Identification and evaluation of a new nucleic acid amplification test target for specific detection of Mycobacterium tuberculosis.
Clin Chem Lab Med
; 48(10): 1501-5, 2010 Oct.
Article
en En
| MEDLINE
| ID: mdl-20658950
ABSTRACT
BACKGROUND:
Accurate and early diagnosis of tuberculosis (TB) is of major importance in the management and control of TB. Because the conventional bacteriological diagnosis of TB has several limitations, nucleic acid amplification (NAA) tests have emerged as promising alternatives. A potential problem with NAA tests is that some strains lack a target, which may be the one of main reasons for the much lower and highly variable accuracy in diagnosis. A possible solution may be to use more valid and applicable targets to increase detection accuracy.METHODS:
In this paper, we designed a two-step program to obtain NAA test targets. Inter-simple sequence repeats (ISSR) based on oligonucleotide (GTG)(5) were first constructed to genotype Mycobacterium strains to obtain Mycobacterium tuberculosis (MTB)-specific fragment. Second, sequence characterized amplified region (SCAR) markers were developed from these species-specific sequences to identify MTB. Some 312 Mycobacterium strains were used to evaluate the efficacy of the SCAR markers, IS6110 element [specific identification of Mycobacterium tuberculosis complex (MTC)] and 16SrRNA gene (specific identification of Mycobacterium) amplification, together with traditional bacteriology testing was used as a control.RESULTS:
MTB-specific sequences located in a gene coding for Rv1508c, as a new NAA test target, were obtained using ISSR-PCR genotyping. Based on these sequences, the SCAR primer pairs MISP1 and MISP2 were designed. All 312 strains from Mycobacterium accurately produced the genus-specific 16SrRNA amplicon. 271 MTB strains and M. africanum were positive. However, all nontuberculous mycobacteria (NTM) strains and 1 MTB strain named 1143 were negative in both SCAR and IS6110 PCR amplification. M. bovis, bacille Calmette-Guérin (BCG) were IS6110-PCR positive, while SCAR-PCR was negative. Strain 1143 was defined as M. arupense with 99% identity by 16SrRNA gene sequencing identification, despite being diagnosed as MTB using traditional testing.CONCLUSIONS:
SCAR markers developed with this two-step program can be used as a new NAA test target to correctly detect MTB.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tuberculosis
/
ARN Ribosómico 16S
/
Técnicas de Amplificación de Ácido Nucleico
/
Mycobacterium tuberculosis
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
/
Prognostic_studies
/
Screening_studies
Límite:
Humans
Idioma:
En
Revista:
Clin Chem Lab Med
Asunto de la revista:
QUIMICA CLINICA
/
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Año:
2010
Tipo del documento:
Article