Validation of a real-time PCR for the quantitative estimation of a G143A mutation in the cytochromebc1 gene ofPyrenophora teres

2007 ◽  
Vol 63 (3) ◽  
pp. 219-224 ◽  
Author(s):  
Arash Kianianmomeni ◽  
Gerhard Schwarz ◽  
Friedrich G Felsenstein ◽  
Gerhard Wenzel
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Estimation

scholarly journals Quantitative estimation of Flavobacterium psychrophilum infected ayu Plecoglossus altivelis altivelis by real-time PCR

2009 ◽  
Vol 75 (2) ◽  
pp. 258-260 ◽  
Author(s):  
KENICHI OHARA ◽  
TETSUSHI KAGEYAMA ◽  
TOMONORI KUWADA ◽  
TETSUYA UMINO ◽  
SHUICHI FURUSAWA ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Estimation ◽  
Plecoglossus Altivelis ◽  
Flavobacterium Psychrophilum

Application of Quantitative Real-Time PCR in the Detection of Prion-Protein Gene Species-Specific DNA Sequences in Animal Meals and Feedstuffs

2006 ◽  
Vol 69 (4) ◽  
pp. 891-896 ◽  
Author(s):  
FEDERICA BELLAGAMBA ◽  
SERGIO COMINCINI ◽  
LUCA FERRETTI ◽  
FRANCO VALFRÈ ◽  
VITTORIO M. MORETTI
Keyword(s):  
Real Time ◽  
Prion Protein ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Animal Feed ◽  
Quantitative Estimation ◽  
Taqman Assay ◽  
Quantitative Real Time Pcr ◽  
The Real ◽  
Species Specific

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130°C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.

scholarly journals Development of an F57 Sequence-Based Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Milk

2005 ◽  
Vol 71 (10) ◽  
pp. 5957-5968 ◽  
Author(s):  
T. Tasara ◽  
R. Stephan
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Quantitative Estimation ◽  
Bacterial Species ◽  
Detection Sensitivity ◽  
Bacterial Strains ◽  
Pcr Assay ◽  
Sequence Element ◽  
Milk Samples

ABSTRACT A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 × 101 to 2 ×106 copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.

scholarly journals Quantitative estimation of Flavobacterium psychrophilum discharged from infected ayu Plecoglossus altivelis altivelis by real-time PCR

2010 ◽  
Vol 76 (4) ◽  
pp. 705-707
Author(s):  
KENICHI OHARA ◽  
TETSUSHI KAGEYAMA ◽  
TOMONORI KUWADA ◽  
TETSUYA UMINO ◽  
SHUICHI FURUSAWA
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Estimation ◽  
Plecoglossus Altivelis ◽  
Flavobacterium Psychrophilum

scholarly journals Conventional and Real-Time PCR-Based Assay for Detecting Pathogenic Alternaria brassicae in Cruciferous Seed

Plant Disease ◽  
2004 ◽  
Vol 88 (5) ◽  
pp. 490-496 ◽  
Author(s):  
Thomas Guillemette ◽  
Béatrice Iacomi-Vasilescu ◽  
Philippe Simoneau
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Estimation ◽  
Pathogenic Fungus ◽  
Black Spot ◽  
Detection Methods ◽  
Morphological Identification ◽  
Alternaria Brassicae ◽  
Polymerase Chain ◽  
Current Detection

Alternaria brassicae is an important seedborne pathogenic fungus responsible for the black spot disease of crucifers. Sanitary control of commercial seed is necessary to limit the spread of this pathogen. Current detection methods, based on culture and morphological identification of the fungus, are time consuming, laborious, and not always reliable. Therefore, a polymerase chain reaction (PCR)-based assay was developed with A. brassicae-specific primers designed on the basis of the sequence of two clustered genes potentially involved in pathogenicity. Two sets of primers were selected for conventional and real-time PCR, respectively. In both cases, A. brassicae was specifically detected using DNA extracted from seed. The real-time PCR-based method presented here can be automated easily and preliminary results indicate that it is efficient for quantitative estimation of seed infection.

scholarly journals The Evaluation of Gastrointestinal Nematodes in Faecal Samples Using Multiplex Real-Time PCR Assays

2021 ◽  
Author(s):  
Nikol Reslová ◽  
Lucie Skorpikova ◽  
Iveta Angela Kyrianova ◽  
Jaroslav Vadlejch ◽  
Johan Höglund ◽  
...  
Keyword(s):  
Real Time ◽  
Species Identification ◽  
Real Time Pcr ◽  
Quantitative Estimation ◽  
False Negative ◽  
Infection Intensity ◽  
Multiplex Assays ◽  
Ashworthius Sidemi ◽  
Faecal Samples ◽  
Pcr Assays

Abstract Background: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods and followed by larval culture techniques. Such an approach is laborious, time-consuming, requires a skilled expert and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies.Methods: Two multiplex real-time PCR assays for specific detection of six main GIN species, including an internal amplification control to avoid false negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi‑quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms and results were compared to those from faecal egg counts (FEC) using the Concentration McMaster technique, and larval cultures (LC).Results: Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing 3 false negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp.Conclusions: Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.

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