Detection of toxigenic Vibrio cholerae with new multiplex PCR

Mehrabadi, Jalil Fallah; Morsali, Parisa; nejad, Hamideh Rohani; Fooladi, Abbas Ali Imani

doi:10.1016/j.jiph.2012.02.004

Cited by 51 publications

(29 citation statements)

References 16 publications

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“…They reported that this developed method is able to detect 8.5 -85 pg of genomic DNA and 10 -100 colony-forming units of V. Cholerea. In addition, the specificity of this technique is 100% (11). In our previous study, we developed a multiplex PCR to detect hlyA, ctxB and tcpI as virulence and regulatory genes.…”

Section: Discussionmentioning

confidence: 99%

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Facile and Rapid Detection of Vibrio cholerae by Multiplex PCR Based on ompU, ctxA, and toxR Genes

Alishahi

Fooladi

Mehrabadi

et al. 2013

Jundishapur J Microbiol

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Background: Several epidemic and endemic cases have been reported involving Vibrio cholerae (V. cholerae) as a causative organism in serious diarrheal diseases with high mortality. Hence, quick identification of this microorganism is critical for health care communities. Objectives: This investigation suggests an accurate, sensitive and rapid test to detect toxigenic and pathogenic species of V. cholerae simultaneously. Materials and methods:The standard bacteriological tests were utilized to isolate V. cholerae from the clinical samples. A multiplex PCR assay was carried out with three separate primers designed for ctxA, toxR and ompU genes, which play basic roles in toxigenicity and pathogenicity. To investigate the specificity of the designed primers, the amplification statuses of Salmonella typhi (S. typhi), Shigella dysenteriae (S. dysenteriae) and Escherichia coli O157:H7, as common intestinal pathogens, were evaluated. In addition, different concentrations of V. cholerae genome were used for determining the test sensitivity. Results: From the total of collected stool samples, 72 V. cholerae isolates were found and separated from the patients. All of them carried toxR and ompU genes, but ctxA gene was detected in only 61 isolates. The specificity and sensitivity of this test was 100%. Conclusions: Obtained data demonstrated that the designed assay is an accurate, easy, rapid, and cost-effective tool for detecting V. cholerae that can serve as an alternate to current bacteriological tests.

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Section: Discussionmentioning

confidence: 99%

“…Furthermore, trained and expert technicians are required to perform the detection processes correctly. Since these requirements are not available in some medical laboratories, eliminating these problems with a quick and simple method is desirable (10,11).…”

Section: Introductionmentioning

confidence: 99%

Facile and Rapid Detection of Vibrio cholerae by Multiplex PCR Based on ompU, ctxA, and toxR Genes

Alishahi

Fooladi

Mehrabadi

et al. 2013

Jundishapur J Microbiol

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“…Up to present, polymerase chain reaction (PCR) is the most powerful analytical method of nucleic acid detection by virtue of its higher sensitivity and specificity . Multiplex PCR is a suitable method for early detection of pathogens .…”

Section: Introductionmentioning

confidence: 99%

“…5 Up to present, polymerase chain reaction (PCR) is the most powerful analytical method of nucleic acid detection by virtue of its higher sensitivity and specificity. [6][7][8] Multiplex PCR is a suitable method for early detection of pathogens. [9][10][11][12] However, owing to the mutual competition and interference between primers, the success rate of obtaining the target DNA varies.…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of foodborne bacterial pathogens using visual high‐throughput microfluidic chip

Jin

Ding²,

et al. 2020

J of Chemical Tech & Biotech

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BACKGROUND Foodborne diseases are a major global public health concern. It is urgent to monitor foodborne pathogens from contaminated foods based on multi‐target detection. RESULTS In this work, a self‐priming compartmentalization (SPC) microdevice made of poly(methyl methacrylate) (PMMA) was developed and integrated into a loop‐mediated isothermal amplification (LAMP) system for performing high‐throughput visual detection. The microfluidic chip is a self‐filling microwell array which can introduce reaction solution only through an access hole in a vacuum pumping system. The independent and closed wells can avoid cross‐contamination and ensure the accuracy of the results. The approach was able to perform the simultaneous identification of six foodborne pathogens (Salmonella typhimurium, Bacillus cereus, Staphylococcus aureus, Yersinia enterocolitica, Vibrio parahaemolyticus and Listeria monocytogenes) within 1 h. The results demonstrated that the method could specifically recognize the six foodborne pathogens. CONCLUSION The use of a LAMP‐based microdevice can be a faster alternative to conventional methods to detect pathogens in food samples, especially for those foods in which no pathogen is allowed (S. aureus), or foods for infant use. The simplicity and portability of the device can provide an alternative platform for foodborne pathogens where food safety needs accurate and timely detection confirmation. © 2020 Society of Chemical Industry

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“…To date, a plethora of real-time PCR assays for the detection of V. cholerae have been published. Three examples are the assays published by Huang et al (2009), Koskela et al (2009) and Mehrabadi et al (2012) While the elaborate multiplex assays typically published in the literature are technologically impressive, almost none of these include a single process control which is carried through both nucleic acid extraction and PCR amplification steps. In addition, most target several virulence genes which may introduce unnecessary complexity when the principal concern is to detect toxigenic V. cholerae in the simplest way possible.…”

Section: Introductionmentioning

confidence: 99%

Development and implementation of a rapid real-time polymerase chain reaction assay for the detection of toxigenic <i>Vibrio cholerae</i> in water

Leat

Grundlingh

2013

WSA

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Assays which use real-time polymerase chain reaction (PCR) technology can be developed for the rapid identification of genetic sequences carried by waterborne pathogens. Rand Water has established facilities within which a selection of PCR assays will be developed. This paper reports on the optimisation and validation of the first assay to be implemented. This assay facilitates the detection of the ctxA gene of toxigenic Vibrio cholerae (V. cholerae) strains. The assay also includes an internal process control in the form of an Escherichia coli (E. coli) strain carrying a single genomic copy of the gfp gene from Aequorea victoria. Establishment of the assay required the selection of suitable PCR primers and probes for both the ctxA and gfp genes. This was followed by an optimisation phase where ideal PCR cycling conditions and primer/probe concentrations were established. A validation phase established the performance parameters of the assay. Parameters assessed included: limit of detection, sensitivity, specificity, reproducibility and robustness. The validation was conducted using potable water, surface water and sewage effluent matrices. The process has resulted in the establishment of a robust assay for the detection of toxigenic V. cholerae strains within 24 hours after samples are received.

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Detection of toxigenic Vibrio cholerae with new multiplex PCR

Abstract: This multiplex PCR method has higher sensitivity and specificity than other methods. The proposed test provides an appropriate and sensitive tool for detecting the presence of toxigenic and pathogenic V. cholerae.

Cited by 51 publications

References 16 publications

Facile and Rapid Detection of Vibrio cholerae by Multiplex PCR Based on ompU, ctxA, and toxR Genes

Facile and Rapid Detection of Vibrio cholerae by Multiplex PCR Based on ompU, ctxA, and toxR Genes

Rapid detection of foodborne bacterial pathogens using visual high‐throughput microfluidic chip

Development and implementation of a rapid real-time polymerase chain reaction assay for the detection of toxigenic <i>Vibrio cholerae</i> in water

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