Validation of two real-time PCRs targeting the PE-PGRS 20 gene and the region of difference 4 for the characterization of Mycobacterium bovis isolates

Sales, Mariana Lázaro; Fonseca, Antônio Augusto; Orzil, Lívia de Lima; Alencar, Andrea Padilha de; Hodon, Mikael Arrais; Issa, M.; Filho, Paulo Martins Soares; Silva, M.R.; Lage, Andrey Pereira; Heinemann, Marcos Bryan

doi:10.4238/2014.june.18.3

Cited by 18 publications

(15 citation statements)

References 26 publications

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“…M. bovis genomic DNA was extracted and the fragment corresponding to the region of difference 4 (RD4) was amplified with the specific primers reported by Sales et al . [ 12 ]. The target fragment was cloned using Escherichia coli XL1 blue strain and TA cloning kit® (Invitrogen) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil

et al. 2016

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BackgroundThe present study aimed to direct detect Mycobacterium bovis in milk (n = 401) and blood (n = 401) samples collected from 401 dairy cows of 20 properties located in the state of Pernambuco, Brazil, by real-time quantitative PCR (qPCR) targeting the region of difference 4 (RD4). Risk factors possibly associated with bovine tuberculosis (BTB) were also evaluated.ResultsOf the 802 samples analyzed, one milk (0.25 %) and eight blood (2 %) samples were positive for M. bovis in the qPCR and their identities were confirmed by sequencing. Animals positive for M. bovis were found in six (30 %) of the 20 properties visited. None of the risk factors evaluated were statistically associated with BTB.ConclusionsM. bovis DNA was detected in one milk sample what may pose a risk to public health because raw milk is commonly consumed in Brazil.

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Section: Methodsmentioning

confidence: 99%

Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil

et al. 2016

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“…M . bovis isolates were collected in Lanagro/MG slaughterhouses from tissues with lesions characteristic of bovine tuberculosis and tested in qPCR previously validated by our group ( Sales et al, 2014a , Sales et al, 2014b ). The mycobacterial DNA was subjected to amplification of the pncA gene ( Scorpio and Zhang, 1996 ) and detection of the pncA polymorphism by cleavage of the amplicon with Eco 065I, as previously described ( Barouni et al , 2004 ) for the genetic identification of M. bovis or M. tuberculosis .…”

Section: Methodsmentioning

confidence: 99%

Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis

Sales¹,

Júnior²,

Orzil³

et al. 2014

Braz. J. Microbiol.

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Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 – 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% – 100%) and 100% (CI = 93.98% – 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

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“…La principal desventaja de estos sistemas radica en que diagnostican falsos positivos con mucha facilidad debido principalmente a la contaminación de los reactivos en el proceso del laboratorio. Aun así, el diagnóstico de TBb mediante las variantes de PCR se ha convertido en una de las técnicas más rápidas, seguras y confiables [82][83][84].…”

Section: Compuestosunclassified

Prevención y diagnóstico veterinario de la tuberculosis bovina. Una revisión de las tendencias globales

et al. 2019

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Introducción: la tuberculosis bovina (TBb) causada por Mycobacterium bovis es una enfermedad bacteriana grave, crónica, reemergente y zoonótica. Afecta a la producción pecuaria, así como a los animales silvestres y afectivos. En los hatos bovinos provoca sustanciales pérdidas económicas relacionadas con las medidas sanitarias para su control, e importantes restricciones comerciales a los productos derivados. Dado que las estrategias de prevención y control deben actualizarse con relación a los desarrollos científicos se realizó el estudio para presentar los avances y tendencias científico-tecnológicas alrededor de las estrategias sanitarias para el control de la TBb. Metodología: Se realizó una amplia consulta de artículos originales y de revisión, sobre vacunas y diagnóstico de TBb entre los años 2002 y 2017, en las bases de datos MEDLINE/PubMed, EBSCO y SciELO. Resultados: se encontró una activa y consistente acción internacional orientada a explorar formulaciones vacunales y avances en herramientas diagnósticas robustas. No se reportaron avances importantes en el desarrollo de inmunógenos para el ganado bovino. Tampoco se reportó una prueba con altos valores de sensibilidad (Se) y especificidad (Es) para el diagnóstico poblacional de la enfermedad. Conclusiones: se estima que los adelantos científicos y tecnológicos obtenidos durante los últimos años, no necesariamente van a contribuir por el momento, con el éxito de los programas nacionales de control y erradicación de la TBb. Por lo tanto, en este contexto la priorización de un abordaje integral que considere las realidades de cada región o país, dentro del concepto “Una sola salud”, el empleo combinado y estratégico de los sistemas diagnósticos el firme compromiso político de los gobiernos y la cooperación internacional son indispensables para avanzar en la lucha contra esta enfermedad.

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Validation of two real-time PCRs targeting the PE-PGRS 20 gene and the region of difference 4 for the characterization of Mycobacterium bovis isolates

Cited by 18 publications

References 26 publications

Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil

Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil

Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis

Prevención y diagnóstico veterinario de la tuberculosis bovina. Una revisión de las tendencias globales

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