2014
DOI: 10.4238/2014.june.18.3
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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

Abstract: ABSTRACT. This study aimed to develop and validate real-time PCR for the diagnosis of Mycobacterium bovis isolates. Two hundred and seventy-four M. bovis isolates and 156 M. tuberculosis isolates were tested. Both qPCRs amplified all of the 274 M. bovis samples, but none of the 156 M. tuberculosis samples. The qPCR for PE-PGRS 20 had 91% efficiency and a detection limit of 0.32 ng (sensitivity and specificity for qPCR "Mbovis.100" were 99.64 and 100%, respectively). The qPCR for RD4 had 100% efficiency, and a … Show more

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Cited by 18 publications
(15 citation statements)
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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%
“…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%
“…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%
“…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