Application of duplex-PCR in rapid and reliable detection of toxigenic Vibrio cholerae in water samples in Thailand

Chomvarin, Chariya; Namwat, Wises; Wongwajana, Suwin; Alam, Munirul; Thaew-Nonngiew, Kesorn; Sinchaturus, Anuchit; Engchanil, Chulapan

doi:10.2323/jgam.53.229

Cited by 16 publications

(14 citation statements)

References 39 publications

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“…This toxin is considered as a major virulence factor. Therefore, the ctx gene is often used to determine the pathogenicity of V. cholerae (Chen et al, 2004;Chomvarin et al, 2007;Huq et al, 2012). This review will discuss the molecular mechanism of cholerae toxin in causing diarrhea.…”

Section: Oceana Biomedicina Journalmentioning

confidence: 99%

Molecular Mechanism of Cholerae Toxin (ctx) in Causing Diarrhea

Praja

Rosalina

2018

Biomed Journal

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Vibrio cholerae is one of the pathogenic bacteria transmitted through contaminated food, especially seafood and contaminated beverages. V. cholerae produces cholerae toxin (ctx) which is encoded by the ctx gene located within its chromosome. This toxin has been recognized as one of the toxins responsible for cholera outbreaks. The mechanism of ctx gene expression is induced by environmental signals such as pH, osmolarity, temperature, bile, amino acids, and CO2. These signals will be a positive transcriptional factor to the ToxR gene that regulates the biogenesis of cholerae toxin. After cholerae toxin has been successfully expressed, V. cholerae uses a type II secretion (T2S) pathway to deliver cholerae toxin to the extracellular environment. Cholerae toxin consists of A and B subunits. The B subunit plays a role in attaching to the receptor Manosialosyl Ganglioside (GM1 ganglioside) and the A subunit plays a role in catalyzing ADP-ribosylation of Gs (stimulatory) proteins and turning them into active condition. The Gs proteins will convert the inactive adenilate cyclase (AC) into active AC. The increase of AC activity will increase the cyclic adenosine 3'5'-monophosphate (cAMP) concentration along the cell membrane. The cAMP then causes the active secretion of sodium (Na+), chloride (Cl-), potassium (K+), bicarbonate (HCO3-), and water (H2O) out of the cell into the intestinal lumen, resulting in large fluid losses and electrolyte imbalances. Keywords: Vibrio cholerae, cholerae toxin (ctx), ToxR gene, type II secretion (T2S), GM1 ganglioside, adenilate cyclase.

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Section: Oceana Biomedicina Journalmentioning

confidence: 99%

Molecular Mechanism of Cholerae Toxin (ctx) in Causing Diarrhea

Praja

Rosalina

2018

Biomed Journal

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“…Although culture-based microbiological methods for the identification of Vibrio species from environmental samples are reliable, they require several days to complete (Kong et al 2002) and may not be suitable for cells that are viable but non-culturable (VBNC) (Chomvarin et al 2007). …”

Section: Introductionmentioning

confidence: 99%

“…However, the efficiency of PCR detection depends on several factors with the selection of an appropriate DNA extraction method and the pre-PCR procedures considered as the most crucial factors (Chomvarin et al 2007). Various detection protocols have been developed based on cell filtration and PCR amplification of target sequences (Bej et al 1991;Lipp et al 2003) without culturing.…”

Section: Introductionmentioning

confidence: 99%

“…Cholera disease is currently recognised to be caused by the serogroups of V. cholerae O1 and O139 (Chomvarin et al 2007) and have been implicated in various diarrhoeal outbreaks around the world. V. parahaemolyticus accounts for the majority of human Vibrio infections and is acquired through consumption of contaminated water and seafood leading to acute gastroenteritis (Tarr et al 2007).…”

Section: Introductionmentioning

confidence: 99%

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Detection of Vibrio cholerae and Vibrio parahaemolyticus by molecular and culture based methods from source water to household container-stored water at the point-of-use in South African rural communities

Ntema

Potgieter

Barnard

2010

Water Science and Technology

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Detection methods for Vibrio cholerae and Vibrio parahaemolyticus which included the culture based approach with polymerase chain reaction (PCR) confirmation, PCR detection without enrichment and PCR with a pre-enrichment were developed and their performance evaluated. PCR assays targeted the SodB (V. cholerae species), Flae (V. parahaemolyticus species), 16S rRNA (Vibrio and Enterobacteriacea species) genes (Multiplex 1) and V. cholerae O1 and V. cholerae O139 rfb genes, ctxA (cholera toxin) gene and 16S rRNA gene (Multiplex 2). These methods were used to determine the occurrence of selected Vibrios in source water as well as in household container-stored water. The combination of filtration, enrichment and PCR method provided a sensitive and specific method for the detection of selected Vibrios in water samples. The PCR with a pre-enrichment method detected as few as 4-10 cfu/100 mL of selected Vibrios and PCR detection without the enrichment method detected as few as 40-100 cfu/100 mL of selected Vibrios. The inclusion of an enrichment period allows detection of culturable bacteria. As an application of the developed methods, V. cholerae and V. parahaemolyticus were detected in the source water used by the population and in the water-storage containers. The results indicate that Vibrio species in the containers could have originated from the source water and survive in biofilms inside the containers.

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“…They are widely used in microbiology for detection of viruses (Agindotan et al 2007), bacteria (Chomvarin et al 2007), and fungi (Guglielmo et al 2007). Real-time polymerase chain reaction (PCR) is an approved method of detection of genetically modiÞed organism components in food (Ciabatti et al 2006).…”

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confidence: 99%

Molecular Techniques for Detection of Tribolium confusum Infestations in Stored Products

Nowaczyk¹,

Obrępalska‐Stęplowska²,

Gawlak³

et al. 2009

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The confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) is a stored-product pest that contaminates a wide range of food products, from flour and cereals to spices. The insect reduces food quality and is responsible for large economic losses every year. Although several methods for detection of stored-product pests are common and widely used, they are time-consuming and expensive. Therefore, establishing molecular methods of detection of stored-product pests could provide a useful alternative method. We have undertaken attempts to establish methods of detection of T. confusum based on molecular biology techniques of standard and real-time polymerase chain reaction (PCR). Total DNA of T. confusum and red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), used as a negative control, was isolated from insects and used as a template in standard and real-time PCR reactions. Specific primers have been designed on the basis of sequences of internal transcribed spacer (ITS) fragment of rDNA and subunit I of mitochondrial cytochrome oxidase of T. confusum available in the GenBank database. Standard PCR reactions with primers specific to the ITS fragment proved to be reliable and sensitive. Real-time PCR reactions with primers specific for mitochondrial DNA are considered to serve as a supplemental detection method for quantitative assessment of the infestation level.

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Application of duplex-PCR in rapid and reliable detection of toxigenic Vibrio cholerae in water samples in Thailand

Cited by 16 publications

References 39 publications

Molecular Mechanism of Cholerae Toxin (ctx) in Causing Diarrhea

Molecular Mechanism of Cholerae Toxin (ctx) in Causing Diarrhea

Detection of Vibrio cholerae and Vibrio parahaemolyticus by molecular and culture based methods from source water to household container-stored water at the point-of-use in South African rural communities

Molecular Techniques for Detection of <I>Tribolium confusum</I> Infestations in Stored Products

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