RESUMEN
Death from tuberculosis has resulted in an increased need for early detection to prevent a tuberculosis (TB) epidemic, especially in closed and crowded populations. Herein, a sensitive electrochemical DNA biosensor based on functionalized iron oxide with mercaptopropionic acid (MPA-Fe3O4) nanoparticle and nanocellulose crystalline functionalized cetyl trimethyl ammonium bromide (NCC/CTAB) has been fabricated for the detection of Mycobacterium tuberculosis (MTB). In this study, a simple drop cast method was applied to deposit solution of MPA-Fe3O4/NCC/CTAB onto the surface of the screen-printed carbon electrode (SPCE). Then, a specific sequence of MTB DNA probe was immobilized onto a modified SPCE surface by using the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling mechanism. For better signal amplification and electrochemical response, ruthenium bipyridyl Ru(bpy)32+ was assigned as labels of hybridization followed by the characteristic test using differential pulse voltammetry (DPV). The results of this biosensor enable the detection of target DNA until a concentration as low as 7.96 × 10-13 M with a wide detection range from 1.0 × 10-6 to 1.0 × 10-12 M. In addition, the developed biosensor has shown a differentiation between positive and negative MTB samples in real sampel analysis.
Asunto(s)
Carbono/química , ADN Bacteriano/análisis , Compuestos Férricos/química , Mycobacterium tuberculosis/aislamiento & purificación , Ácido 3-Mercaptopropiónico/química , Técnicas Biosensibles , Cetrimonio/química , Técnicas Electroquímicas , Electrodos , Mycobacterium tuberculosis/genéticaRESUMEN
Salmonella enterica serovar Typhi with reduced susceptibility to ciprofloxacin is increasingly being reported globally. An outbreak caused by Salmonella Typhi with decreased ciprofloxacin susceptibility has not been reported before in Malaysia. We present here the annotated draft genome of a Salmonella Typhi strain involved in a typhoid outbreak.
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Here, we report of the annotated genome sequence of Mycobacterium tuberculosis MTB221/11. The organism was isolated from the cerebrospinal fluid of a patient in Malaysia.
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This is a report of an annotated genome sequence of Mycobacterium tuberculosis MTBR1/09. The organism was isolated from a sputum sample from a male patient in Malaysia.
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Mycobacterium tuberculosis MTBR2/09 was isolated from a sputum sample from a male patient in Malaysia. This is a report of an annotated genome sequence of M. tuberculosis MTBR2/09.
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This is a report of the annotated genome sequence of Mycobacterium tuberculosis MTBR3/09. The organism was isolated from a sputum sample in Malaysia.
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A quantitative real-time PCR (qPCR) followed by high resolution melting (HRM) analysis was developed for the differentiation of Mycobacterium species. Rapid differentiation of Mycobacterium species is necessary for the effective diagnosis and management of tuberculosis. In this study, the 16S rRNA gene was tested as the target since this has been identified as a suitable target for the identification of mycobacteria species. During the temperature gradient and primer optimization process, the melting peak (Tm) analysis was determined at a concentration of 50 ng DNA template and 0.3, 0.4 and 0.5 µM primer. The qPCR assay for the detection of other mycobacterial species was done at the Tm and primer concentration of 62 °C and 0.4 µM, respectively. The HRM analysis generated cluster patterns that were specific and sensitive to distinguished small sequence differences of the Mycobacterium species. This study suggests that the 16S rRNA-based real-time PCR followed by HRM analysis produced unique cluster patterns for species of Mycobacterium and could differentiate the closely related mycobacteria species.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Técnicas de Diagnóstico Molecular/métodos , Infecciones por Mycobacterium/diagnóstico , Mycobacterium/clasificación , Mycobacterium/genética , Temperatura de Transición , ADN Ribosómico/química , ADN Ribosómico/genética , Humanos , Datos de Secuencia Molecular , Mycobacterium/aislamiento & purificación , Infecciones por Mycobacterium/microbiología , ARN Ribosómico 16S/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
A real-time quantitative polymerase chain reaction (qPCR) was developed for detection and discrimination of Mycobacterium tuberculosis (H37Rv and H37Ra) and M. bovis bacillus Calmette-Guérin (BCG) of the Mycobacterium tuberculosis complex (MTBC) from mycobacterial other than tuberculosis (MOTT). It was based on the melting curve (Tm) analysis of the gyrB gene using SYBR(®) Green I detection dye and the LightCycler 1.5 system. The optimal conditions for the assay were 0.25 µmol/L of primers with 3.1 mmol/L of MgCl(2) and 45 cycles of amplification. For M. tuberculosis (H37Rv and H37Ra) and M. bovis BCG of the MTBC, we detected the crossing points (Cp) at cycles of 16.96 ± 0.07, 18.02 ± 0.14, and 18.62 ± 0.09, respectively, while the Tm values were 90.19 ± 0.06 °C, 90.27 ± 0.09 °C, and 89.81 ± 0.04 °C, respectively. The assay was sensitive and rapid with a detection limit of 10 pg of the DNA template within 35 min. In this study, the Tm analysis of the qPCR assay was applied for the detection and discrimination of MTBC from MOTT.