PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions - PubMed (original) (raw)

PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions

Richard C Huard et al. J Clin Microbiol. 2003 Apr.

Abstract

The classical Mycobacterium tuberculosis complex (MtbC) subspecies include Mycobacterium tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M. bovis bacillus Calmette-Guérin [BCG]), and Mycobacterium microti; increasingly recognized MtbC groupings include Mycobacterium bovis subsp. caprae and "Mycobacterium tuberculosis subsp. canettii." Previous investigations have documented each MtbC subspecies as a source of animal and/or human tuberculosis. However, study of these organisms is hindered by the lack of a single protocol that quickly and easily differentiates all of the MtbC groupings. Towards this end we have developed a rapid, simple, and reliable PCR-based MtbC typing method that makes use of MtbC chromosomal region-of-difference deletion loci. Here, seven primer pairs (which amplify within the loci 16S rRNA, Rv0577, IS1561', Rv1510, Rv1970, Rv3877/8, and Rv3120) were run in separate but simultaneous reactions. Each primer pair either specifically amplified a DNA fragment of a unique size or failed, depending upon the source mycobacterial DNA. The pattern of amplification products from all of the reactions, visualized by agarose gel electrophoresis, allowed immediate identification either as MtbC composed of M. tuberculosis (or M. africanum subtype II), M. africanum subtype I, M. bovis, M. bovis BCG, M. caprae, M. microti, or "M. canettii" or as a Mycobacterium other than MtbC (MOTT). This MtbC PCR typing panel provides an advanced approach to determine the subspecies of MtbC isolates and to differentiate them from clinically important MOTT species. It has proven beneficial in the management of Mycobacterium collections and may be applied for practical clinical and epidemiological use.

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Figures

FIG. 1.

The composite MtbC PCR typing panel. Illustrated are the typical MtbC PCR panel typing results for a single representative of each MtbC subspecies as well as MOTT (M. avium subsp. avium is shown). A total of 71 MtbC isolates and 44 MOTT isolates were tested. PCR products and a 100-bp ladder were visualized by agarose gel electrophoresis and ethidium bromide staining. Images were captured with the Nighthawk imaging system (PDI Inc.) and Quality One software package (PDI Inc.). Lanes: 1, 16S rRNA; 2, Rv0577; 3, IS_1561_′; 4, Rv1510; 5, Rv1970; 6, Rv3877/8; 7, Rv3120. Unlabeled lanes in each panel contain the 100-bp ladder.

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PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions - PubMed (original) (raw)