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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.

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Related in: MedlinePlus

Diagrammatic representation of primers location on pvrbp-2 gene. Gray and black boxes indicate intron and exon respectively, and arrows indicate location of primers. F and R: forward and reverse primers of primary PCR respectively, NF and NR: forward and reverse primers of nested PCR respectively. IR1 and IR2 are internal sequencing primers.
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Figure 2: Diagrammatic representation of primers location on pvrbp-2 gene. Gray and black boxes indicate intron and exon respectively, and arrows indicate location of primers. F and R: forward and reverse primers of primary PCR respectively, NF and NR: forward and reverse primers of nested PCR respectively. IR1 and IR2 are internal sequencing primers.

Mentions: Nested PCR primers for pvrbp-2 gene were designed manually using pvrbp-2 sequence available in GenBank (AY501887). These primers are RBP2-F (5’-gatgatcaatttttatgcctgac-3’), RBP2-R (5’-cagaatccgcaataatagag-3’), RBP2-NF (5’-ttcccgcacacacaaggtag-3’), RBP2-NR (5’-gcgtagtgtttagctgccac-3’), RBP2-IR1 (5’-tggaaccgtatgcgattc-3’) and RBP2-IR2 (5’-ttttgcagataagatagc-3’). Internal primers used for sequencing this fragment are IR1 and IR2 and the schematic diagram of gene showing location of primers is given in Figure 2. Optimized PCR conditions for primary PCR for amplification of pvrbp-2 were:-initial denaturation 95°C/5 minute, denaturation 95°C/30 S annealing 50°C/30 S and extension at 68°C/2 minute for 35 cycles, and a final extension of 68°C/5 minute. The cycling conditions of nested PCR were similar to primary PCR except annealing temperature, which was 55°C. All PCR amplifications were carried out in a 20 μl reactions volume (Qiagen’s Master Mix) with 10 pM of each primer and.1-2 μl (~ 3–5 ng) of genomic DNA in primary PCR and 0.5-1 μl of primary PCR product in nested PCR.


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

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

Diagrammatic representation of primers location on pvrbp-2 gene. Gray and black boxes indicate intron and exon respectively, and arrows indicate location of primers. F and R: forward and reverse primers of primary PCR respectively, NF and NR: forward and reverse primers of nested PCR respectively. IR1 and IR2 are internal sequencing primers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Diagrammatic representation of primers location on pvrbp-2 gene. Gray and black boxes indicate intron and exon respectively, and arrows indicate location of primers. F and R: forward and reverse primers of primary PCR respectively, NF and NR: forward and reverse primers of nested PCR respectively. IR1 and IR2 are internal sequencing primers.
Mentions: Nested PCR primers for pvrbp-2 gene were designed manually using pvrbp-2 sequence available in GenBank (AY501887). These primers are RBP2-F (5’-gatgatcaatttttatgcctgac-3’), RBP2-R (5’-cagaatccgcaataatagag-3’), RBP2-NF (5’-ttcccgcacacacaaggtag-3’), RBP2-NR (5’-gcgtagtgtttagctgccac-3’), RBP2-IR1 (5’-tggaaccgtatgcgattc-3’) and RBP2-IR2 (5’-ttttgcagataagatagc-3’). Internal primers used for sequencing this fragment are IR1 and IR2 and the schematic diagram of gene showing location of primers is given in Figure 2. Optimized PCR conditions for primary PCR for amplification of pvrbp-2 were:-initial denaturation 95°C/5 minute, denaturation 95°C/30 S annealing 50°C/30 S and extension at 68°C/2 minute for 35 cycles, and a final extension of 68°C/5 minute. The cycling conditions of nested PCR were similar to primary PCR except annealing temperature, which was 55°C. All PCR amplifications were carried out in a 20 μl reactions volume (Qiagen’s Master Mix) with 10 pM of each primer and.1-2 μl (~ 3–5 ng) of genomic DNA in primary PCR and 0.5-1 μl of primary PCR product in nested PCR.

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