scholarly journals Quantitative Detection of Trichloroethylene-Degrading Mycobacterium sp. TA27 with a Real-time PCR Product Detection System.

2001 ◽  
Vol 16 (2) ◽  
pp. 109-116 ◽  
Author(s):  
Akiko Hashimoto ◽  
Kazuhiro Iwasaki ◽  
Mutsuyasu Nakajima ◽  
Osami Yagi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection System ◽  
Quantitative Detection ◽  
Pcr Product

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.

Real-Time PCR for Quantitative Detection of Bovine Tissues in Food and Feed

2008 ◽  
Vol 71 (3) ◽  
pp. 564-572 ◽  
Author(s):  
IRENE MARTÍN ◽  
TERESA GARCÍA ◽  
VIOLETA FAJARDO ◽  
MARÍA ROJAS ◽  
PABLO E. HERNÁNDEZ ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Detection System ◽  
Total Content ◽  
18S Rrna Gene ◽  
Universal Primers ◽  
Quantitative Detection ◽  
12S Rrna ◽  
Rrna Gene

A real-time PCR approach with the SYBR Green detection system has been developed for the quantitative detection of bovine tissues in food and feedstuffs. The method combines the use of bovine-specific primers, which amplify an 84-bp fragment of the mitochondrial 12S rRNA gene, and universal primers, which amplify a 140-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. The 18S rRNA primers are used as endogenous controls for the total content of PCR-amplifiable DNA in the sample. The specificity of the primers was tested against 18 animal species, including mammals, birds, and fish, as well as 6 plant species. Analysis of experimental bovine tissues–oats mixtures demonstrated the suitability of the assay for the detection of bovine DNA in mixtures containing as low as 0.1% of bovine tissues. The performance of the method is not affected by severe heat treatment (up to 133°C for 20 min at 300 kPa). The reported PCR assay could be very useful for detecting bovine-derived ingredients in raw and heat-treated food and feedstuffs.

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.

scholarly journals Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa
Keyword(s):  
Gastrointestinal Tract ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Necrotic Enteritis ◽  
Analytical Sensitivity ◽  
Quantitative Real Time Pcr ◽  
Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

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

Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

2005 ◽  
Vol 68 (6) ◽  
pp. 1217-1221 ◽  
Author(s):  
PAVEL KRCMAR ◽  
EVA RENCOVA
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Single Species ◽  
Absolute Quantification ◽  
Quantitative Detection ◽  
Vertebrate Species ◽  
Target Species ◽  
Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

scholarly journals Detection and characterisation of papillomavirus in skin lesions of giraffe and sable antelope in South Africa

2011 ◽  
Vol 82 (2) ◽  
Author(s):  
E. Van Dyk ◽  
A-M Bosman ◽  
E. Van Wilpe ◽  
J. H. Williams ◽  
R. G. Bengis ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Skin Lesions ◽  
Immunoperoxidase Staining ◽  
Bovine Papillomavirus ◽  
Virus Particles ◽  
Tissue Sections ◽  
Equine Sarcoid ◽  
Pcr Product ◽  
Papillomavirus Dna

Papillomavirus was detected electron microscopically in cutaneous fibropapillomas of a giraffe (Giraffa camelopardalis) and a sable antelope (Hippotragus niger). The virus particles measured 45 nm in diameter. Histopathologically, the lesions showed histopathological features similar to those of equine sarcoid as well as positive immunoperoxidase-staining of tissue sections for papillomavirus antigen. Polymerase chain reaction (PCR) detected bovine papillomavirus (BPV) DNA. Bovine papillomavirus-1 was characterised by real-time PCR in the sable and giraffe, and cloning and sequencing of the PCR product revealed a similarity to BPV-1. As in the 1st giraffe, the lesions from a 2nd giraffe revealed locally malignant pleomorphism, possibly indicating the lesional end-point of papilloma infection. Neither virus particles nor positively staining papillomavirus antigen could be demonstrated in the 2nd giraffe but papillomavirus DNA was detected by real-time PCR which corresponded with BPV-1 and BPV-2.

scholarly journals Application of real-time PCR for quantitative detection ofCampylobacter jejuniin poultry, milk and environmental water

2003 ◽  
Vol 38 (3) ◽  
pp. 265-271 ◽  
Author(s):  
Chengbo Yang ◽  
Yuan Jiang ◽  
Kehe Huang ◽  
Changqing Zhu ◽  
Yulong Yin
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Water ◽  
Quantitative Detection
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