Simultaneous detection of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in sliced fruits using multiplex PCR

Microbial water-borne diseases still affect developing countries and are major water quality concerns throughout the world. Routine culture-based methods of identifying bacterial pathogens in water sources are laborious and time-consuming. Recently, the use of molecular techniques such as the polymerase chain reaction (PCR) has provided rapid and highly promising detection methods. In this study, we developed two multiplex PCR assays for simultaneous detection of six water-borne bacteria. Two triplex PCR protocols were developed to detect six target genes. The first protocol targets uidA (Escherichia coli), int (Shigella spp.), and gyrB (Pseudomonas aeruginosa) genes, while invA (Salmonella spp.), ompW (Vibrio cholera), and lacZ (coliforms) were amplified by the second protocol. Specificity testing was carried out for 12 reference strains. Furthermore, the applicability of the multiplex PCR assays for detection of these bacteria was investigated for 52 surface water samples. The results indicated that all primer pairs showed specificities only for their corresponding target organisms. The detection sensitivity of both multiplex PCR assays was 3 × 102 − 3 × 103 colony forming units. The developed assays represent simple and efficient diagnostic procedures for co-detection of water-borne bacteria and have the potential to provide earlier warnings of possible public health threats and more accurate surveillance of these organisms.

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Multiplex PCR for Simultaneous Detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in Meat Samples

Journal of Food Protection ◽

10.4315/0362-028x-68.3.551 ◽

2005 ◽

Vol 68 (3) ◽

pp. 551-556 ◽

Cited By ~ 95

Author(s):

SUSUMU KAWASAKI ◽

NAOKO HORIKOSHI ◽

YUKIO OKADA ◽

KAZUKO TAKESHITA ◽

TAKASHI SAMESHIMA ◽

...

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Simultaneous Detection ◽

Detection Sensitivity ◽

Rapid Screening ◽

Escherichia Coli O157 ◽

Salmonella Spp ◽

E Coli ◽

Meat Samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 103 CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.

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Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR

Food Control ◽

10.1016/j.foodcont.2008.09.010 ◽

2009 ◽

Vol 20 (8) ◽

pp. 733-738 ◽

Cited By ~ 69

Author(s):

Andrea Germini ◽

Annalisa Masola ◽

Paola Carnevali ◽

Rosangela Marchelli

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Simultaneous Detection ◽

Salmonella Spp

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Simultaneous detection of Salmonella spp and Escherichia coli O157:H7 by multiplex PCR

Journal of Industrial Microbiology & Biotechnology ◽

10.1038/sj.jim.2900520 ◽

1998 ◽

Vol 21 (3) ◽

pp. 92-98 ◽

Cited By ~ 43

Author(s):

P M Fratamico ◽

T P Strobaugh

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Simultaneous Detection ◽

Escherichia Coli O157 ◽

Salmonella Spp

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Simultaneous Detection of Listeria monocytogenes, Escherichia coli O157:H7, Bacillus cereus, Salmonella spp., and Staphylococcus aureus in Low-fatted Milk by Multiplex PCR

Korean Journal for Food Science of Animal Resources ◽

10.5851/kosfa.2014.34.5.717 ◽

2014 ◽

Vol 34 (5) ◽

pp. 717-723 ◽

Cited By ~ 8

Author(s):

Ji-Hyun Kim ◽

Seong-Ryul Rhim ◽

Kee-Tae Kim ◽

Hyun-Dong Paik ◽

Joo-Yeon Lee

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Listeria Monocytogenes ◽

Bacillus Cereus ◽

Multiplex Pcr ◽

Simultaneous Detection ◽

Escherichia Coli O157 ◽

Salmonella Spp ◽

And Staphylococcus Aureus

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Multiplex PCR for the simultaneous detection of Salmonella spp. and Salmonella Enteritidis in food

International Journal of Food Science & Technology ◽

10.1111/j.1365-2621.2011.02646.x ◽

2011 ◽

Vol 46 (7) ◽

pp. 1502-1507 ◽

Cited By ~ 25

Author(s):

Daniel Santos Pinto Silva ◽

Thayse Canato ◽

Marciane Magnani ◽

Juliane Alves ◽

Elisa Yoko Hirooka ◽

...

Keyword(s):

Multiplex Pcr ◽

Salmonella Enteritidis ◽

Simultaneous Detection ◽

Salmonella Spp

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Reverse Transcription-Multiplex PCR Assay for Simultaneous Detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi

Clinical Chemistry ◽

10.1373/clinchem.2004.036814 ◽

2004 ◽

Vol 50 (11) ◽

pp. 2037-2044 ◽

Cited By ~ 41

Author(s):

Nicole J Morin ◽

Zhilong Gong ◽

Xing-Fang Li

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Vibrio Cholerae ◽

Reverse Transcription ◽

Simultaneous Detection ◽

Salmonella Typhi ◽

Escherichia Coli O157 ◽

Single Tube ◽

E Coli ◽

Vibrio Cholerae O1

Abstract Background: Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi are pathogenic bacteria that can be found in contaminated water supplies throughout the world. No currently available assays can simultaneously detect and identify all three pathogens. Our aim was to develop a rapid and reliable technique for simultaneous detection of these pathogens. Methods: Four unique genes were chosen as the targets of detection. Forward and reverse primers were designed to specifically amplify different sizes of these target genes: a 239-bp region of the E. coli O157 lipopolysaccharide (LPS) gene (rfbE); a 179-bp region of the H7 flagellin gene (fliC); a 419-bp region of the V. cholerae O1 LPS gene (rfbE); and a 329-bp region of Salmonella Typhi LPS gene (tyv). To ensure the detection of only viable replicating bacteria, RNA was extracted for analysis. After reverse transcription, cDNAs were simultaneously amplified in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. To characterize the assay we analyzed, in a blinded fashion, seven unknown RNA samples containing various combinations of total RNA from these bacteria as well as clinical isolates. Results: All seven unknown RNA samples were correctly identified. The assay was able to detect and identify as few as 30 cells of E. coli O157:H7 and Salmonella Typhi in clinical isolates, and the presence of other bacteria did not interfere with the analysis. Conclusion: An assay combining reverse transcription with single-tube multiplex PCR was successfully developed and validated for simultaneous detection of viable E. coli O157:H7, V. cholerae O1, and Salmonella Typhi.

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