Rapid and Sensitive Detection of<i>Mycobacterium avium</i>subsp.<i>paratuberculosis</i>in Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

Khare, Sangeeta; Ficht, Thomas A.; Santos, Renato L.; Romano, Juan E.; Ficht, Allison; Zhang, Shuping; Grant, Irene R.; Libal, Melissa C.; Hunter, David M.; Adams, L. Garry

doi:10.1128/jcm.42.3.1075-1081.2004

Cited by 97 publications

(93 citation statements)

References 29 publications

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“…By recording the amount of fluorescent emission at each cycle, it is possible to monitor the PCR reaction during the exponential phase when the first significant increase in the amount of PCR product correlates to the initial amount of target template. This method has been used for MAP detection in milk since 2002 (Khare et al, 2004;Hill, 2002, 2004). The "classic" detection target IS900 is also used as a template for Real-Time PCR, but it is evident that this detection target is not suitable for accurate quantification.…”

Section: Real-time Pcrmentioning

confidence: 99%

Detection methods for Mycobacterium avium subsp paratuberculosis in milk and milk products: a review

Slaná¹,

Paolicchi²,

Janštová³

et al. 2008

Vet. Med. - Czech

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Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a disease with considerable economic impact, principally on dairy cattle herds. Animals with paratuberculosis shed viable MAP especially in their milk, faeces and semen. MAP may have a role in the development of Crohn's disease in humans via the consumption of contaminated milk and milk products. The current methods of milk pasteurization are not sufficient to kill all MAP cells present in milk and MAP has been cultured from raw or pasteurized milk and isolated from cheese. The purpose of the present study was to review the different methods used for detection of MAP in milk and milk products. We analyze the current methods for direct or non direct identification of MAP and culture and molecular biology methods that can be applied to milk and milk products.

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Section: Real-time Pcrmentioning

confidence: 99%

Detection methods for Mycobacterium avium subsp paratuberculosis in milk and milk products: a review

Slaná¹,

Paolicchi²,

Janštová³

et al. 2008

Vet. Med. - Czech

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“…The DNA purity (A260/A280 5 2) obtained using the MVRDL method was also much higher than what had been reported by other groups (A260/ A280 5 1.3-1.7). 1,12 The effectiveness of this DNA extraction method enabled the downstream real-time PCR to achieve a remarkably high analytical sensitivity, approximately 3 CFU per ml culture (or per g feces).…”

Section: Discussionmentioning

confidence: 99%

“…To date, various in-house protocols and commercial kits have been used to purify MAP DNA. 1,3,7,12,16 While all of them are useful, each has its own inherent limitations in terms of DNA yield and purity, the specificity and sensitivity of downstream PCR, cost-effectiveness, and practicability. The current study aimed to develop an efficient and cost-effective DNA extraction method for PCR-based detection of MAP organisms in bovine feces.…”

Section: Discussionmentioning

confidence: 99%

An Efficient DNA Extraction Method for Polymerase Chain Reaction–Based Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Fecal Samples

Zhang

2011

J VET Diagn Invest

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Abstract. Due to the lipid rich cell wall of Mycobacterium avium subspecies paratuberculosis (MAP), the complex nature of bovine feces, and intermittent organism shedding by infected cattle, it is difficult to recover a sufficient amount of high-quality MAP DNA from fecal samples, directly affecting the sensitivity of downstream polymerase chain reaction (PCR) tests. In the current study, a DNA extraction method, designated the Mississippi Veterinary Research and Diagnostic Laboratory (MVRDL) method, was developed for PCR-based detection of MAP in bovine fecal samples. The MVRDL method combined multiple procedures, including chemical pretreatment, 1-tube cell lysis and extraction, chelex matrix absorption, and mini-column purification. The DNA yield and purity, as measured by spectrophotometry, was 3.36 fg per colony forming unit (CFU) MAP and A260/280 absorbance ratio of 2, respectively. This method was further evaluated by real-time PCR. A linear correlation was found between cycle-threshold (Ct) and log input CFU (ranging from 7.2 to 7.2 3 10 7 CFU per ml or CFU per g). The detection limit of the realtime PCR assay was 3 CFU per ml of MAP culture or per g of MAP-spiked feces. In addition, the MVRDL method was validated by performing 7 Johne's direct fecal PCR proficiency tests administered by the National Veterinary Service Laboratories. Based on culture results as the ''gold standard,'' the specificity of MVRDL PCR was 100%, and the sensitivity was 98.46% for samples containing more than 1.5 CFU per tube of fecal cultures. To the authors' knowledge, this is the most efficient MAP DNA extraction method in comparison with all previously published protocols.

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“…Desde la aplicación del PCR en los estudios de MAP, la secuencia de inserción IS900 ha sido el principal blanco de amplifi cación utilizado, ya que sus 14 a 18 copias en el genoma bacteriano permiten resultados de alta sensibilidad. Aunque se ha reportado la exis-tencia de secuencias muy similares en organismos no relacionados 54,55 , la IS900 sigue considerándose de una especifi cidad aceptable como biomarcador de MAP 5,19,56,57 .…”

Section: El Diagnóstico De Mapunclassified

Mycobacterium avium subsp paratuberculosis y enfermedad de Crohn: evidencias de una zoonosis

et al. 2011

Rev. méd. Chile

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L a paratuberculosis bovina, también conocida como enfermedad de Johne, es una patología causada por la bacteria Mycobacterium avium subsp. paratuberculosis (MAP), que se caracteriza por una lesión intestinal crónica de carácter proliferativa 1 . Debido a las similitudes fi siopatológicas entre la infección de los bovinos y la enfermedad de Crohn (EC) en el humano, una asociación causal del MAP con ambas patologías ha sido sugerida desde hace algunos años, lo que ha generado controversia en la comunidad científi ca relacionada a la transmisión inter-especies 1,2 . En este trabajo se revisan exhaustivamente los hallazgos más representativos y recientes que dan cuenta de esta controversia, y se describen brevemente los antecedentes en Chile que explican la necesidad de realizar estudios en este ámbito. El agenteMAP es un patógeno intracelular facultativo, Gram positivo, ácido alcohol resistente, aerobio y cuya temperatura óptima de crecimiento es 37ºC. Con un tiempo de generación mayor a 20 h, es una bacteria de crecimiento lento que requiere varias semanas de incubación antes que sus colonias sean detectables en los medios de cultivo 1,3,4 . Como las bacterias del género Mycobacterium, MAP posee un genoma rico en nucleótidos G+C y su pared celular tiene un alto contenido de ácidos micólicos. Como característica especie-específi ca destacan su dependencia del sideróforo micobactina para el crecimiento in vitro y la presencia del segmento de inserción IS900 en su genoma, que se ha utilizado para la detección específi ca de este patógeno 1,5 .MAP es capaz de infectar una amplia variedad

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Rapid and Sensitive Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

Cited by 97 publications

References 29 publications

Detection methods for Mycobacterium avium subsp paratuberculosis in milk and milk products: a review

Detection methods for Mycobacterium avium subsp paratuberculosis in milk and milk products: a review

An Efficient DNA Extraction Method for Polymerase Chain Reaction–Based Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Fecal Samples

Mycobacterium avium subsp paratuberculosis y enfermedad de Crohn: evidencias de una zoonosis

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