ABSTRA ABSTRA ABSTRA ABSTRA ABSTRACT CT CT CT CT: R : R : R : R : Rapid detection of pathogenic and spoilage micr apid detection of pathogenic and spoilage micr apid detection of pathogenic and spoilage micr apid detection of pathogenic and spoilage micr apid detection of pathogenic and spoilage microor oor oor oor oorganisms is essential for ensur ganisms is essential for ensur ganisms is essential for ensur ganisms is essential for ensur ganisms is essential for ensuring the safety and ing the safety and ing the safety and ing the safety and ing the safety and quality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive, quality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive, quality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive, quality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive, quality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive, and specific detection of these micr and specific detection of these micr and specific detection of these micr and specific detection of these micr and specific detection of these microor oor oor oor oorganisms in food. I ganisms in food. I ganisms in food. I ganisms in food. I ganisms in food. In this r n this r n this r n this r n this review eview eview eview eview, w , w , w , w , we discuss r e discuss r e discuss r e discuss r e discuss real-time PCR technologies in eal-time PCR technologies in eal-time PCR technologies in eal-time PCR technologies in eal-time PCR technologies in use today use today use today use today use today, applications of r , applications of r , applications of r , applications of r , applications of real-time PCR in food systems eal-time PCR in food systems eal-time PCR in food systems eal-time PCR in food systems eal-time PCR in food systems, and some of the associated challenges and limitations , and some of the associated challenges and limitations , and some of the associated challenges and limitations , and some of the associated challenges and limitations , and some of the associated challenges and limitations. . . . .

A TaqMan real-time PCR assay was developed to quantify the tetS gene pool present in retail cheeses. This protocol offers a rapid, specific, sensitive, and culture-independent method for assessing antibiotic resistance genes in food samples rich in fats and proteins.Identification of the main routes of the evolution of antibiotic resistance (AR) and its transmission to humans is essential for effective mitigation. The recent findings of large AR gene pools in commensal bacteria in many ready-to-eat retail products indicates that the food production and processing environment might be involved in AR evolution and that food might have become a direct source of dissemination of various AR gene-containing bacteria to the general public on a daily basis (2,4,5,6). Indeed, up to 10 7 CFU of antibiotic-resistant (ART) bacteria per gram of food was found in many retail block cheeses and salads examined, with various AR genes detected in at least 10% of the ART isolates (4, 6). Representative AR genes from several food isolates were successfully transmitted to and caused increased resistance in Streptococcus mutans, indicating the mobility and functionality of the AR genes from the food microbiota (6). These studies, however, used conventional plating methods under given incubation conditions and antibiotic concentrations. Thus, the numbers reported represent only a portion of the total ART bacterial load in these foods. The real magnitude of the AR gene reservoir in food ecosystems is yet to be revealed. Here we report the development of a specific, TaqMan-based real-time quantitative PCR (qPCR) assay to detect and quantify the total tetS gene pools in dairy food products. The tetS gene was chosen because it has been found in both retail cheeses (6) and host oral microbiota (3) and may pose a potential risk to consumers.Assay development. The tetS-specific primer pair (tetSrealFP, 5Ј-GTATGTTCATCTTTCTAAG-3Ј, and tetS-realRP, 5Ј-GCAATAACATCTTTTCAAC-3Ј) and the fluorescencelabeled tetS-specific probe (5Ј FAM-CCATGTGTCCAGGAG TATCTAC-BHQ3Ј) were designed and synthesized according to published procedures (1). The primer pair results in a 190-bp tetS fragment. The qPCR thermoprofiles consist of 95°C for 30 s, followed by 40 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 30 s, on an iCycler (Bio-Rad Laboratories, Hercules, CA).The 667-bp tetS gene fragment was obtained by PCR using Lactococcus sp. strain CZ-T4 DNA as the template (6) and was used as the real-time PCR standard. The copy number concentration of the original standard was calculated based on the mass concentration and the molecular weight of the tetS gene fragment. Tenfold serial dilutions of the standard were made in sterile water and stored at Ϫ80°C until use. A linear standard curve between 10 1 and 10 8 gene copies per reaction was established using the tetS standard DNA (r 2 Ͼ 0.99) (Fig. 1). The standard ranging from 4.86 ϫ 10 8 to 48 copies per reaction was always run in parallel with the samples. qPCR validation. A tetS ϩ cheese isolate, Lactococcus l...

Several virulence factors are involved in Listeria monocytogenes pathogenicity. L. monocytogenes internalins, particularly internalin A, are required for bacterial adhesion to and invasion of human intestinal epithelial cells. The expression of internalins is thus related to virulence. Identification of conditions involved in regulating the expression of L. monocytogenes virulence factors is essential for developing targeted strategies to control listeriosis incidence and improving therapeutic approaches. The primary aim of this study was to develop a quantitative real-time reverse transcriptase PCR platform to study the impact of environmental factors on L. monocytogenes Scott A virulence factor expression, particularly in potentially complex ecosystems. A Taqman PCR-based, rapid quantitative gene expression evaluation method was established with the L. monocytogenes ribosomal protein L4 encoding gene used as an internal standard. Our data suggest that inlA expression is influenced by food composition and temperature, indicating that certain food processing or storage conditions, such as the use of lactic and acetic acids at common storage temperatures, could affect the expression of L. monocytogenes virulence factor.

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