poultry industry, the Pseudomonads not only cause the majority of this spoilage, but are also primary biofilm formers which may harbor and spread pathogens. Currently, only biochemical assays are available in the food industry for detection and differentiation of Pseudomonads. However, biochemical assays require 5 days for results, are limited to detection of a few species, and often produce erroneous results. The PCR assays developed in this work can reduce detection time to a few hours. Furthermore, these nucleic acid‐based assays have the potential to simplify the identification process, reduce food spoilage and foodborne illness, and speed the diagnosis of ill birds.

Microbiome Profiles of Commercial Broilers Through Evisceration and Immersion Chilling During Poultry Slaughter and the Identification of Potential Indicator Microorganisms

“…The organisms present in this study have also been previously reported by other researchers analyzing meat sample microbiomes or from meat spoilage investigations ( Borch et al, 1996 ; Patsias et al, 2006 ; Nychas et al, 2008 ; Handley et al, 2010 ; Rothrock et al, 2016 ; Kim et al, 2017 ). The presence of Pseudomonas in fresh carcasses is consistent with observations made by Hanning et al (2009) when they used PCR to detect and differentiate Pseudomonas spp. from retail poultry carcasses.…”

Salmonella and the Potential Role for Methods to Develop Microbial Process Indicators on Chicken Carcasses

“…PCR has been well established to be able to detect low levels of bacterial cells; Lampel et al (2000) reported detection of Shigella flexneri, Salmonella Typhimurium, and Listeria monocytogenes as low as 30, 50, and 200 Colony Forming Units (CFU), respectively. Hanning et al (2009a) detected Pseudomonas species immediately after processing, where traditional culturing methods did not yield results until day 4. Hanning et al (2009a) detected Pseudomonas species immediately after processing, where traditional culturing methods did not yield results until day 4.…”

“…Using real-time PCR, Bohaychuk et al (2007) were able to detect as low as 1 CFU of Salmonella in artificially contaminated chicken carcasses. However, while PCR targeting DNA can enumerate both viable and nonviable bacterial cellular DNA, the use of mRNA and rRNA as the PCR target will detect only actively growing cells (Hanning et al, 2009a;Maukonen and Saarela, 2009;Park et al, 2014;de Medici et al, 2015). This lower detection limit can reduce the amount of preenrichment time, thus becoming closer to a real-time aspect of data collection.…”

“…This lower detection limit can reduce the amount of preenrichment time, thus becoming closer to a real-time aspect of data collection. An already low amount of DNA present may be lost with multiple wash steps, preventing identification of a potential pathogen or spoilage organism (Miller et al, 1999;Hanning et al, 2009a). However, rRNA and mRNA do have a shorter half-life compared to DNA and require more labor because they are more difficult to isolate in pure form (Scheu et al, 1998;Ye et al, 2012).…”

Metagenomic assessment of the microbial diversity in ground pork products from markets in the North Central Region of South Korea

“…pork-15 is from the pork samples located the first from the top of Fig. 1 et al [20] evaluated the extent of spoilage on raw chicken carcasses via PCR on pseudomonads, and similar assays could be implemented to assess spoilage in ground pork products. Future research can assess fluctuations in Pseudomonas species, suggest the optimal percentage of Pseudomonas species in ground pork that have the longest shelf stability, and how to genetically engineer a beneficial species of Pseudomonas that reduces spoilage and/or foodborne illness to lead intervention strategies.…”