scholarly journals Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

2006 ◽  
Vol 72 (9) ◽  
pp. 6424-6428 ◽  
Author(s):  
Aneta J. Gubala ◽  
David F. Proll
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Molecular Beacon ◽  
Environmental Water ◽  
Molecular Beacons ◽  
Pcr Assay ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACT A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

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 Detection and enumeration of Cryptosporidium oocysts in environmental water samples by Real-time PCR assay

2017 ◽  
Vol 4 (3) ◽  
pp. 42-47
Author(s):  
Mohammad-Reza Mahmoudi ◽  
Mojgan Bandepour ◽  
Bahram Kazemi ◽  
Asad Mirzaei ◽  
◽  
...  
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Pcr Assay ◽  
Cryptosporidium Oocysts

Evaluation of a molecular beacon real-time PCR assay for detection of Baylisascaris procyonis in different soil types and water samples

2009 ◽  
Vol 106 (2) ◽  
pp. 499-504 ◽  
Author(s):  
Rachel R. Gatcombe ◽  
Narayanan Jothikumar ◽  
Sriveny Dangoudoubiyam ◽  
Kevin R. Kazacos ◽  
Vincent R. Hill
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Molecular Beacon ◽  
Soil Types ◽  
Pcr Assay ◽  
Baylisascaris Procyonis

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Molecular Detection of Adenovirus in Tap Water from Coastal Areas Of Karachi Pakistan:The Real-Time PCR Assay

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S140-S140
Author(s):  
A Kalam
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Low Income ◽  
Water Samples ◽  
Real Time Pcr ◽  
Tap Water ◽  
Watery Diarrhea ◽  
Acid Extraction ◽  
Pcr Assay ◽  
Viral Contamination

Abstract Introduction/Objective Diarrhea is a major source of morbidity and mortality in low-income and middle-income countries. In underdeveloped countries, diseases caused by viruses identified in environmental samples cause major health problems. Little knowledge about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. Adenovirus which causes watery diarrhea, particular has been recognized as important causal pathogen. Adenovirus remains a global threat to public health and an indicator of inequity and lack of social development. Tap water samples from coastal sites in Karachi between 2019 and 2020 over a period of 11 months. The total of 40 tap water sample was examined for infectious Adenovirus by a real time polymerase chain reaction (PCR) amplification. Methods/Case Report This Pilot study is conducted on tap water samples from Karachi Pakistan, n=40 are processed. Extraction of nucleic acid from all filtered water samples collected with Sterivex filter units by using Qiagen DNeasy Power Water Sterivex Kit. As per the manufacturer’s instruction. Phocine herpesvirus(PhHV) is added as an external positive control to monitor the efficiency of nucleic acid extraction and amplification. TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) is being used in probe based real time PCR assay,the below 35 Ct value is considered as a positive sample. Results (if a Case Study enter NA) Results showed the total of 37.7% of the sources were positive for adenovirus.The level of viral contamination was moderate to high. Conclusion The results has been showed that no seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Further the Real time PCR assay increases the sensitivity and provides the high resolution of pathogen detection.

Digoxigenin-Labeled Probe-Based Colony Blotting Assay for Rapid Quantification of Salmonella Serovars in Seafood and Water

2012 ◽  
Vol 95 (6) ◽  
pp. 1652-1655 ◽  
Author(s):  
Rakesh Kumar ◽  
K V Lalitha
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Gene Probe ◽  
Pcr Assay ◽  
Nylon Membrane ◽  
Rapid Enumeration ◽  
Salmonella Serovars ◽  
Labeled Probe

Abstract A non-radio-labeled probe-based detection method was developed for rapid enumeration of Salmonella in seafood and water samples. A Salmonella-specific invA gene probe was developed using a digoxigenin-based non-radio labeling assay, which was evaluated with naturally contaminated seafood and water samples. The probe-based technique was further compared with the quantitative PCR assay. The method was specific for detection of different Salmonella serovars without any nonspecific hybridization with other Salmonella-related Enterobacteriaceae. The optimum labeling efficiency was determined for the labeled probe, and 10 pg/μL probe concentration was observed to be most efficient for detection of Salmonella colonies on nylon membrane. Quantification of Salmonella in naturally contaminated seafood and water samples (n = 21) was in the range 10–102 CFU/mL. The assay successfully quantified Salmonella in spiked seafood and water samples in the presence of background flora, and the entire assay was completed within 48 h. The probe-based assay was further evaluated with real-time PCR, and results showed that the assay was comparable to real-time PCR assay. Thus, this probe-based assay can be a rapid, useful, and alternative technique for quantitative detection of Salmonella in food, feed, and water samples.

Sign in / Sign up

Export Citation Format

Share Document