Detection of PCR amplicons from bacterial pathogens using microsphere agglutination

Wu, Shaw Jye; Chan, Alex W; Kado, Clarence I.

doi:10.1016/j.mimet.2003.11.005

Cited by 17 publications

(11 citation statements)

References 27 publications

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“…According to Toma et al (2003) , the primer stx was designed for the specific identification of E. coli O157:H7 which detects both stx1 or/and stx2 genes, and this has also been applied in different studies (Aranda et al 2007 ; Tobias and Vutukuru 2012 ; Toma et al 2003 ). The inv A gene of S. enterica selected in the study has already been proven to be an important diagnostic tool for detection, as it contains a sequence unique to this genus (Rahn et al 1992 ; Shanmugasamy et al 2011 ). While the hly A gene codes for the action of listeriolysin protein ( hly A), which was mainly the potential of virulence pathogenic of L. monocytogenes , this was considered as the target gene for PCR detection (Dharmendra et al 2013 ; Kaur et al 2007 ).…”

Section: Discussionmentioning

confidence: 99%

Multiplex PCR for simultaneous identification of E. coli O157:H7, Salmonella spp. and L. monocytogenes in food

Nguyen

Giau

Vo³

2016

3 Biotech

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The rapid detection of pathogens in food is becoming increasingly critical for ensuring the safety of consumers, since the majority of food-borne illnesses and deaths are caused by pathogenic bacteria. Hence, rapid, sensitive, inexpensive and convenient approaches to detect food-borne pathogenic bacteria is essential in controlling food safety. In this study, a multiplex PCR assay for the rapid and simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes was established. The invA, stx and hlyA genes specifically amplified DNA fragments of 284, 404 and 510 bp from Salmonella spp., L. monocytogenes and E. coli O157:H7, respectively. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity of the multiplex PCR were performed by testing different strains. The multiplex PCR assay was able to specifically simultaneously detect ten colony-forming unit/mL of each pathogen in artificially inoculated samples after enrichment for 12 h. The whole process took less than 24 h to complete, indicating that the assay is suitable for reliable and rapid identification of these three food-borne pathogens, which could be suitable in microbial epidemiology investigation.

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Section: Discussionmentioning

confidence: 99%

Multiplex PCR for simultaneous identification of E. coli O157:H7, Salmonella spp. and L. monocytogenes in food

Nguyen

Giau

Vo³

2016

3 Biotech

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“…Specific primers (Ecoli670‐F/R, LM404‐F/R, and Sal284‐F/R) used to detect the E. coli , L. monocytogenes , and Salmonella spp. are from the references (McDaniels and others 1996; Wu and others 2004), respectively. Compound specific primers (Ecoli706‐F/R, LM440‐F/R, and Sal320‐F/R) used in UP‐M‐PCR for the specific detection of E. coli , L. monocytogenes , and Salmonella spp.…”

Section: Methodsmentioning

confidence: 99%

Universal Primer‐Multiplex PCR Approach for Simultaneous Detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in Food Samples

Yang¹,

Xu²,

Zhai³

et al. 2009

Journal of Food Science

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Escherichia coli, Listeria monocytogenes, and Salmonella spp. are 3 kinds of the most important food-borne human pathogens. Traditional microbiological analysis is labor-intensive, time-consuming, and easily contaminated, thus producing false positive signals; it also involves much subjectivity judgments. Multiplex-PCR could be applied to detect multiple target organisms simultaneously to save time and labor, but there is always disproportionate amplification resulting from the disparity of different primers. To gain a rapid and sensitive method, a universal primer-multiplex PCR system (UP-M-PCR) was developed and applied for simultaneous detection of the 3 organisms. This method simplified traditional multiplex-PCR reaction system and overcame its amplification disparities among different primers; moreover, it got a high specificity and sensitivity (85, 155, and 104 copies/reaction for E. coli O157, L. monocytogenes, and Salmonella spp., respectively). Compared with the time-consuming and laborious microbiological analysis, UP-M-PCR had a lower risk of cross-contamination without inoculation and incubation. Test results for 36 food samples showed that UP-M-PCR method got a relative accuracy of 91.77% when compared with traditional microbiological analysis. It could serve as a rapid screening method for pathogen detection and could detect target genes even in dead pathogenic cells. In addition, it has the potential to be performed in an automation mode and might find broader application in simultaneous detection of other multiple pathogens.

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“…The primers for detecting E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica were targeted specifically on the rfbE (This study), hlyA (Wu et al ) , nuc (Brakstad et al ) and invA genes (Hoorfar et al ), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The primers for detecting E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica were targeted specifically on the rfbE (This study), hlyA (Wu et al 2004), nuc (Brakstad et al 1992) and invA genes (Hoorfar et al 2000), respectively. The rfbE gene encoding O157 lipopolysaccharide and for E. coli O157: H7 serogroup is unique (Oberst et al 1998), hlyA encoding listeriolysin for phagosomal escape into the host cell's cytosol (Wu et al 2004), the nuc (thermonuclease) gene in S. aureus (Brakstad et al 1992) and invA encoding an invasion protein in S. enterica (Hoorfar et al 2000). The primers were synthesized by Macrogen Company from South Korea.…”

Section: Primer Designmentioning

confidence: 99%

Multiplex touchdown PCR assay to enhance specificity and sensitivity for concurrent detection of four foodborne pathogens in raw milk

et al. 2019

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Aims The aim of this study was to develop a multiplex touchdown PCR (multiplex TD‐PCR) for rapid and simultaneous detection of four major foodborne pathogens to avoid mispriming and unwanted production during gene amplification. Touchdown PCR is the modified form of standard PCR, which enhances specificity, sensitivity. Methods and Results For this reason, a multiplex TD‐PCR assay with a pre‐enrichment step was developed to detect four foodborne pathogens namely Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica serovar Enteritidis in pure culture and raw milk samples. The results showed that this protocol can eliminate the unwanted band or reduce significantly. The detection sensitivity of the single and multiplex TD‐PCR was one cell per ml in pure culture. Furthermore, the detection limit of multiplex TD‐PCR was one cell per 25 ml for artificially contaminated raw milk. We obtained similar results for detection of aforementioned pathogens in raw milk, after comparing the multiplex TD‐PCR method with the traditional culture, except in one or two samples. Conclusions Hence, the proposed multiplex TD‐PCR method could be confirmed as an effective way for rapid optimization of PCR reactions to increase specificity, sensitivity during gene amplification. Significance and Impact of the Study Hence, due to its simplicity, cost‐effectiveness and being time‐saving, it seems that this method is reasonable and economical for rapid optimization of PCR reactions.

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Detection of PCR amplicons from bacterial pathogens using microsphere agglutination

Cited by 17 publications

References 27 publications

Multiplex PCR for simultaneous identification of E. coli O157:H7, Salmonella spp. and L. monocytogenes in food

Multiplex PCR for simultaneous identification of E. coli O157:H7, Salmonella spp. and L. monocytogenes in food

Universal Primer‐Multiplex PCR Approach for Simultaneous Detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in Food Samples

Multiplex touchdown PCR assay to enhance specificity and sensitivity for concurrent detection of four foodborne pathogens in raw milk

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