scholarly journals Rapid and Accurate Identification by Real-Time PCR of Biotoxin-Producing Dinoflagellates from the Family Gymnodiniaceae

Marine Drugs ◽  
2014 ◽  
Vol 12 (3) ◽  
pp. 1361-1376 ◽  
Author(s):  
Kirsty Smith ◽  
Miguel de Salas ◽  
Janet Adamson ◽  
Lesley Rhodes
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Accurate Identification ◽  
The Family

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.

scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

A real-time PCR toolbox for accurate identification of invasive fruit fly species

2015 ◽  
Vol 140 (7) ◽  
pp. 536-552 ◽  
Author(s):  
M. K. Dhami ◽  
D. N. Gunawardana ◽  
D. Voice ◽  
L. Kumarasinghe
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fruit Fly ◽  
Accurate Identification

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 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 A New Approach for Accurate Identification of Allele State Based on Real-Time PCR for Biomedical Tasks

2022 ◽  
Author(s):  
Ernest Benaguev ◽  
Ivan Vladimirov ◽  
Olga Pavlova ◽  
Denis Bogomaz
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic System ◽  
Significant Snps ◽  
Nucleotide Polymorphisms ◽  
Accurate Identification ◽  
New Approach ◽  
Conventional Systems ◽  
Fine Tune

Genotyping of single nucleotide polymorphisms (SNPs) is an important task in medicine, veterinary medicine and biology. Precise differentiation of SNPs can be challenging. Methods based onTaqman can lead to false positive results due to nonspecific annealing of the probe. The aim of this research was to develop a new approach for the accurate differentiation of SNPs based on real-time PCR with Taqmanprobes and their rivals.The rivals competed with the Taqmanprobes for annealing to the site. The rivals blocked the nonspecific allele so that the Taqmanprobe could not anneal to it. Thus,the Taqmanprobe only detected specific alleles.This approach madeit possible to fine-tune the diagnostic system by selecting the ratio of Taqmanprobes and rivals (in non-equimolar amounts too).The new approach was tested on several diagonally significant SNPs in veterinary medicine.Using Taqman probes and rival probes showed a significantly greater specificity and efficiency in the determination of both homozygotes and heterozygotes than when conventional systems based only on Taqmanwere used. Keywords: SNP, allele identification, real-time PCR, fluorescent dye

Fluorescent PCR Based Gene Dose Assessment for Detection of Deletion Mutations in the FIX Gene Among Carriers of Hemophilia B.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3486-3486
Author(s):  
Eunice Sindhuvi Edison ◽  
G. Sankari Devi ◽  
G. Jayandharan ◽  
Shaji R Velayudhan ◽  
Auro Viswabandya ◽  
...  
Keyword(s):  
Linkage Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Gene Dosage ◽  
Family Members ◽  
Peak Height ◽  
Hemophilia B ◽  
Carrier Status ◽  
Fluorescent Pcr ◽  
The Family

Abstract Abstract 3486 Poster Board III-423 Haemophilia B (HB), an X linked inherited disorder is caused by heterogeneous mutations in the F9 gene. Approximately 3% of hemophilia B patients have major deletions in the F9 gene. Gross and small deletions in the F9 gene in HB affected males are easily detected by PCR but detecting the carrier state of females in the family is challenging due to the presence of the normal allele. Different methods like linkage analysis, real time PCR and MLPA have been used to assess the carrier status in this situation. Linkage analysis is limited by the availability of informative markers and adequate number of family members. Real time PCR involves standardisation and preparation of calibration curves for each run. Although MLPA is a better alternative, it can be time consuming and involves multiple steps. We have therefore developed a fluorescent PCR based gene dosage approach which is simple, rapid and cost-effective for determining the carrier status of females in families with deletions in the F9 gene. 200ng of DNA extracted by standard protocols was used for amplification with primers designed to amplify a 160bp product from exon h in the F9 gene. One of the primers was fluorescently labelled. Amplification was carried out using these primers for 20 cycles only and the amplified product was subjected to capillary electrophoresis on an ABI 310 genetic analyser. A 230 bp fragment in the albumin gene was used as the control. Analysis was done using Genescan and Genotyper software. The ratio between the peak heights of the exon h in the F9 and control genes in the patient samples were normalised to a normal control. Five families with deletional HB were analysed (in toto deletion-1; Ex g-h – 1; Ex g-poly A-1; Ex h-poly A-2). The ratios in the probands and the family members are presented in the table. Out of eight females analysed, 6 were carriers and 2 were normal. The mean ratio in the carriers was 0.49±0.08 and 0.75±0.05 in the normal. Deletions are not uncommon in HB and deletions involving the exon g and h constitute a major group. Among 212 families with HB assessed at our centre, we have identified large deletions in 8 families (3.7%). It is interesting to note that all except one of these deletional mutations involved exon h. This method confirmed the presence of these deletions in the males and helped us to identify the carrier status of the females in the family. Identification of carrier status of females with deletions in F9 gene by gene dosage Subject ID Peak height of Exh in F9 Peak height of albumin Normalised Ratio Interpretation HB5 284 442 0.59 Carrier HB6 305 489 0.57 Carrier HB22 188 372 0.47 Carrier HB63 85 165 0.48 Carrier HB129 247 295 0.78 Normal HB238 94 326 0.4 Carrier HB280 372 679 0.77 Normal HB384 202 670 0.4 Carrier Disclosures: No relevant conflicts of interest to declare.

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.

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