Use of a Multiplex PCR System for the Simultaneous Detection of Heat Labile Toxin I and Heat Stable Toxin II Genes of Enterotoxigenic Escherichia coli in Skim Milk and Porcine Stool

Tsen, Hau‐Yang; Jian, L Z; Chi, Wan-Rong

doi:10.4315/0362-028x-61.2.141

Cited by 19 publications

(6 citation statements)

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“…In addition to these, EHEC strain was not detected in any farms that were contaminated with E. coli ( Table 2). This observation is consistent with previous reports claiming that enterotoxigenic E. coli strains that are lt2+ are rarely found [17], and so far, ETEC strains that are lt2+ have been isolated only in Brazil, Thailand and [7,8,18,19]. Roy et al [8] were able to identify the virulent strains lt2 genes of ETEC group and EAE gene of EPEC group from shrimp farms.…”

Section: Pcr Analysis Of Water Sediment Of Shrimp Farms and Shrimp Ssupporting

confidence: 81%

“…Roy et al [8] were able to identify the virulent strains lt2 genes of ETEC group and EAE gene of EPEC group from shrimp farms. In the current experiment, the PCR was performed without screening and removing detritus to avoid target loss [17] and to reduce extra time needed assuming that PCR is able to amplify target DNA if present in the sample. Overall, the PCR analyses confirmed that st1+, lt2+ (of ETEC origin) and vt+ E. coli strains were present in water, sediment and shrimp samples of unhygienic farms.…”

Section: Pcr Analysis Of Water Sediment Of Shrimp Farms and Shrimp Smentioning

confidence: 99%

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Molecular Approach in Detection of Enterovirulent Escherichia coli Strains from Traditional Shrimp Farms of Bangladesh

Raseduzzaman¹,

Sayeduzzaman²,

Shams³

et al. 2016

AJLSR

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Introduction: This study was conducted to detect the presence of enterovirulent Escherichia coli strains using PCR (polymerase chain reaction) technique in some selected unhygienic and hygienic traditional shrimp farms in Bangladesh. The target genes chosen for this investigation included: the phoa housekeeping gene (present in all E. coli); the lt1, lt2 and st1 genes of ETEC (enterotoxigenic); the vtverotoxin, and eae virulence genes of EHEC (enterohaemorrhagic) and EPEC (enteropathogenic), respectively. Methods: Six pairs of oligonucleotide primers were designed to amplify internal fragments of these genes by PCR to generate PCR products that were analyzed by gel electrophoresis. The presence of E. coli strains were determined by visualization of strain specific band comparing with a 100 bp DNA size marker in UV transilluminator. Results:The results revealed that water of both hygienic and unhygienic farms were highly contaminated with E. coli followed by sediment and shrimp. Whereas, only water, sediment and shrimp of unhygienic farms were found to be contaminated with virulent E. coli strains. Conclusions: Among the virulent E. coli strains, ETEC and EPEC strains were detected in unhygienic farms, but no EHEC strain was detected. However, no contamination with virulent E. coli strains was found in hygienic farms.

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Section: Pcr Analysis Of Water Sediment Of Shrimp Farms and Shrimp Ssupporting

confidence: 81%

Section: Pcr Analysis Of Water Sediment Of Shrimp Farms and Shrimp Smentioning

confidence: 99%

Molecular Approach in Detection of Enterovirulent Escherichia coli Strains from Traditional Shrimp Farms of Bangladesh

Raseduzzaman¹,

Sayeduzzaman²,

Shams³

et al. 2016

AJLSR

View full text Add to dashboard Cite

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“…Relevant techniques include use of PCR and/or hybridization probes to detect E. coli-specific genes encoding invasion proteins (12,13), toxins (17,23,24), catabolic enzymes (4,8), or structural (lipo)proteins (10,12). A drawback to these approaches has been that when cross-reactivity tests were done, target sites were often detected not only in E. coli but also in closely related organisms (14,17,18).…”

mentioning

confidence: 99%

Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil

Sabat¹,

Rose

Hickey

et al. 2000

Appl Environ Microbiol

139

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A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminating E. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72°C, which is 10°C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl 2 concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detecting E. coli DNA in heterogeneous DNA samples, such as those extracted from soil.

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“…ETEC, an important cause of travelers' diarrhea and infantile diarrhea, is detected by mPCR by targeting the genes that encode the ETEC enterotoxins, LT (heat-labile enterotoxin) and ST (heat-stable enterotoxin). Tsen et al (1998) developed an mPCR assay specific for eltA (heat-labile enterotoxin subunit A) and the porcine variant of estA (heat-stable enterotoxin) for detection of ETEC in porcine stool and feed samples. Grant et al (2006) used primer-TaqMan probe sets targeting eltB and both human and porcine variants of estA to detect ETEC in raw vegetables, water and salsa.…”

Section: Single-pathogen Detection By Mpcrmentioning

confidence: 99%

Application of Multiplex Polymerase Chain Reaction to the Detection of Pathogens in Food

Perry¹,

Heard²,

Kane³

et al. 2007

Rapid Methods Automation Mic

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Multiplex polymerase chain reaction (mPCR), the simultaneous amplification of two or more polymerase chain reaction (PCR) products in the same reaction tube, can be applied to the rapid detection of pathogenic microorganisms in food matrices. mPCR can be used to test for multiple pathogens simultaneously or to test for multiple virulence factors associated with a single pathogen. Heterogeneous pathogens such as Escherichia coli cannot only be detected by mPCR but can also be differentiated by pathotype or serotype based on amplicon profiles. Sensitive detection of pathogens in food by mPCR (≤1 cfu/g) can be achieved if a high concentration of target DNA with minimal contamination by PCR inhibitors can be prepared. This process generally requires enrichment, which adds 6–24 h to the assay time. Even so, pathogens that grow slowly, are difficult to isolate or are difficult to identify can be detected more rapidly by mPCR than by culture‐based methods. This review covered methods and application of mPCR assays for detection of pathogens in the food production and distribution process.

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Use of a Multiplex PCR System for the Simultaneous Detection of Heat Labile Toxin I and Heat Stable Toxin II Genes of Enterotoxigenic Escherichia coli in Skim Milk and Porcine Stool

Cited by 19 publications

References 0 publications

Molecular Approach in Detection of Enterovirulent Escherichia coli Strains from Traditional Shrimp Farms of Bangladesh

Molecular Approach in Detection of Enterovirulent Escherichia coli Strains from Traditional Shrimp Farms of Bangladesh

Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil

Application of Multiplex Polymerase Chain Reaction to the Detection of Pathogens in Food

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