Comparison of two different PCR amplification products (the 18-kDa protein gene vs. RLEP repetitive sequence) in the diagnosis of Mycobacterium leprae.

To determine the best molecular method for diagnosing leprosy, two sets of Mycobacterium leprae-specific primers were compared. Fresh biopsies and slit skin smear samples were obtained from 67 leprosy patients and examined by touchdown (TD) PCR using primers amplifying either a 129-bp fragment of the RLEP repetitive sequence or a 360-bp fragment of the 18-kDa protein gene of M. leprae. Seventeen of 30 (56.7%) biopsy specimens and four of 37 (10.8%) slit skin smear specimens were positive using the primer for the 18-kDa protein gene, whereas 24 of 30 (80%) biopsy and 27 of 37 (73%) slit skin smear samples showed detectable PCR products in the RLEP repetitive sequence. Twenty-one of 31 cases (67.7%) with a bacterial index of zero were PCR positive for the primer RLEP repetitive sequence. These results demonstrate that detection of M. leprae using PCR with primers to a RLEP sequence is more sensitive and specific than PCR with the 18-kDa protein gene primers and also slit smears with acid fast staining. PCR of RLEP repetitive sequences is therefore a useful means of detecting M. leprae DNA even when it is present at very low levels.

Comparison of two different PCR amplification products (the 18-kDa protein gene vs. RLEP repetitive sequence) in the diagnosis of Mycobacterium leprae

To determine the best molecular method for diagnosing leprosy, two sets of Mycobacterium leprae-specific primers were compared. Fresh biopsies and slit skin smear samples were obtained from 67 leprosy patients and examined by touchdown (TD) PCR using primers amplifying either a 129-bp fragment of the RLEP repetitive sequence or a 360-bp fragment of the 18-kDa protein gene of M. leprae. Seventeen of 30 (56.7 per cent) biopsy specimens and four of 37 (10.8 per cent) slit skin smear specimens were positive using the primer for the 18-kDa protein gene, whereas 24 of 30 (80 per cent) biopsy and 27 of 37 (73 per cent) slit skin smear samples showed detectable PCR products in the RLEP repetitive sequence. Twenty-one of 31 cases (67.7 per cent) with a bacterial index of zero were PCR positive for the primer RLEP repetitive sequence. These results demonstrate that detection of M. leprae using PCR with primers to a RLEP sequence is more sensitive and specific than PCR with the 18-kDa protein gene primers and also slit smears with acid fast staining. PCR of RLEP repetitive sequences is therefore a useful means of detecting M. leprae DNA even when it is present at very low levels.

Interactions of intercalative and minor groove binding ligands with triplex poly(dA).[poly(dT)]2 and with duplex poly(dA).poly(dT) and poly[d(A-T)]2 studied by CD, LD, and normal absorption.

The binding of 9-aminoacridine and one bis-acridine compound to double helical poly(dA).poly-(dT) and poly[d(A-T)]2 and triple helical poly(dA).[poly(dT)]2 has been investigated using linear dichroism (LD) and circular dichroism (CD). A close examination of the negative reduced LD and the induced CD for the first pi-->pi* transition absorption region leads us to conclude that the acridine moiety of the 9-aminoacridine and bis-acridine molecule intercalates with both duplex and triplex DNA. Binding geometries of the acridine moieties in the examined polynucleotides are similar to those found for the ligands with DNA (Hansen et al. (1984) J. Chem. Soc., Chem. Commun., 509-511). It is also found that both 9-aminoacridine and bis-acridine effectively enhance the thermal stability of the triplex DNA. The corresponding spectra for the complexes of the minor groove binders DAPI and Hoechst with poly-(dA).[poly(dT)]2 were studied for comparison. They both show a positive LD and a mixing ratio dependent positive CD in the ligand absorption region, similar to those of their duplex complexes. This indicates that these ligands bind in the grooves of the triplex, probably to the one corresponding to the minor groove of the template duplex.

Production of monoclonal antibody to a phenolic glycolipid of Mycobacterium tuberculosis and its use in detection of the antigen in clinical isolates.

A monoclonal antibody (MAbIII604) specific to phenolic glycolipid Tb (PGL-Tb), a Mycobacterium tuberculosis-specific antigen, was produced and used in the detection of the antigen. MAbIII604 reacted with the PGL-Tb antigen but not with other phenolic glycolipids from Mycobacterium leprae, M. bovis, and M. kansasii, thus indicating the specificity of the monoclonal antibody to PGL-Tb. A dot enzyme-linked immunosorbent assay with MAbIII604 was employed to detect the PGL-Tb antigen in lipids purified from M. tuberculosis clinical isolates. Of 50 isolates, 32 (64.0%) showed clear evidence of the PGL-Tb antigen by the dot enzyme-linked immunosorbent assay, but there were marked variations in the intensities and sizes of spots. This suggests differences in PGL-Tb antigen production among M. tuberculosis strains even when they are grown in the same culture media and conditions. This was most evident from the fact that in only eight (16.0%) of the isolates examined was the PGL-Tb antigen detectable by thin-layer chromatography, which is much less sensitive for the detection of glycolipid antigens. This study shows that monoclonal antibodies specific to PGL-Tb are useful in detecting the antigen in lipid extracts and that there is a marked variation in the PGL-Tb production among M. tuberculosis clinical isolates.