scholarly journals Direct detection of Mycobacterium avium SUBSP. paratuberculosis in bovine milk by multiplex real-time PCR

2013 ◽  
Vol 29 (3) ◽  
pp. 513-525 ◽  
Author(s):  
A. Selim ◽  
M. El-Haig ◽  
E.S. Galila
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Detection Sensitivity ◽  
Mycobacterium Avium Subsp ◽  
Pcr Assay ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Pcr Targeting

This study aimed to direct detection of Mycobacterium avium subsp. paratuberculosis (MAP) in milk by evaluating a multiplex real-time PCR assay targeting IS900 and ISMAV2 sequences including the amplification of PUC19-plasmid as internal control. The sensitivity of the assays was evaluated by testing MAP isolates in broad linear range of DNA (50 ng - 5 fg/?l). For the validation of the specificity, 6 MAP isolates and 22 isolates of genus Mycobacteriacea were tested. Results revealed that reproducible detection limit for real-time PCR targeting IS900 and ISMAV2 was 5 fg/?l and 50 fg/?l respectively. By targeting ISMAV2 sequence, 100% specificity was detected. However, a cross reaction with 5 ng/?l of genome of 3 M. avian subspecies avium strains was detected by targeting IS900 and negative in lower genome quantity (5pg/?l). To maximize the assay?s detection sensitivity, an efficient strategy for MAP-DNA extraction from spiked milk was assessed. Targeting of IS900 was sensitive and targeting ISMAV2 was very specific. Therefore, a multiplex real-time PCR assay targeting IS900 and ISMAV2 in combination with two commercial DNA extraction kits could be an ideal sensitive and specific protocol for routine large scale analysis of milk samples and other clinical specimens from man and animals.

Quantification of Mycobacterium avium subsp. paratuberculosis from the tissues of challenged mice using SYBR Green real time PCR assay for the assessment of vaccine efficacy

2018 ◽  
Author(s):  
R. K. Chaitanya ◽  
Y.Krishnamohan Reddy ◽  
G.Dhinakar Raj ◽  
A. Thangavelu
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Protective Efficacy ◽  
Absolute Quantification ◽  
Sybr Green ◽  
Mycobacterium Avium Subsp ◽  
Bacterial Burden ◽  
Pcr Assay ◽  
Mycobacterium Avium Subsp Paratuberculosis

A SYBR Green real time PCR assay amplifying F57 gene of Mycobacterium avium subsp. paratuberculosis (MAP) was developed to evaluate the protective efficacy of the inactivated vaccine to prevent the colonization of MAP in the tissues of BALB/c mice challenge model. Bacterial burden in the liver, spleen and intestine of vaccinated and sham immunized control mice, after intra peritoneal challenge with MAP were quantified by real time PCR assay. A 195 bp fragment of MAP F57 sequence was cloned into TA cloning vector and the resultant recombinant plasmid was used to generate a series of quantification standards with copy number ranging from 109 to 1 copy. This absolute quantification technique is rapid and able to detect as few as 10 MAP organisms per gram of tissue and the assay has the efficiency of 0.982.

Real-time PCR-based methods for detection of Mycobacterium avium subsp. paratuberculosis in water and milk

2005 ◽  
Vol 101 (1) ◽  
pp. 93-104 ◽  
Author(s):  
David Rodríguez-Lázaro ◽  
Martin D'Agostino ◽  
Arnold Herrewegh ◽  
Maria Pla ◽  
Nigel Cook ◽  
...  
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Mycobacterium Avium Subsp ◽  
Mycobacterium Avium Subsp Paratuberculosis

scholarly journals Validation of an in-house real-time PCR fecal assay and comparison with two commercial assays for the antemortem detection of Mycobacterium avium subsp. paratuberculosis infection in culled sheep

2018 ◽  
Vol 31 (1) ◽  
pp. 58-68 ◽  
Author(s):  
Julie Arsenault ◽  
Jagdip Singh Sohal ◽  
Anne Leboeuf ◽  
Pierre Hélie ◽  
Gilles Fecteau ◽  
...  
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Positive Culture ◽  
Cost Effective ◽  
Mycobacterium Avium Subsp ◽  
Fecal Culture ◽  
Convenience Sample ◽  
Promising Tool ◽  
Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is a chronic infectious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). In sheep, the antemortem detection of the infection is challenging given the slow progression of the disease and the lack of sensitive, specific, and cost-effective validated tests. We adapted an in-house real-time PCR (rtPCR) assay targeting the multi-copy IS 900 element of MAP. The sensitivity and specificity of this essay for the detection of MAP infection were estimated in a convenience sample of culled ewes from 7 infected flocks and compared to a commercial fecal rtPCR, a commercial ELISA, and fecal culture. An infected ewe was defined as a ewe with a positive culture of the ileum and/or mesenteric lymph node. A non-infected ewe was defined as a ewe negative in intestinal tissue culture, negative in fecal culture, and with no lesions consistent with paratuberculosis. The in-house rtPCR had a sensitivity estimate of 84% (95% confidence interval [CI]: 59%, 97%) among the 44 infected ewes, which was significantly higher ( p ⩽ 0.05) than the sensitivity of a commercial fecal rtPCR (52%, 95% CI: 27%, 76%; or 63%, 95% CI: 35%, 87% depending on the cutoff used), an ELISA (14%, 95% CI:2.0%, 41%), and fecal culture (21%, 95% CI: 2.7%, 59%). No statistical difference in assay specificities was observed for the 30 non-infected ewes. The in-house rtPCR is a promising tool that could be used advantageously for the antemortem detection of MAP infection in sheep.

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.

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