Sensitivity of multiplex real-time PCR reactions, using the LightCycler and the ABI PRISM 7700 Sequence Detection System, is dependent on the concentration of the DNA polymerase

2002 ◽  
Vol 16 (5) ◽  
pp. 351-357 ◽  
Author(s):  
M.M. Exner ◽  
M.A. Lewinski
Keyword(s):  
Real Time ◽  
Dna Polymerase ◽  
Real Time Pcr ◽  
Detection System ◽  
Sequence Detection

scholarly journals Different Real-Time PCR Formats Compared for the Quantitative Detection of Human Cytomegalovirus DNA

1999 ◽  
Vol 45 (11) ◽  
pp. 1932-1937 ◽  
Author(s):  
Andreas Nitsche ◽  
Nina Steuer ◽  
Christian Andreas Schmidt ◽  
Olfert Landt ◽  
Wolfgang Siegert
Keyword(s):  
Real Time ◽  
Human Cytomegalovirus ◽  
Real Time Pcr ◽  
Detection System ◽  
Quantitative Detection ◽  
Fluorescence Signal ◽  
Hybridization Probe ◽  
Sequence Detection ◽  
Hybridization Probes ◽  
Assay Time

Abstract Background: The aim of this study was to compare the ABI PRISM 7700 Sequence Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable for routine hospital application. Methods: We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments. Results: The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 101 to 107 HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation. Conclusions: The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.

scholarly journals Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

2021 ◽  
Vol 9 (5) ◽  
pp. 1031
Author(s):  
Roberto Zoccola ◽  
Alessia Di Blasio ◽  
Tiziana Bossotto ◽  
Angela Pontei ◽  
Maria Angelillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Bacterial Load ◽  
Microbial Control ◽  
Hospital Setting ◽  
Sample Volume ◽  
Analytical Sensitivity ◽  
Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system

2004 ◽  
Vol 30 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Sani Hussein Aliyu ◽  
Muktar Hassan Aliyu ◽  
Hamisu M Salihu ◽  
Surendra Parmar ◽  
Hamid Jalal ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Detection System ◽  
Hepatitis B Virus Dna ◽  
B Virus

scholarly journals Species identification of Anguilla japonica by real-time PCR based on a sequence detection system: a practical application to eggs and larvae

2009 ◽  
Vol 66 (9) ◽  
pp. 1915-1918 ◽  
Author(s):  
Yuki Minegishi ◽  
Tatsuki Yoshinaga ◽  
Jun Aoyama ◽  
Katsumi Tsukamoto
Keyword(s):  
Real Time ◽  
Species Identification ◽  
Developmental Stages ◽  
Detection System ◽  
Anguilla Japonica ◽  
Sequence Detection ◽  
Target Region ◽  
System A ◽  
Sequence Variations ◽  
Eggs And Larvae

Abstract Minegishi, Y., Yoshinaga, T., Aoyama, J., and Tsukamoto, K. 2009. Species identification of Anguilla japonica by real-time PCR based on a sequence detection system: a practical application to eggs and larvae. – ICES Journal of Marine Science, 66: 1915–1918. To develop a practical method for identifying Japanese eel Anguilla japonica eggs and larvae to species by a sequence detection system using a real-time polymerase chain reaction (PCR), we examined (i) the sensitivity of the system using samples at various developmental stages, and (ii) influences of intra- and interspecific DNA sequence variations in the PCR target region. PCR amplifications with extracted DNA solution at 7.0 ng µl−1 or lower were efficient at distinguishing A. japonica from other anguillids. A single egg at the gastrula or later developmental stages could also be identified. Two sequence variations in the PCR target region were observed in 2 out of 35 A. japonica collected from three localities, and from four year classes at a single locality. These mutations, however, did not affect the result of species identification achieved by A. japonica-specific PCR primers and probe. The accuracy of this PCR-based method of species identification will help in field surveys of the species.

scholarly journals Characterization and Event Specific-Detection by Quantitative Real-Time PCR of T25 Maize Insert

2005 ◽  
Vol 88 (2) ◽  
pp. 536-546 ◽  
Author(s):  
Cécile Collonnier ◽  
Alexandra Schattner ◽  
Georges Berthier ◽  
Francine Boyer ◽  
Géraldine Coué-Philippe ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection System ◽  
Integration Site ◽  
Ribosomal Gene ◽  
Cauliflower Mosaic Virus ◽  
Plant Genome ◽  
35S Promoter ◽  
Specific Detection ◽  
A Genome

Abstract T25 is one of the 4 maize transformation events from which commercial lines have so far been authorized in Europe. It was created by polyethylene glycol-mediated transformation using a construct bearing one copy of the synthetic pat gene associated with both promoter and terminator of the 35S ribosomal gene from cauliflower mosaic virus. In this article, we report the sequencing of the whole T25 insert and the characterization of its integration site by using a genome walking strategy. Our results confirmed that one intact copy of the initial construct had been integrated in the plant genome. They also revealed, at the 5′ junction of the insert, the presence of a second truncated 35S promoter, probably resulting from rearrangements which may have occurred before or during integration of the plasmid DNA. The analysis of the junction fragments showed that the integration site of the insert presented high homologies with the Huck retrotransposon family. By using one primer annealing in the maize genome and the other in the 5′ end of the integrat ed DNA, we developed a reliable event-specific detection system for T25 maize. To provide means to comply with the European regulation, a real-time PCR test was designed for specific quantitation of T25 event by using Taqman® chemistry.

scholarly journals Evaluation of Applied Biosystems MicroSEQ® Real-Time PCR System for Detection of Salmonella spp. in Food

2011 ◽  
Vol 94 (4) ◽  
pp. 1106-1116 ◽  
Author(s):  
Priya Balachandran ◽  
Yanxiang Cao ◽  
Lily Wong ◽  
Manohar R Furtado ◽  
Olga V Petrauskene ◽  
...  
Keyword(s):  
Real Time ◽  
Study Design ◽  
Real Time Pcr ◽  
Detection System ◽  
Peanut Butter ◽  
Reference Method ◽  
Black Pepper ◽  
Salmonella Spp ◽  
Culture Methods ◽  
Significant Difference

Abstract Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time-to-results compared to traditional culture methods. In this study, the MicroSEQ® real-time PCR system was evaluated for detection of Salmonella spp. in 10 different food matrixes following the AOAC Research Institute's Performance Tested MethodSM validation program. In addition, the performance of the MicroSEQ system was evaluated for the detection of Salmonella in peanut butter as a part of the Emergency Response Validation Program sponsored by the AOAC Research Institute. The system was compared to the ISO 6579 reference method using a paired-study design for detecting Salmonella spp. in raw ground beef, raw chicken, raw shrimp, Brie cheese, shell eggs, cantaloupe, chocolate, black pepper, dry infant formula, and dry pet food. For the peanut butter study, the system was compared to the U.S. Food and Drug Administration's Bacteriological Analytical Manual procedures using an unpaired-study design. No significant difference in performance was observed between the MicroSEQ Salmonella spp. detection system and the corresponding reference methods for all 11 food matrixes. The MicroSEQ system detected all Salmonella strains tested, while showing good discrimination against detection of an exclusivity panel of 30 strains, with high accuracy.

scholarly journals Influence of PCR Reagents on DNA Polymerase Extension Rates Measured on Real-Time PCR Instruments

2014 ◽  
Vol 60 (2) ◽  
pp. 334-340 ◽  
Author(s):  
Jesse L Montgomery ◽  
Carl T Wittwer
Keyword(s):  
Real Time ◽  
Melting Temperature ◽  
Dna Polymerase ◽  
Real Time Pcr ◽  
Monovalent Cation ◽  
Polymerase Activity ◽  
Simple Extension ◽  
Extension Rate ◽  
Nucleotide Analogs ◽  
Dna Dyes

Abstract BACKGROUND Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR. METHODS A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed. Extension rates were measured in nucleotides per second per molecule of polymerase. To initiate the reaction, a nucleotide analog was heat activated at 95 °C for 5 min, the temperature decreased to 75 °C, and fluorescence monitored until substrate exhaustion in 30–90 min. RESULTS The assay was linear with time for over 40% of the reaction and for polymerase concentrations over a 100-fold range (1–100 pmol/L). Extension rates decreased continuously with increasing monovalent cation concentrations (lithium, sodium, potassium, cesium, and ammonium). Melting-temperature depressors had variable effects. DMSO increased rates up to 33%, whereas glycerol had little effect. Betaine, formamide, and 1,2-propanediol decreased rates with increasing concentrations. Four common noncovalent DNA dyes inhibited polymerase extension. Heat-activated nucleotide analogs were 92% activated after 5 min, and hot start DNA polymerases were 73%–90% activated after 20 min. CONCLUSIONS Simple DNA extension rate assays can be performed on real-time PCR instruments. Activity is decreased by monovalent cations, DNA dyes, and most melting temperature depressors. Rational inclusion of PCR components on the basis of their effects on polymerase extension is likely to be useful in PCR, particularly rapid-cycle or fast PCR.

scholarly journals High-throughput HTLV-1 proviral DNA detection system using a nucleic acid extraction robot and real-time PCR detection.

2001 ◽  
Vol 47 (3) ◽  
pp. 378-383 ◽  
Author(s):  
Chieko Matsumoto ◽  
Rieko Shiozawa ◽  
Shigeki Mitsunaga ◽  
Akiko Ichikawa ◽  
Rika Ishiwatari ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Detection System ◽  
Dna Detection ◽  
Pcr Detection ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Proviral Dna

scholarly journals Development of a high-throughput real-time PCR system for detection of enzootic pathogens in pigs

2019 ◽  
Vol 32 (1) ◽  
pp. 51-64 ◽  
Author(s):  
Nicole B. Goecke ◽  
Charlotte K. Hjulsager ◽  
Jesper S. Krog ◽  
Kerstin Skovgaard ◽  
Lars E. Larsen
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Detection System ◽  
Working Hours ◽  
Negative Influence ◽  
Optimal Choice ◽  
National Veterinary Institute ◽  
Wide Range

Respiratory and intestinal diseases in pigs can have significant negative influence on productivity and animal welfare. A wide range of real-time PCR (rtPCR) assays are used in our laboratory (National Veterinary Institute, Technical University of Denmark) for pathogen detection, and PCR analyses are performed on traditional rtPCR platforms in which a limited number of samples can be analyzed per day given limitations in equipment and personnel. To mitigate these restrictions, rtPCR assays have been optimized for the high-throughput rtPCR BioMark platform (Fluidigm). Using this platform, we developed a high-throughput detection system that can be used for simultaneous examination of 48 samples with detection specificity for 18 selected respiratory and enteric viral and bacterial pathogens of high importance to Danish pig production. The rtPCR assays were validated and optimized to run under the same reaction conditions using a BioMark 48.48 dynamic array (DA) integrated fluidic circuit chip, and the sensitivity and specificity were assessed by testing known positive samples. Performance of the 48.48DA was similar to traditional rtPCR analysis, and the specificity of the 48.48DA was high. Application of the high-throughput platform has resulted in a significant reduction in cost and working hours and has provided production herds with a new innovative service with the potential to facilitate the optimal choice of disease control strategies such as vaccination and treatment.

scholarly journals Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR

2000 ◽  
Vol 38 (5) ◽  
pp. 1747-1752 ◽  
Author(s):  
C. E. Corless ◽  
M. Guiver ◽  
R. Borrow ◽  
V. Edwards-Jones ◽  
E. B. Kaczmarski ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Dna Polymerase ◽  
Real Time Pcr ◽  
Rrna Gene ◽  
Restriction Digestion ◽  
Bacterial Dna ◽  
The Real ◽  
Log Reduction ◽  
Highly Sensitive

A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.

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