RESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Hanseníase/diagnóstico , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase/métodos , Anticorpos Antibacterianos/sangue , Primers do DNA , Humanos , Hanseníase/microbiologia , Mycobacterium leprae/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , Pele/microbiologiaRESUMO
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.
Assuntos
Humanos , Anticorpos Antibacterianos/sangue , Hanseníase/diagnóstico , Hanseníase/microbiologia , Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , Pele/microbiologia , Primers do DNA , Proteínas de Bactérias/genética , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Ácido NucleicoRESUMO
Although there is no genetic diversity in isolates of Mycobacterium leprae, the variance of tandem repeats in the rpoT gene was recently demonstrated. We have typed clinical isolates of M. leprae in Korea using difference of the tandem repeats in the rpoT gene. Among 69 patients, 65 Korean isolates (94.2%) demonstrated four copies of the 6 bp tandem repeat (GACATC) in the rpoT gene, and incidences of three copies were found in only two Koreans and two foreigners (2.9%, respectively).