Limits...
Molecular analysis of reticulocyte binding protein-2 gene in Plasmodium vivax isolates from India.

Prajapati SK, Kumari P, Singh OP - BMC Microbiol. (2012)

Bottom Line: ApoI-RFLP was found to be more efficient in identifying the extent of genetic polymorphism in pvrbp-2 compared to AluI-RFLP.The study suggests that pvrbp-2 is highly polymorphic genetic marker which can be used for population genetic analyses.RFLP analysis suggests presence of nearly similar proportion of Sal-1 and Belem alleles in Indian P. vivax populations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Division, National Institute of Malaria Research, Sector 8, Dwarka, Delhi, India. surendramrc@gmail.com

ABSTRACT

Background: Plasmodium vivax reticulocyte binding protein-2 (PvRBP-2) is a promising candidate for development of vaccine against parasite. DNA sequence polymorphism in pvrbp-2 which may hamper the vaccine development program has been identified in laboratory strains. Therefore, unraveling genetic polymorphism in pvrbp-2 from field isolates is a prerequisite for success in vaccine development. This study was designed with a primary aim to uncover genetic polymorphism in pvrbp-2 among P. vivax field isolates.

Results: Using virtual restriction mapping of pvrbp-2 sequences, two restriction enzymes (AluI and ApoI) were selected for the development of pvrbp-2 as a PCR-RFLP marker. Restriction fragment length polymorphism (RFLP) analysis revealed a high degree of genetic polymorphism in the pvrbp-2 gene among field isolates of P. vivax. ApoI-RFLP was found to be more efficient in identifying the extent of genetic polymorphism in pvrbp-2 compared to AluI-RFLP. Combined genotyping/haplotyping of RFLP pattern revealed a total of 36 distinct RFLP patterns among 83 P. vivax isolates analyzed. DNA sequence analysis also supports high degree of genetic polymorphism among field isolates of P. vivax. Pvrbp-2 PCR-RFLP method is able to distinguish multiple infection up to 16.86% and it revealed a low level of shared genetic pool between more than two populations.

Conclusion: The study suggests that pvrbp-2 is highly polymorphic genetic marker which can be used for population genetic analyses. RFLP analysis suggests presence of nearly similar proportion of Sal-1 and Belem alleles in Indian P. vivax populations. The larger extent of genetic polymorphism identified from limited samples advocates to screen genetic polymorphism in pvrbp-2 from malaria endemic geographical regions and countries for designing pvrbp-2 based anti-malarial control measures.

Show MeSH

Related in: MedlinePlus

Gel pictures of PCR and RFLP of pvrbp-2 gene, a) PCR amplification, b) AluI digestion, c) ApoI digestion. Name above the each well represents sample identity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3539960&req=5

Figure 3: Gel pictures of PCR and RFLP of pvrbp-2 gene, a) PCR amplification, b) AluI digestion, c) ApoI digestion. Name above the each well represents sample identity.

Mentions: A total of 90 P. vivax samples were analyzed where in all samples gave single clear amplification of ~2.0 kb fragment size and none of the PCR fragments showed size variation (Figure 3a). Amplified PCR fragment covers both coding and non-coding regions. The coding regions are marked by a 449–503 bp and 705–1946 bp in the amplified PCR product (Figure 2). Virtual restriction mapping of pvrbp-2 sequence suggests the use of ApoI and AluI restriction enzymes for RFLP analysis. Initially, five samples were digested with the above two enzymes to make sure that these enzymes can identify genetic polymorphism from field isolates. Interestingly, genetic polymorphism in AluI and ApoI digestion was observed in selected five samples. Further, PCR products obtained from 83 P. vivax isolates were digested with AluI and ApoI enzymes separately. RFLP pattern of pvrbp-2 gene with AluI and ApoI has been shown in figure 3b & c respectively.


Molecular analysis of reticulocyte binding protein-2 gene in Plasmodium vivax isolates from India.

Prajapati SK, Kumari P, Singh OP - BMC Microbiol. (2012)

Gel pictures of PCR and RFLP of pvrbp-2 gene, a) PCR amplification, b) AluI digestion, c) ApoI digestion. Name above the each well represents sample identity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539960&req=5

Figure 3: Gel pictures of PCR and RFLP of pvrbp-2 gene, a) PCR amplification, b) AluI digestion, c) ApoI digestion. Name above the each well represents sample identity.
Mentions: A total of 90 P. vivax samples were analyzed where in all samples gave single clear amplification of ~2.0 kb fragment size and none of the PCR fragments showed size variation (Figure 3a). Amplified PCR fragment covers both coding and non-coding regions. The coding regions are marked by a 449–503 bp and 705–1946 bp in the amplified PCR product (Figure 2). Virtual restriction mapping of pvrbp-2 sequence suggests the use of ApoI and AluI restriction enzymes for RFLP analysis. Initially, five samples were digested with the above two enzymes to make sure that these enzymes can identify genetic polymorphism from field isolates. Interestingly, genetic polymorphism in AluI and ApoI digestion was observed in selected five samples. Further, PCR products obtained from 83 P. vivax isolates were digested with AluI and ApoI enzymes separately. RFLP pattern of pvrbp-2 gene with AluI and ApoI has been shown in figure 3b & c respectively.

Bottom Line: ApoI-RFLP was found to be more efficient in identifying the extent of genetic polymorphism in pvrbp-2 compared to AluI-RFLP.The study suggests that pvrbp-2 is highly polymorphic genetic marker which can be used for population genetic analyses.RFLP analysis suggests presence of nearly similar proportion of Sal-1 and Belem alleles in Indian P. vivax populations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Division, National Institute of Malaria Research, Sector 8, Dwarka, Delhi, India. surendramrc@gmail.com

ABSTRACT

Background: Plasmodium vivax reticulocyte binding protein-2 (PvRBP-2) is a promising candidate for development of vaccine against parasite. DNA sequence polymorphism in pvrbp-2 which may hamper the vaccine development program has been identified in laboratory strains. Therefore, unraveling genetic polymorphism in pvrbp-2 from field isolates is a prerequisite for success in vaccine development. This study was designed with a primary aim to uncover genetic polymorphism in pvrbp-2 among P. vivax field isolates.

Results: Using virtual restriction mapping of pvrbp-2 sequences, two restriction enzymes (AluI and ApoI) were selected for the development of pvrbp-2 as a PCR-RFLP marker. Restriction fragment length polymorphism (RFLP) analysis revealed a high degree of genetic polymorphism in the pvrbp-2 gene among field isolates of P. vivax. ApoI-RFLP was found to be more efficient in identifying the extent of genetic polymorphism in pvrbp-2 compared to AluI-RFLP. Combined genotyping/haplotyping of RFLP pattern revealed a total of 36 distinct RFLP patterns among 83 P. vivax isolates analyzed. DNA sequence analysis also supports high degree of genetic polymorphism among field isolates of P. vivax. Pvrbp-2 PCR-RFLP method is able to distinguish multiple infection up to 16.86% and it revealed a low level of shared genetic pool between more than two populations.

Conclusion: The study suggests that pvrbp-2 is highly polymorphic genetic marker which can be used for population genetic analyses. RFLP analysis suggests presence of nearly similar proportion of Sal-1 and Belem alleles in Indian P. vivax populations. The larger extent of genetic polymorphism identified from limited samples advocates to screen genetic polymorphism in pvrbp-2 from malaria endemic geographical regions and countries for designing pvrbp-2 based anti-malarial control measures.

Show MeSH
Related in: MedlinePlus