Design and Validation of a Novel Multiplex Real-Time PCR Assay for Vibrio Pathogen Detection

2011 ◽  
Vol 74 (6) ◽  
pp. 939-948 ◽  
Author(s):  
ROBERT S. TEBBS ◽  
PIUS M. BRZOSKA ◽  
MANOHAR R. FURTADO ◽  
OLGA V. PETRAUSKENE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Nearest Neighbor ◽  
Vibrio Vulnificus ◽  
Molecular Techniques ◽  
Pcr Assay ◽  
Accurate Identification ◽  
Vibrio Pathogens ◽  
Pathogenic Vibrios

Three species—Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus—account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers.

scholarly journals Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Yousun Chung ◽  
Taek Soo Kim ◽  
Young Gi Min ◽  
Yun Ji Hong ◽  
Jeong Su Park ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Methicillin Resistance ◽  
Blood Cultures ◽  
Pcr Assay ◽  
Accurate Identification ◽  
Essential Information ◽  
Methicillin Resistant

Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targetsmecA,femAspecific forS. aureus,femAspecific forS. epidermidis,16S rRNAfor universal bacteria, and16S rRNAspecific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC). For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA), the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistantS. aureus(MRSA), methicillin-susceptibleS. aureus(MSSA), methicillin-resistantS. epidermidis(MRSE), methicillin-susceptibleS. epidermidis(MSSE), methicillin-resistant non-S. epidermidisCoNS (MRCoNS), and methicillin-susceptible non-S. epidermidisCoNS (MSCoNS) (κ=0.9313). The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings.

scholarly journals Detection of Peanut Allergen Traces with a Real Time PCR Assay - The Challenge to Protect Food-Allergic Consumers

2017 ◽  
Vol 7 (1) ◽  
pp. 32 ◽  
Author(s):  
Dimitra Houhoula ◽  
Stamatios Koussissis ◽  
Vladimiros Lougovois ◽  
John Tsaknis ◽  
Dimitra Kassavita ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Food Products ◽  
Molecular Techniques ◽  
Food Allergens ◽  
Pcr Assay ◽  
Peanut Allergen ◽  
Pcr Method ◽  
Detection And Quantification

The aim of the present study was the implementation of molecular techniques in the detection and quantification of allergic substances of peanut in various kinds of food products, e.g., breakfast cereals, chocolates and biscuits that are frequently related to allergies. In some cases, the presence of peanuts can be due to contamination during production and are not declared on the label. A total of 152 samples were collected from supermarkets and were analysed by a Real Time PCR method. The results indicated that 125 samples (83,3%) were found positive in peanut traces but the most important finding is that from the 84 samples that had no allergen declaration for peanuts, 48 (57,1%) of them were found positive. In conclusion, Real Time PCR can be a very important tool for the rapid detection and quantification of food allergens.

scholarly journals Duplex Real-Time PCR Assay for Rapid Detection of Ampicillin-Resistant Enterococcus faecium

2004 ◽  
Vol 48 (2) ◽  
pp. 556-560 ◽  
Author(s):  
Stein Christian Mohn ◽  
Arve Ulvik ◽  
Roland Jureen ◽  
Rob J. L. Willems ◽  
Janetta Top ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Enterococcus Faecium ◽  
Rapid Detection ◽  
Resistant Bacteria ◽  
Pcr Assay ◽  
Culture Methods ◽  
Accurate Identification ◽  
Antibiotic Resistant ◽  
Highly Correlated

ABSTRACT Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the d-Ala-d-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.

Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats

2008 ◽  
Vol 56 (4) ◽  
pp. 451-458 ◽  
Author(s):  
Jitu Patel ◽  
Arvind Bhagwat
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Molecular Beacon ◽  
False Negative ◽  
Detection Methods ◽  
Time Data ◽  
Pcr Assay ◽  
Serovar Typhimurium ◽  
Meat Samples

A real-time PCR assay was evaluated for the rapid detection (10 h) ofSalmonellain meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated withSalmonella entericaserovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.

scholarly journals Development of qualitative methods of bifidobacterium bifidum in probiotic with Real-time PCR method

2018 ◽  
Vol 1 (1) ◽  
pp. 13-17
Author(s):  
Trong Pham Nhu ◽  
Long Le Thanh ◽  
Trung Nguyen Thanh ◽  
Yen Ta Thi ◽  
Loan Pham Thi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dairy Products ◽  
Food Additives ◽  
Bifidobacterium Bifidum ◽  
Pcr Assay ◽  
Accurate Identification ◽  
Powdered Milk ◽  
Pcr Method

Bifidobacterium strains with probiotic effects have been widely used in dairy products, food additives and pharmaceuticals. Especially, Bifidobacterium bifidum (B. bifidum) is usually presented into food products such as functional food. However, it is difficult to detect, and quantify B. bifidum in a sample with a combination of different probiotics. In Vietnam, there is no official standard method to identify and quantify B. bifidum in the sample with the mix of probiotic species. To fulfil the requirements of a robust quality management, we have developed a quantitative real-time PCR assay based on groEL gene for accurate identification and quantification of Bifidobacterium bifidum. The developed assay allows an unambiguous speciesspecific detection. We built the real-time PCR method to detect and identify B. bifidum in functional and supplemented food with specific up to 100% and reproducibility (SR<0.25) suitable with Annex F AOAC: 2016. This real-time PCR method is rapidly and effectively than conventional method. It takes only 24 hours to detect and identify B. bifidum in compare with at least a period of 3-5 days for conventional methods. The low quantitative limit is 105 CFU/g/mL, which is consistent with probiotic and powdered milk products with a declared quality of more than 106 CFU/g/mL.

scholarly journals A real-time PCR for the differentiation of typhoidal and non-typhoidal Salmonella

2019 ◽  
Author(s):  
Satheesh Nair ◽  
Vineet Patel ◽  
Tadgh Hickey ◽  
Clare Maguire ◽  
David R Greig ◽  
...  
Keyword(s):  
Public Health ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Analysis ◽  
Rapid Identification ◽  
Specific Gene ◽  
Whole Genome ◽  
Pcr Assay ◽  
Gene Encoding ◽  
Accurate Identification

AbstractRapid and accurate differentiation of Salmonella spp. causing enteric fever from non-typhoidal Salmonella is essential for clinical management of cases, laboratory risk management and implementation of public health measures. Current methods used for confirmation of identification including biochemistry and serotyping as well as whole genome sequencing analyses, takes several days. Here we report the development and evaluation of a real-time PCR assay that can be performed directly on crude DNA extracts from bacterial colonies, for the rapid identification of typhoidal and non-typhoidal Salmonella.This novel two-hour assay identifies the genus Salmonella by detecting the ttr gene, encoding tetrathionate reductase, and defines typhoidal Salmonella by the detection of S. Typhi and Paratyphi-specific gene combinations. PCR assay performance was determined using 211 clinical cultures of Salmonella (114 non-typhoidal and 97 Typhoidal strains) and 7 clinical non-Salmonella cultures. In addition, the specificity of the assay was evaluated in silico using a diverse in-house collection of 1882 Salmonella whole genome sequences. The real-time PCR results for 218 isolates and the genomic analysis of the 1882 isolates produced 100% sensitivity and 100% specificity (based on a 7 gene profile) for identifying typhoidal Salmonella compared to the Salmonella whole genome sequening identification methods currently used at Public Health England.This paper describes a robust real-time PCR assay for the rapid, accurate identification of typhoidal and non-typhoidal Salmonella which will be invaluable for the urgent screening of isolates from symptomatic individuals, the safe processing of isolates in laboratories and for assisting the management of public health risks.

scholarly journals Rapid and Accurate Identification of Candida albicans and Candida dubliniensis by Real-Time PCR and Melting Curve Analysis

2018 ◽  
Vol 27 (6) ◽  
pp. 543-548 ◽  
Author(s):  
Mohammad Asadzadeh ◽  
Suhail Ahmad ◽  
Noura Al-Sweih ◽  
Ziauddin Khan
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gel Electrophoresis ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Pcr Assay ◽  
Candida Spp ◽  
Accurate Identification

Objective: Candida albicans and Candida dubliniensis are germ tube-positive pathogenic yeast species. Accurate identification of these two species is warranted since C. albicans is a highly pathogenic species while C. dubliniensis exhibits increased adherence to buccal epithelial cells, reduced susceptibility to azoles and resistance to flucytosine. We have developed a duplex real-time PCR assay for rapid detection and differentiation between clinical C. albicans and C. dubliniensis isolates. Materials and Methods: A duplex real-time PCR assay was developed by using two species-specific primer pairs and SYBR Green dye to differentiate C. albicans and C. dubliniensis isolates via melting curve analysis of real-time PCR amplicons. Amplification products were also analyzed by agarose gel electrophoresis to confirm real-time PCR results. Results: Melting temperatures (Tm) for reference strains of C. albicans and C. dubliniensis were 86.55 and 82.75°C, respectively. No amplicon was obtained with DNA from reference strains of 8 other common Candida spp. When real-time PCR was applied on 226 clinical isolates previously identified by the Vitek 2 system and/or PCR sequencing of rDNA, Tm values for C. albicans (n = 113) and C. dubliniensis (n = 98) were 86.68 ± 0.529 and 82.616 ± 0.535°C, respectively. The results were confirmed by agarose gel electrophoresis. No amplicon was obtained from 15 isolates belonging to 9 other Candida spp. Conclusions: The real-time PCR assay described here does not require prior identification of clinical yeast isolates as C. albicans/C. dubliniensis by germ tube formation and accurately reports results within 2 h. Detection of amplicons by agarose gel electrophoresis is also suitable for resource-poor settings devoid of real-time PCR facilities.

A real-time PCR assay for the rapid determination of 16S rRNA genotype in Vibrio vulnificus

2007 ◽  
Vol 68 (2) ◽  
pp. 376-384 ◽  
Author(s):  
Michael C.L. Vickery ◽  
William B. Nilsson ◽  
Mark S. Strom ◽  
Jessica L. Nordstrom ◽  
Angelo DePaola
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Vulnificus ◽  
Rapid Determination ◽  
Pcr Assay

Soil Baiting, Rapid PCR Assay and Quantitative Real Time PCR to Diagnose Late Blight of Potato in Quarantine Programs

2018 ◽  
Vol 4 ◽  
pp. 25-32 ◽  
Author(s):  
Touseef Hussain ◽  
Bir Pal Singh ◽  
Firoz Anwar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Soil Samples ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Pcr Assay ◽  
Diagnostic Laboratory ◽  
Rapid Pcr ◽  
Accurate Detection

Phytophthora infestans (mont) de Bary is a pathogen of great concern across the globe, and accurate detection is an important component in responding to the outbreaks of potential disease. Although the molecular diagnostic protocol used in regulatory programs has been evaluated but till date methods implying direct comparison has rarely used. In this study, a known area soil samples from potato fields where light blight appear every year (both A1 and A2 mating type) was assayed by soil bait method, PCR assay detection and quantification of the inoculums. Suspected disease symptoms appeared on bait tubers were further confirmed by rapid PCR, inoculums were quantified through Real Time PCR, which confirms presence of P. infestans. These diagnostic methods can be highly correlated with one another. Potato tuber baiting increased the sensitivity of the assay compared with direct extraction of DNA from tuber and soil samples. Our study determines diagnostic sensitivity and specificity of the assays to determine the performance of each method. Overall, molecular techniques based on different types of PCR amplification and Real-time PCR can lead to high throughput, faster and more accurate detection method which can be used in quarantine programmes in potato industry and diagnostic laboratory.

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