scholarly journals PP-040 Real-time PCR assay for identification of Brucella abortus in bulk milk samples in Iran

2010 ◽  
Vol 14 ◽  
pp. S36-S37 ◽  
Author(s):  
S. Moshkelani ◽  
M. Javaheri Koupaei ◽  
S. Rabiei ◽  
A. Doosti
Keyword(s):  
Real Time ◽  
Brucella Abortus ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Milk Samples ◽  
Bulk Milk

scholarly journals The Sensitivity of the Real-time PCR and Nested-PCR for Detection of Coxiella burnetii in Milk Samples

2017 ◽  
Vol 4 (2) ◽  
pp. 11
Author(s):  
Mojtaba Bonyadian ◽  
Hamdollah Moshtaghi ◽  
Hamidreza Kazemeini
Keyword(s):  
Real Time ◽  
Coxiella Burnetii ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Pcr Assay ◽  
Milk Samples ◽  
Bulk Milk ◽  
Significant Difference ◽  
Pcr Assays ◽  
Nested Pcr Assays

Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis. In humans serology is the gold standard for diagnosis but is inadequate for early case detection, so real-time PCR and nested-PCR assays were developed in this study to measure amounts of C.burnetii shed in milk. Our study was to assess the sensitivity of the realtime PCR and nested-PCR for detection of Coxiella burnetii in bovine bulk milk samples from dairy herds in 3 provinces (Chaharmahal and Bakhtiari , Isfahan and Yazd) of Iran. In the present study, 300 bulk milk samples from 89 dairy cattle herds were tested for C. burnetii using real-time PCR and nested-PCR assays. The animals which their milk samples collected for this study were clinically healthy. In total, 74 of 300 (24.7%) cow milk samples were positive in real-time PCR assay and 26 of 300 (8.7%) samples were positive in nested-PCR assay. McNemar test shows a significant difference in detection of C. burnetii between real-time PCR and nested-PCR. Also the results of this study indicate those clinically healthy dairy cows are important sources of C. burnetii infection in Iran.

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.

Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture

2015 ◽  
Vol 82 (2) ◽  
pp. 200-208 ◽  
Author(s):  
Heidi Hiitiö ◽  
Rauna Riva ◽  
Tiina Autio ◽  
Tarja Pohjanvirta ◽  
Jani Holopainen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Bovine Mastitis ◽  
Antimicrobial Treatment ◽  
Reference Laboratory ◽  
Pcr Assay ◽  
Coagulase Negative Staphylococci ◽  
Staph Aureus ◽  
Milk Samples

Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85·7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83·0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97·0 and 95·8% compared with BC. For Staphylococcus spp., the corresponding figures were 86·7 and 75·4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37·0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.

scholarly journals Evidence of Brucella abortus in Non-Preferred Caprine and Ovine Hosts by Real-time PCR Assay

2019 ◽  
Vol 51 (3) ◽  
Author(s):  
Muhammad Zain Saleem ◽  
Raheela Akhtar ◽  
Asim Aslam ◽  
Muhammad Imran Rashid ◽  
Zafar Iqbal Chaudhry ◽  
...  
Keyword(s):  
Real Time ◽  
Brucella Abortus ◽  
Real Time Pcr ◽  
Pcr Assay

scholarly journals Detection of Brucella Abortus in Caprine and Ovine by Real-Time PCR Assay

2021 ◽  
Vol 9 (5) ◽  
pp. 141
Author(s):  
Muhammad Bilawal Arain ◽  
Abdullah Babar ◽  
Muhammad Ibrahim Panhwar ◽  
Khush Hal ◽  
Muhammad Mubashir Farooq ◽  
...  
Keyword(s):  
Real Time ◽  
Brucella Abortus ◽  
Real Time Pcr ◽  
Pcr Assay

Application of an F57 Sequence-Based Real-Time PCR Assay for Mycobacterium paratuberculosis Detection in Bulk Tank Raw Milk and Slaughtered Healthy Dairy Cows

2006 ◽  
Vol 69 (7) ◽  
pp. 1662-1667 ◽  
Author(s):  
C. BOSSHARD ◽  
R. STEPHAN ◽  
T. TASARA
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Raw Milk ◽  
Mesenteric Lymph Nodes ◽  
Pcr Assay ◽  
Milk Samples ◽  
Light Cycler ◽  
Bulk Tank ◽  
Mycobacterium Avium Subsp Paratuberculosis

A light cycler–based real-time PCR assay that targets the F57 sequence was used to collect data on the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) in 100 bulk tank raw milk samples and in a population of 101 slaughtered dairy cattle. The assay's reproducible detection limit in total genomic DNA templates isolated from 10-ml samples of MAP-spiked raw milk was 100 cells per ml. Similarly, the evaluation of MAP-spiked bovine feces also demonstrated that the assay had a reproducible detection limit of 100 cells if they were contained within 200 mg of fecal sample material. Among the 100 bulk tank milk samples that were tested, we found 3 samples (3%) to be positive for MAP. In the slaughterhouse part of the study, 8.9% (9 of 101) of the cows were positive for MAP DNA in fecal samples, 4.9% (5 of 101) in mesenteric lymph nodes, 0.9% (1 of 101) in ileum tissue, and 3.6% (3 of 84) in milk. Meanwhile, for 2.9% (3 of 101) of the culled cows, MAP DNA was detected in samples of diaphragmatic muscles.

Real-Time PCR Method for Quantification of Staphylococcus aureus in Milk

2007 ◽  
Vol 70 (1) ◽  
pp. 90-96 ◽  
Author(s):  
M. GOTO ◽  
H. TAKAHASHI ◽  
Y. SEGAWA ◽  
H. HAYASHIDANI ◽  
K. TAKATORI ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Pure Culture ◽  
High Specificity ◽  
Pcr Assay ◽  
Milk Samples ◽  
High Temperature Short Time ◽  
Specificity And Sensitivity ◽  
Pcr Method

A reproducible real-time PCR method that targets the putative transcriptional regulator gene of Staphylococcus aureus was developed to quantify this microorganism in milk samples. On the basis of partial sequences of this gene determined from S. aureus strains, we designed the specific primers and probe for use in a quantitative PCR assay. These specificities were confirmed with 25 strains of S. aureus and 35 strains of other bacteria. A real-time PCR assay with serial 10-fold dilutions of purified DNA and pure culture was conducted. It was possible to construct standard curves with a high correlation coefficient (r2 = 0.99) in the range of 50 ng to 50 fg for purified DNA and 107 to 101 CFU/ml for a pure culture. The constructed standard curve for milk samples was similar to that for the pure culture, and the quantification of S. aureus in the range of 107 to 101 CFU/ml was possible. Moreover, to determine how our real-time PCR method would perform under actual analytical conditions, we quantified the DNA from S. aureus after two types of heat treatments were used for the pasteurization of milk. The amount of DNA found was affected after heat treatment at 63°C for 30 min (low-temperature long-time method) but not at 72°C for 15 s (high-temperature short-time method). The results indicate that the real-time PCR method developed in this study is effective for monitoring S. aureus contamination in milk because of its high specificity and sensitivity.

Duplex real-time PCR assay for rapid identification ofStaphylococcus aureusisolates from dairy cow milk

2013 ◽  
Vol 80 (2) ◽  
pp. 223-226 ◽  
Author(s):  
Rachel Pilla ◽  
Gustavo G M Snel ◽  
Michela Malvisi ◽  
Renata Piccinini
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Dairy Cow ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Rapid Identification ◽  
Pcr Assay ◽  
Staph Aureus ◽  
Bacteriological Analysis ◽  
Milk Samples

Staphylococcus aureusisolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification ofStaph. aureusisolates, targeting bothnucandSa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detectnucorSa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124Staph. aureusisolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent withnucandSa442were revealed, while 2 isolates showed only the peak consistent withSa442. Four isolates bacteriologically identified asStaph. aureus, were PCR-negative and were further identified asStaph. pseudintermediusby sequencing.Staph. pseudintermediusand coagulase-negative staphylococci did not carrynucorSa442. The results showed the correct identification of all isolates, comprehending also coagulase—or nuc-negativeStaph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, onStaph. aureus-positive or—negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypicalStaph. aureusisolates causing dairy cow mastitis. Also, it could be applied directly to milk samples, to detectStaph. aureusmammary infections avoiding bacteriological analysis.

scholarly journals Development of a Real-Time PCR for Detection of Mycoplasma Bovis in Bovine Milk and Lung Samples

2005 ◽  
Vol 17 (6) ◽  
pp. 537-545 ◽  
Author(s):  
Hugh Y. Cai ◽  
Patricia Bell-Rogers ◽  
Lois Parker ◽  
John F. Prescott
Keyword(s):  
Real Time ◽  
Lung Tissue ◽  
Real Time Pcr ◽  
Bovine Milk ◽  
Melting Peak ◽  
Clinical Samples ◽  
Mycoplasma Bovis ◽  
Pcr Assay ◽  
Milk Samples ◽  
Culture Positive

A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6°C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1°C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 × 104 and 7.7 × 108 cfu/ml and the M. bovis culture-positive lungs between 1 × 103 and 1 × 109 cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.

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