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Low frequency of moaA3 gene among the clinical isolates of Mycobacterium tuberculosis from Tamil Nadu and Pondicherry--south eastern coastal states of India.

Sekar B, Arunagiri K, Selvakumar N, Preethi KS, Menaka K - BMC Infect. Dis. (2009)

Bottom Line: In BCG it was absent as expected, but a 386 bp fragment was amplified.The second PCR amplified the flanking sequence of moaA3 and yielded the expected amplicon of 1254 bp in all those 10.3% of clinical isolates which had the 386 bp fragment.However the earlier study carried out in Kerala, reported the presence of moaA3 gene in majority (97%) of their clinical isolates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Laboratories, Central Leprosy Teaching and Research Institute, Chengalpattu, Tamil Nadu, India. drbsekar@yahoo.com

ABSTRACT

Background: Comparative genomic analysis of M. tuberculosis H37Rv and M. bovis BCG have shown that 16 RDs (Regions of Differences) are deleted in BCG and have shown six deletion regions in M. tuberculosis H37Rv. RD1, is present in M. tuberculosis but is absent in all M. bovis BCG sub-strains. A study from Kerala, a south-western coastal state of India aimed to find out differences in RD1 region showed for the first time the presence of moaA3 gene in majority of their clinical isolates, that was absent in type strain H37Rv. We attempted to find out such polymorphism between type strains and the clinical isolates within RD1, targeting moaA3 gene among the clinical isolates of Tamil Nadu & Pondicherry, south-eastern coastal states of India

Methods: One hundred and sixteen clinical isolates of M. tuberculosis were included in the study. PCR using RD1DLa and RD1DRa primers was carried out to amplify a 652 bp fragment, encoding for cfp10 and esat 6 proteins of RD1. A second PCR using primers designed from the surrounding regions of moaA3 gene was done to confirm the presence of the full Open Reading Frame (ORF) in clinical isolates.

Results: In M. tuberculosis H37Rv the expected 652 bp band was present. In BCG it was absent as expected, but a 386 bp fragment was amplified. Around 12/116 (10.3%) of our clinical isolates showed both 652 and 386 bp fragments. The additional 386 bp amplicon is a part of the moaA3 gene which codes for molybdopterin cofactor protein A in M. bovis. The second PCR amplified the flanking sequence of moaA3 and yielded the expected amplicon of 1254 bp in all those 10.3% of clinical isolates which had the 386 bp fragment. However the earlier study carried out in Kerala, reported the presence of moaA3 gene in majority (97%) of their clinical isolates.

Conclusion: This finding showed that there was regional variation presenting polymorphism in moA3 gene, among the strains of M. tuberculosis and further strengthens the speculation of genetic differences among the strains of Kerala and Tamil Nadu & Pondicherry, the South Indian states.

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Related in: MedlinePlus

PCR for amplification of cfp 10 and esat 6 of RD1. Lane-1 – Molecular weight marker (100 bp ladder). Lane-2 – M. tuberculosis H37Rv, showing only 652 bp (RD1). Lane-3 – M. bovis BCG showing only 386 bp. Lane-4 & 5 – Clinical isolates showing 652 bp & 386 bp extra amplicon (moaA3). Lane-6 to 11 – Clinical isolates showing 652 bp only. Lane12 – Negative control.
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Figure 1: PCR for amplification of cfp 10 and esat 6 of RD1. Lane-1 – Molecular weight marker (100 bp ladder). Lane-2 – M. tuberculosis H37Rv, showing only 652 bp (RD1). Lane-3 – M. bovis BCG showing only 386 bp. Lane-4 & 5 – Clinical isolates showing 652 bp & 386 bp extra amplicon (moaA3). Lane-6 to 11 – Clinical isolates showing 652 bp only. Lane12 – Negative control.

Mentions: The PCR primers designed to amplify regions within RD1 to find out the polymorphism between the type strain and the clinical isolates amplified the expected 652 bp fragment (comprising of Rv 3874 and Rv 3875, coding for cfp10 and esat 6) in M. tuberculosis H37Rv. In M.bovis BCG, the 652 bp was absent as expected, but a 386 bp fragment was amplified. All the 116 clinical isolates showed 652 bp products. However in 8 out of 74 (10.8%) of Tamil Nadu isolates and 4 out of 42 (9.5%) of Pondicherry isolates showed both 652 and 386 bp fragments (Figure 1). Thus, 12 out of the 116(10.3%) samples showed the extra amplicon of 386 bp. (Table 1)


Low frequency of moaA3 gene among the clinical isolates of Mycobacterium tuberculosis from Tamil Nadu and Pondicherry--south eastern coastal states of India.

Sekar B, Arunagiri K, Selvakumar N, Preethi KS, Menaka K - BMC Infect. Dis. (2009)

PCR for amplification of cfp 10 and esat 6 of RD1. Lane-1 – Molecular weight marker (100 bp ladder). Lane-2 – M. tuberculosis H37Rv, showing only 652 bp (RD1). Lane-3 – M. bovis BCG showing only 386 bp. Lane-4 & 5 – Clinical isolates showing 652 bp & 386 bp extra amplicon (moaA3). Lane-6 to 11 – Clinical isolates showing 652 bp only. Lane12 – Negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2721839&req=5

Figure 1: PCR for amplification of cfp 10 and esat 6 of RD1. Lane-1 – Molecular weight marker (100 bp ladder). Lane-2 – M. tuberculosis H37Rv, showing only 652 bp (RD1). Lane-3 – M. bovis BCG showing only 386 bp. Lane-4 & 5 – Clinical isolates showing 652 bp & 386 bp extra amplicon (moaA3). Lane-6 to 11 – Clinical isolates showing 652 bp only. Lane12 – Negative control.
Mentions: The PCR primers designed to amplify regions within RD1 to find out the polymorphism between the type strain and the clinical isolates amplified the expected 652 bp fragment (comprising of Rv 3874 and Rv 3875, coding for cfp10 and esat 6) in M. tuberculosis H37Rv. In M.bovis BCG, the 652 bp was absent as expected, but a 386 bp fragment was amplified. All the 116 clinical isolates showed 652 bp products. However in 8 out of 74 (10.8%) of Tamil Nadu isolates and 4 out of 42 (9.5%) of Pondicherry isolates showed both 652 and 386 bp fragments (Figure 1). Thus, 12 out of the 116(10.3%) samples showed the extra amplicon of 386 bp. (Table 1)

Bottom Line: In BCG it was absent as expected, but a 386 bp fragment was amplified.The second PCR amplified the flanking sequence of moaA3 and yielded the expected amplicon of 1254 bp in all those 10.3% of clinical isolates which had the 386 bp fragment.However the earlier study carried out in Kerala, reported the presence of moaA3 gene in majority (97%) of their clinical isolates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Laboratories, Central Leprosy Teaching and Research Institute, Chengalpattu, Tamil Nadu, India. drbsekar@yahoo.com

ABSTRACT

Background: Comparative genomic analysis of M. tuberculosis H37Rv and M. bovis BCG have shown that 16 RDs (Regions of Differences) are deleted in BCG and have shown six deletion regions in M. tuberculosis H37Rv. RD1, is present in M. tuberculosis but is absent in all M. bovis BCG sub-strains. A study from Kerala, a south-western coastal state of India aimed to find out differences in RD1 region showed for the first time the presence of moaA3 gene in majority of their clinical isolates, that was absent in type strain H37Rv. We attempted to find out such polymorphism between type strains and the clinical isolates within RD1, targeting moaA3 gene among the clinical isolates of Tamil Nadu & Pondicherry, south-eastern coastal states of India

Methods: One hundred and sixteen clinical isolates of M. tuberculosis were included in the study. PCR using RD1DLa and RD1DRa primers was carried out to amplify a 652 bp fragment, encoding for cfp10 and esat 6 proteins of RD1. A second PCR using primers designed from the surrounding regions of moaA3 gene was done to confirm the presence of the full Open Reading Frame (ORF) in clinical isolates.

Results: In M. tuberculosis H37Rv the expected 652 bp band was present. In BCG it was absent as expected, but a 386 bp fragment was amplified. Around 12/116 (10.3%) of our clinical isolates showed both 652 and 386 bp fragments. The additional 386 bp amplicon is a part of the moaA3 gene which codes for molybdopterin cofactor protein A in M. bovis. The second PCR amplified the flanking sequence of moaA3 and yielded the expected amplicon of 1254 bp in all those 10.3% of clinical isolates which had the 386 bp fragment. However the earlier study carried out in Kerala, reported the presence of moaA3 gene in majority (97%) of their clinical isolates.

Conclusion: This finding showed that there was regional variation presenting polymorphism in moA3 gene, among the strains of M. tuberculosis and further strengthens the speculation of genetic differences among the strains of Kerala and Tamil Nadu & Pondicherry, the South Indian states.

Show MeSH
Related in: MedlinePlus