Identification of a Novel DNA Probe for Strain Typing Mycobacterium bovis by Restriction Fragment Length Polymorphism Analysis

Abstract

Bovine tuberculosis caused by Mycobacterium bovisremains a significant disease of farmed cattle in many countries despite ongoing tuberculosis eradication programs. Molecular typing methods such as restriction fragment length polymorphism (RFLP) analysis and spoligotyping have been used to identify related herd breakdowns in an attempt to identify more precisely the route of infection into cattle herds and to trace the transmission of bovine tuberculosis. A recent geographical survey of Irish M. bovis isolates demonstrated that a significant proportion of isolates (∼20%) exhibit a common strain type, limiting the value of current strain typing methods as an epidemiological tool. We have identified and cloned a region of the M. bovis genome, pUCD, which generates a clear, highly polymorphic banding pattern when used as an RFLP probe on AluI restriction-digested M. bovis genomic DNA and which effectively subdivides this common strain type. When used to type 60 Irish M. bovis isolates, pUCD exhibited greater discriminatory power than the commonly used mycobacterial RFLP probes IS6110, PGRS, and DR and detected an equivalent number of strain types to a combination of these three probes. pUCD also detected significantly more strain types than the spoligotyping technique, while maintaining a high level of concordance between epidemiologically related and unrelated herd breakdowns. The polymorphic element within pUCD remains to be fully characterized, however the potential for this probe to greatly decrease the workload necessary to genotype M. bovis by RFLP analysis is compelling.

Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets

“…Therefore, the accepted IS 6110 -RFLP protocol agreed for M. tuberculosis (van Embden et al , 1993 ) is not appropriate for M. bovis . To identify M. bovis strains, additional discrimination is required with further RFLP procedures, such as PGRS-RFLP analysis and direct repeat(DR)-RFLP analysis (Skuce et al , 1996 ), or alternatives such as pUCD probing (O’Brien et al , 2000a ). However, these are not ideally suited to inter-laboratory typing studies.…”

Direct DNA Modified CTAB Preparation from Nasal Exudate in Live M. bovis Infected Cattle in Mexico Provide with a Valuable Assay Extrapolated to Humans TB Diagnostic Test

“…Nowadays, there are several molecular methods for the detection of M. bovis that goes from simple PCR to multiplex real time PCR using either milk, semen, urine or nasal exudate [16,23,[24][25][26][27][28][29][30][31], some of them fill one or two of the basic requirements mentioned above, but not all. In some of them, the results should be corroborated throughout histopathology and bacteriology, which would imply, time, and costs; and therefore, it can not be used extensively in epidemiological surveys [37,38,[39][40][41][42]. Thus, in the present work, we aimed to design a dual experimental protocol that can be used as routine assay for the M. bovis or M. tuberculosis detection that involves DNA extraction prepared from fast growing colonies (7 to 8 days) (Figures 2 and 3III) or from direct from nasal exudate using cetyl-trimethyl-ammonium bromide (CTAB) cationic buffer which improved the quality of DNA for several reasons: a) denatures proteins, b) solubilize cell wall and lipid proteins [26] and c) PCR mediated molecular detection [25][26][27][28][29][30][31]38,39,[43][44].…”

Characterization of the Mycobacterium bovis Restriction Fragment Length Polymorphism DNA Probe pUCD and Performance Comparison with Standard Methods

“…A significant advantage of this approach is that it allows the DR oligonucleotide probe to potentially be incorporated directly into the same hybridization. This would allow the discrimination obtainable from a pUCD-DR probe combination to be achieved from a single round of RFLP analysis, as the restriction enzyme used for the preparation of samples for analysis with pUCD, AluI, is the same as that used for analysis with DR (17). It must be noted that, in this study, pUCD and DR were not included together in such a mixed hybridization and that the results reported here for a pUCD-DR combination were obtained by using these two probes independently and subsequently combining the results.…”

“…Characterization of pUCD. It was reported previously (17) that pUCD bears a high degree of homology with a region of the M. tuberculosis genome (segment 85/162 of the M. tuberculosis H37Rv complete genome [3]; GenBank accession no. Z97193).…”

“…Recently, a newly identified DNA probe for RFLP typing of M. bovis isolates, pUCD, was described (17). This probe was found to detect higher levels of M. bovis polymorphism in cultured isolates from Ireland, based on the results obtained for 60 isolates, than analysis with the mycobacterial RFLP probes IS6110, PGRS, and DR and the spoligotyping method.…”