scholarly journals Detection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification

2007 ◽  
Vol 73 (5) ◽  
pp. 1457-1466 ◽  
Author(s):  
Else M. Fykse ◽  
Gunnar Skogan ◽  
William Davies ◽  
Jaran Strand Olsen ◽  
Janet M. Blatny
Keyword(s):  
Nucleic Acid ◽  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Environmental Water ◽  
Nucleic Acid Sequence ◽  
Environmental Water Samples ◽  
Dnase Treatment ◽  
Nasba Assay ◽  
Toxigenic Strains

ABSTRACT A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.

scholarly journals Isolation of viable but nonculturable Vibrio cholerae O1 from environmental water samples in Kolkata, India, in a culturable state

2014 ◽  
Vol 3 (2) ◽  
pp. 239-246 ◽  
Author(s):  
Mitsutoshi Senoh ◽  
Jayeeta Ghosh‐Banerjee ◽  
Tamaki Mizuno ◽  
Sumio Shinoda ◽  
Shin‐ichi Miyoshi ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Water Samples ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Viable But Nonculturable ◽  
Vibrio Cholerae O1

scholarly journals Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

2005 ◽  
Vol 71 (11) ◽  
pp. 7113-7116 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Nucleic Acid Sequence ◽  
The Real ◽  
Nasba Assay ◽  
Environmental Pathogens

ABSTRACT A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.

scholarly journals Real-Time PCR Assays for Quantification ofqnrGenes in Environmental Water Samples and Chicken Feces

2012 ◽  
Vol 79 (5) ◽  
pp. 1743-1745 ◽  
Author(s):  
Elisabet Marti ◽  
José Luis Balcázar
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Environmental Water ◽  
Quinolone Resistance ◽  
Environmental Water Samples ◽  
Complex Samples ◽  
Chicken Feces ◽  
Pcr Assays

ABSTRACTReal-time PCR assays were developed for the enumeration of plasmid-mediated quinolone resistance (PMQR) determinants, such as theqnrA,qnrB, andqnrSgenes, in different water samples and chicken feces. The results indicate that the developed assays are specific and sensitive for the quantification ofqnrgenes in complex samples.

scholarly journals Detection of Vibrio cholerae O1 and O139 in Environmental Water Samples by an Immunofluorescent-Aggregation Assay

2010 ◽  
Vol 76 (16) ◽  
pp. 5520-5525 ◽  
Author(s):  
Duochun Wang ◽  
Xuebin Xu ◽  
Xiaoling Deng ◽  
Changyi Chen ◽  
Baisheng Li ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
High Specificity ◽  
Culture Method ◽  
Environmental Water ◽  
Estuarine Water ◽  
Conventional Culture ◽  
Conventional Culture Method

ABSTRACT Environmental waters are an important reservoir for Vibrio cholerae, and effective surveillance of the pathogen can help to warn of and prevent infection with this potentially fatal pathogen. An immunofluorescent-aggregation (IFAG) assay to detect V. cholerae O1 and O139 was established and evaluated with estuarine water samples. The practical application of this assay was compared with the conventional culture method and real-time PCR. The IFAG method had a sensitivity of 103 CFU/ml for detection of V. cholerae O1 and O139 strains in a suspension containing 10 different species of enterobacterial strains (total, 105 CFU/ml). Ten fluorescent bacterial aggregate colonies were randomly picked and tested positive in serum agglutination tests for the V. cholerae O1 and O139 strains, showing a high specificity. The enrichment broths of 146 samples of estuarine water were tested, and the percentage positive by the IFAG assay was 19.9% (29/146), which was significantly higher than that of the conventional culture method (10.3%, 15/146; P < 0.01) but lower than that of real-time PCR (29.5%, 43/146; P < 0.01). The coincidence rates of real-time PCR and IFAG detection were decreased with the reduction of the V. cholerae concentration. The IFAG method, with a high specificity and a relatively high sensitivity, may be used for detection and isolation of V. cholerae in environmental water samples.

scholarly journals Particle Diffusometry: An Optical Detection Method for Vibrio cholerae Presence in Environmental Water Samples

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Katherine N. Clayton ◽  
Taylor J. Moehling ◽  
Dong Hoon Lee ◽  
Steven T. Wereley ◽  
Jacqueline C. Linnes ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Water Samples ◽  
Detection Method ◽  
Optical Detection ◽  
Environmental Water ◽  
Environmental Water Samples
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