Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids

Floyd, Margaret M.; Silcox, Vella A.; Jones, Wilbur Devereux; Butler, W. Ray; Kilburn, James O.

doi:10.1128/jcm.30.5.1327-1330.1992

Cited by 68 publications

(30 citation statements)

References 9 publications

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“…Among these, only the oxyR polymorphism at nucleotide 285 has proved to be an accurate and consistent M. bovisspecific marker (9). Other targets that have been investigated include the 16S rRNA gene region with a TB complex-specific signature sequence, targets identified by randomly amplified polymorphic DNA analysis (18,26,28,29), and mycolic acid profiles identified by the high-performance liquid chromatography (13). The high-performance liquid chromatography method requires a considerable bacterial inoculum, thereby requiring mycobacterial culture, which takes 3 to 6 weeks, before application.…”

Section: Discussionmentioning

confidence: 99%

A Multiplex Approach to Molecular Detection ofBrucella abortusand/orMycobacterium bovisInfection in Cattle

et al. 2000

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A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortusinfection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a “gold standard,” was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus(samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortusshedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and ofB. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.

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Section: Discussionmentioning

confidence: 99%

A Multiplex Approach to Molecular Detection ofBrucella abortusand/orMycobacterium bovisInfection in Cattle

et al. 2000

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“…BCG strains can be distinguished from other M. tuberculosis complex strains on the basis of differences in certain biochemical and growth characteristics and on the basis of the resis-tance and sensitivity to cycloserine. However, these methods and other, nonstandardized methods (5,8,12,13) are timeconsuming, may be laborious, sometimes require specialized and expensive equipment, and do not always provide definitive proof that a clinical isolate is BCG. The insertion sequence IS1081 has been proposed as a useful tool that can be used to recognize BCG by restriction fragment length polymorphism analysis (26).…”

Section: Discussionmentioning

confidence: 99%

Specific Differentiation between Mycobacterium bovis BCG and Virulent Strains of the Mycobacterium tuberculosis Complex

1998

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A PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system, named SenX3-RegX3, was developed and was shown to be suitable for identifying Mycobacterium bovis BCG. ThesenX3-regX3 IR contains a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). All tested BCG strains exclusively contained 77-bp MIRUs within the senX3-regX3 IR, whereas all non-BCGM. tuberculosis complex strains contained a 53-bp MIRU, in addition to the 77-bp MIRUs. All 148 strains analyzed so far could be divided into eight different groups according to the copy numbers of the 77-bp MIRU and to the presence or absence of the 53-bp MIRU. BCG strains contained either one, two, or three 77-bp MIRUs. The other strains contained one to five 77-bp MIRUs invariably followed by a 53-bp MIRU. The consistent absence of the 53-bp MIRU in BCG strains and its presence in virulent strains allowed us to develop an enzyme-linked immunosorbent assay using specific capture oligonucleotide probes to distinguish between BCG and other M. tuberculosis complex strains.

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“…By conventional biochemical methods (52,57), the identification of the species to which the cultured mycobacterial strain belongs may require an additional 2 to 4 weeks. More rapid methods for the identification of cultured mycobacteria are the analysis of lipid composition (7,11,33), the use of species-specific antibodies (44,56), and species-specific DNA or RNA probes (14,16,32).…”

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PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples

et al. 1995

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A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.

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Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids

Cited by 68 publications

References 9 publications

A Multiplex Approach to Molecular Detection ofBrucella abortusand/orMycobacterium bovisInfection in Cattle

A Multiplex Approach to Molecular Detection ofBrucella abortusand/orMycobacterium bovisInfection in Cattle

Specific Differentiation between Mycobacterium bovis BCG and Virulent Strains of the Mycobacterium tuberculosis Complex

PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples

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