Random amplified polymorphic DNA analysis of Mycobacterium tuberculosis isolates resistant to Isoniazid in Indonesia

Maidin, Asaad; Lidjaja, Agnes; Hatta, Mochammad

doi:10.4236/jtr.2013.12003

Cited by 1 publication

(4 citation statements)

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“…The spatial cluster of OPN20 positive isolates in Central Sulawesi formed by the two very similar genotypes MT-A and the MT-B indicates that in that particular area either of these genotypes was introduced and that the other genotype evolved from its progeny. Furthermore, the OPN 0.2 loci was rarely prevalent in genotype MT-C, in line with findings from a previous study (17).…”

Section: Resultssupporting

confidence: 92%

“…In a previous study (Maidin, et al 2013), genetic diversity of 7 strains from the same areas in Sulawesi were analyzed by numerical analysis of RAPD PCR finger printings with five oligonucleotide primers (17).It was found that RAPD is faster and technically less demanding than most other molecular typing methods and furthermore, no DNA sequence information is necessary. Also, much smaller amounts of purified DNA are required than for methods such as RFLP.…”

Section: Discussionmentioning

confidence: 99%

“…PCR using the RAPD method was performed with 11 primers according to Tazi et al (16), Singh et al [19] and Assad M et al (17). DNA amplification reaction was performed in a total volume of 25 µl.…”

Section: Molecular Fingerprintmentioning

confidence: 99%

“…Currently, RAPD is increasingly appliedin epidemiologic typing of a wider range of microorganisms including MTC (15,16). Although the technique is simple and rapid, reproducibility issues have been reported due to its sensitivity to primer variation, DNA concentration, DNA template quality, gel electrophoresis, and type of DNA polymerase (17,18).Previous studies have shown that multiplex RAPD with ten, seven and five random decameroligonucleotide primers, respectively,in a single PCR was particularly useful for strain differentiation of Mycobacteria isolates because it is able to increase the number ofinformative genetic markers in comparison with only one round of amplification (16)(17)(18). In this prospective genotyping study covering tuberculosis transmission in the island of Sulawesi, Eastern Indonesia, we were able to show that Multiplex RAPD-PCR using 11 primers is reproducible; this technique revealed a high diversity of MTC isolates.…”

Section: Introductionmentioning

confidence: 99%

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The Reproducibility of the Multiplex RAPD-PCR Assay in Genotyping of Mycobacterium Tuberculosis Isolates from Sulawesi, Indonesia

Hatta¹

2014

AJCEM

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Background: Random Amplified Polymorphic DNA (RAPD) assay has recently emerged as a genotyping method which is both robust and highly discriminatory for bacterial strain differentiation. However, RAPD assessment for Mycobacterium tuberculosiscomplex (MTC) isolates is still limited. Despite its simplicity and rapidity, conventional RAPD also has low reproducibility due to its sensitivity to several factors. Therefore we studied the feasibility of an RAPD-PCR assay to define the genetic diversity of MTC isolates and to evaluate its reproducibility.Methods:493clinical MTC isolates from the island of Sulawesi in Eastern Indonesia, collected from 2005-2012were subjected to Multiplex RAPD assay using 11 random decamer primers instead of one primer which is common in conventional RAPD. All 11 primers were found to be differentiated and produced specific RAPD profiles. The polymorphic amplicons served as RAPD markers for MTC. The dendrograms, obtained by different primers, showed the discriminatory ability of the primers.Results:Multiplex RAPD-PCR results show that the majority of the isolates from South Sulawesi, Southeast Sulawesi andCentral Sulawesiin eastern region of Indonesia belong to group MT-C (80.7%, 80.0% and 62.6 % respectively) with result reproducibility as high as 100%. Conclusion:Molecular typing with multiplex RAPD-PCR is a powerful approach to show the genetic heterogeneity of MTC isolates. The discrimination power and reproducibility of this multiple loci-based RAPD was higher than conventional fewer loci-targeted RAPD.

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Section: Resultssupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%