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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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Map of India showing study sites. N indicates number of sample from individual geographical region.
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Figure 1: Map of India showing study sites. N indicates number of sample from individual geographical region.

Mentions: Ninety P. vivax field isolates collected between 2003–2006 from six geographical regions of the Indian subcontinent were analyzed (Figure 1). Finger prick blood from the symptomatic patients in active case detection surveys as well as from patient attending the clinics, was spotted on autoclaved Whatman filter paper strips (Number 3). The six geographical regions are Delhi (N=13), Nadiad of Gujarat (N=26), Panna of Madhya Pradesh (N=18), Rourkela of Odisa (N=16), Chennai of Tamil Nadu (N=10), and Kamrup of Assam (N=7). Details of individual study sites such as location, parasite and vector species prevalence, and disease transmission pattern are reported elsewhere [23] as well as given in Additional file 1. Genomic DNA was isolated from microscopically diagnosed vivax-positive blood spotted on Whatman filter paper (3 mm) strips using QIAamp mini DNA kit (Qiagen, Germany). Three punches (5 mm diameter) of dried blood spots were used for DNA isolation, as per the manufacturer’s instructions. DNA was eluted in 120 μl triple distilled autoclaved water and stored at −20°C for future use.


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

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

Map of India showing study sites. N indicates number of sample from individual geographical region.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Map of India showing study sites. N indicates number of sample from individual geographical region.
Mentions: Ninety P. vivax field isolates collected between 2003–2006 from six geographical regions of the Indian subcontinent were analyzed (Figure 1). Finger prick blood from the symptomatic patients in active case detection surveys as well as from patient attending the clinics, was spotted on autoclaved Whatman filter paper strips (Number 3). The six geographical regions are Delhi (N=13), Nadiad of Gujarat (N=26), Panna of Madhya Pradesh (N=18), Rourkela of Odisa (N=16), Chennai of Tamil Nadu (N=10), and Kamrup of Assam (N=7). Details of individual study sites such as location, parasite and vector species prevalence, and disease transmission pattern are reported elsewhere [23] as well as given in Additional file 1. Genomic DNA was isolated from microscopically diagnosed vivax-positive blood spotted on Whatman filter paper (3 mm) strips using QIAamp mini DNA kit (Qiagen, Germany). Three punches (5 mm diameter) of dried blood spots were used for DNA isolation, as per the manufacturer’s instructions. DNA was eluted in 120 μl triple distilled autoclaved water and stored at −20°C for future use.

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