Use of Touch-Down Polymerase Chain Reaction to Enhance the Sensitivity of <i>Mycobacterium Bovis</i> Detection

Zumárraga, Martín José; Meikle, Virginia; Bernardelli, Amelia; Abdala, Alejandro Ariel; Tarabla, Héctor D.; Romanó, Maria; Cataldi, Angel

doi:10.1177/104063870501700303

Cited by 56 publications

(43 citation statements)

References 34 publications

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“…Nevertheless, it has already been observed with culture and PCR of M. bovis in bovine milk (Zumarraga et al, 2005), PCR that is more sensitive than culture, a fact that may be attributable to the decontamination method before culture that may kill a high proportion of bacteria (Meikle et al, 2007).…”

Section: Molecular Diagnosismentioning

confidence: 99%

“…However, together, these tests can be slow, cumbersome, inaccurate, not reproducible and time-consuming, can give an ambiguous result and cannot be performed in any laboratory. Nevertheless, polymerase chain reaction (PCR) has been successfully applied to detect members of the M. tuberculosis complex and is especially useful for the direct detection of M. bovis in bovine tissue samples (Zumarraga et al, 2005).…”

Section: Molecular Diagnosismentioning

confidence: 99%

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Diagnosis of bovine tuberculosis: review of main techniques

2015

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Bovine tuberculosis (BTB) remains an important economic and zoonotic problem in Latin America. Traditionally, the fight against BTB is initiated by the implementation of routine diagnostic tests for certification of free properties. The diagnosis of BTB can be made by direct and indirect methods, in which we can mention clinical, post mortem, histopathological, immunological, bacteriological and molecular methods. The renewal of scientific interest in tuberculosis in recent year has led to develop and improve methods of diagnosis, prevention, control and eradication of BTB. The aim of this review is to present and discuss different diagnosis methods of BTB.Keywords: tuberculosis bovine, diagnosis, Mycobacterium bovis, new techniques. Diagnóstico de tuberculose bovina: revisão das principais técnicas ResumoA tuberculose bovina (BTB) continua sendo um importante problema econômico na América Latina, com potenciais consequências zoonóticas. Tradicionalmente, a luta contra a tuberculose bovina tem sido iniciada pela execução de testes de diagnóstico de rotina para a certificação de propriedades livres da doença. O diagnóstico de BTB pode ser feito através de métodos diretos e indiretos, nos quais podemos citar os métodos clínicos, post mortem, histopatológicos, imunológicos, bacteriológicos e moleculares. A renovação do interesse científico em tuberculose nos últimos anos tem levado à necessidade de desenvolver e melhorar os métodos de diagnóstico, prevenção, controle e erradicação da BTB. O objetivo deste artigo é analisar e discutir sobre os diferentes métodos de diagnóstico de BTB.Palavras-chave: tuberculose bovina, diagnóstico, Mycobacterium bovis, novas técnicas.

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Section: Molecular Diagnosismentioning

confidence: 99%

Section: Molecular Diagnosismentioning

confidence: 99%

Diagnosis of bovine tuberculosis: review of main techniques

2015

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“…The presence of a divalent cation (Mg2+ and/or Ca2+) can inhibit DNA polymerase, and the presence of proteins and fat globules can obstruct the accessibility of DNA polymerase to the DNA template. Furthermore, the abundance of host epithelial cells in milk could make the bacterial DNA a very small fraction of the total DNA extracted from the sample (ZUMARRAGA et al, 2005). Despite these difficulties, the extraction method reported in the present study produced DNA from spiked milk, which can be used as a template for a PCR assay.…”

Section: Discussionmentioning

confidence: 99%

“…Previously reported PCR results in milk samples are variable, showing detection limits ranging from 80 to 1000 CFU of M. bovis in samples inoculated with the bacterium (ZANINI et al, 1998;JORDÃO JÚNIOR et al, 2005;ZUMARRAGA et al, 2005). The estimated detection limit of 100 CFU/mL is suitable, since an udder infection can result in the shedding of tuberculosis bacilli ranging from 500 to 500,000 CFU /mL of milk (ZANINI et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

“…The use of the PCR method in spiked milk samples does not guarantee that it would perform equally effectively in the analysis of naturally infected samples. One could expect that in the latter, the interaction between the bacilli and the milk matrix could be more complex, and even that bacilli in milk might have already been killed by mammary macrophages (ZUMARRAGA et al, 2005), and the DNA partially degraded.…”

Section: Discussionmentioning

confidence: 99%

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Use of PCR for detection of bovine tuberculosis bacillus in milk of positive skin test cows

Carvalho

Castro

Fernandes

et al. 2014

Braz. J. Vet. Res. Anim. Sci.

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The causative agent of bovine tuberculosis (BTB) is Mycobacterium bovis, a bacterium belonging to the M. tuberculosis complex (MTC). The definitive diagnosis is achieved through isolation and identification of M. bovis from clinical samples, using a combination of traditional culture and biochemical methods, which is considered the "gold standard". This procedure is cumbersome and time-consuming. We evaluated a PCR assay for the direct detection of MTC DNA in milk of positive skin test cows, using primers that were previously tested and proven reliable to target the IS6110 element. Milk previously seeded with M. bovis was used as the starting material, for standardization of the technique. The procedure involved extracting the DNA by enzymatic lysis (proteinase K and lysozyme), phenol, chloroform, isoamyl alcohol, followed by ethanol precipitation and PCR. The PCR assay allowed us to detect BTB in artificially contaminated milk, with a detection limit of 100 CFU/mL, and was also able to detect the bacillus in 50% (75/150) of the milk samples tested. This procedure could be used to assist the in vivo diagnosis of BTB, complementing sorological or microbiological tests, becoming an alternative option for epidemiological studies of BTB transmission and preventing contaminated milk from entering the food supply.Keywords: Bovine tuberculosis. Molecular biology. Mycobacterium tuberculosis complex. Milk. ResumoO agente causador da tuberculose bovina (BTB) é o Mycobacterium bovis, uma bactéria pertencente ao complexo M. tuberculosis (CMT). O diagnóstico definitivo é realizado através do isolamento e identificação de M. bovis em amostras clínicas, utilizando uma combinação de cultura bacteriológica e métodos bioquímicos, que são considerados como "padrão de ouro". Entretanto, esses procedimentos são trabalhosos e demorados. No presente estudo, foi avaliado um ensaio de PCR para a detecção direta de DNA de CMT em leite de vacas positivas ao teste cutâneo, utilizando primers previamente testados e comprovadamente confiáveis, para amplificação da região de interesse IS6110. Leite previamente contaminado com M. bovis foi utilizado como material de partida para a padronização da técnica. O procedimento envolveu a extração do DNA por lise enzimática (proteinase K e lisozima), fenol, clorofórmio, álcool isoamílico, seguido de precipitação com etanol e PCR. O ensaio de PCR detectou BTB em leite artificialmente contaminado, com um limite de detecção de 100 UFC/mL, e também foi capaz de detectar o bacilo em 50% (75/150) das amostras de leite testadas. A técnica de PCR pode ser utilizada para auxiliar o diagnóstico in vivo da BTB, além de complementar os testes sorológicos ou microbiológicos, tornando-se uma alternativa para estudos epidemiológicos da transmissão de BTB, prevenindo que o leite contaminado entre na cadeia de alimentos.Palavras-chave: Tuberculose bovina. Biologia molecular. Complexo Mycobacterium tuberculosis. Leite.

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Milk Processing and Quality Management

Tamime¹

2008

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Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The Publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought.

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Use of Touch-Down Polymerase Chain Reaction to Enhance the Sensitivity of Mycobacterium Bovis Detection

Cited by 56 publications

References 34 publications

Diagnosis of bovine tuberculosis: review of main techniques

Diagnosis of bovine tuberculosis: review of main techniques

Use of PCR for detection of bovine tuberculosis bacillus in milk of positive skin test cows

Milk Processing and Quality Management

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