A NOVEL MULTIPLEX PCR SYSTEM FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B, TSST, NUC AND FEM GENES OF STAPHYLOCOCCUS AUREUS IN FOOD SYSTEM

Abstract

Staphylococcal enterotoxin B (SEB), toxic shock syndrome, the superantigens are responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. An easy and quick system is desirable to detect toxin‐producing strains. In this report, we described standardization of a novel multiplex‐polymerase chain reaction (PCR) assay for simultaneous detection of four important genes associated with the Staphylococcus aureus viz., SEB, Tsst, genus‐specific nuclease (nuc) and Fem genes along with an internal amplification control (IAC), which has now become mandatory in diagnostic PCRs, particularly when tested on environmental or food samples. This mPCR method is sensitive enough to detect cells as low as 103 cfu/mL or /g of the food samples. When evaluated on 136 food and environmental samples, the system detected 4 SEB‐positive S. aureus strains. The S. aureus strains that have been identified to contain the SEB gene in the mPCR were unequivocally detected for the toxin expression by the TECRA kit. mPCR produced a 100% correlation with conventional identification method. As SEB and Tsst are qualified as biowarfare molecules, this system is of immense help in detecting them during emergencies of biological war and suspected outbreaks of S. aureus food poisoning directly from the food and environmental samples. PRACTICAL APPLICATIONS The virulence‐associated genes targeted in this study are super antigenic in nature and are responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome, and these molecules are also qualified as biowarfare agents. The high throughput and cost‐effective multiplex PCR method reported here could identify all these genes successfully from both artificially spiked and natural food samples and may also find its application in detection of these toxin‐producing Staphylococcus aureus from clinical and environmental samples.

Propidium monoazide combined with real-time PCR for selective detection of viable Staphylococcus aureus in milk powder and meat products

“…A variety of heat-resistant staphylococcal enterotoxins can contribute to symptoms of food poisoning, including diarrhea. When ingested, staphylococcal enterotoxins can induce gastroenteric syndrome, which is characterized by nausea, vomiting, diarrhea, and abdominal cramps (Zouharova and Rysanek, 2008;Shylaja et al, 2010). A dose less than 1 μg of enterotoxin, which can be produced by >10 5 cfu/g of Staph.…”

“…A dose less than 1 μg of enterotoxin, which can be produced by >10 5 cfu/g of Staph. aureus, in contaminated food can cause symptoms after 1 to 6 h in susceptible persons (Alarcon et al, 2006;Shylaja et al, 2010). Therefore, accurate and easy-to-perform methods for detection of Staph.…”

Development and evaluation of a novel combinatorial selective enrichment and multiplex PCR technique for molecular detection of major virulence-associated genes of enterotoxigenic Staphylococcus aureus in food samples

“…; Shylaja et al . ). The primers for conserved region of sea, seg, sei, coa and mecA were designed using Gene Runner version 3.01 software (Hastings Software Inc., Hastings on Hudson, NY, USA) with reference to GenBank sequences of respective genes (Table ).…”

Water dynamics and microbial communities of bigeye tuna (Thunnus obesus) during simulated cold chain logistics

“…Furthermore, the development of metagenomic technology has enabled the isolation and culture of microorganisms whose total DNA is directly extracted from test samples using traditional methods. The results from using metagenomic technology thoroughly reflect the characteristics of a sample microbial community, enabling a more comprehensive monitoring of bacterial diversity (Shylaja, Murali, Batra, & Bawa, ). A considerable amount of scientific research has summarized the application of high‐throughput sequencing to food science, the medical industry, and environmental science (Andersen & Rinnan, ; Li, Zhang, Song, Feng, & Luo, ; Silbande et al, ; Tong, Miaowen, Juhui, Jinxian, & Baofeng, ; Wu et al, ).…”

“…Furthermore, the development of metagenomic technology has enabled the isolation and culture of microorganisms whose total DNA is directly extracted from test samples using traditional methods. The results from using metagenomic technology thoroughly reflect the characteristics of a sample microbial community, enabling a more comprehensive monitoring of bacterial diversity (Shylaja, Murali, Batra, & Bawa, 2010).…”