Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax

Abstract

The 28S rRNA gene was amplified and sequenced from P. falciparum and P. vivax isolates collected from northwest India. Based upon the sequence diversity of the Plasmodium 28SrRNA gene in comparison with its human counterpart, various nested polymerase chain reaction (PCR) primers were designed from the 3R region of the 28SrRNA gene and evaluated on field isolates. This is the first report demonstrating the utility of this gene for species-specific diagnosis of malaria for these two species, prevalent in India. The initial evaluation on 363 clinical isolates indicated that, in comparison with microscopy, which showed sensitivity and specificity of 85·39% and 100% respectively, the sensitivity and specificity of the nested PCR assay was found to be 99·08% and 100% respectively. This assay was also successful in detecting mixed infections that are undetected by microscopy. Our results demonstrate the utility of the 28S rRNA gene as a diagnostic target for the detection of the major plasmodial species infecting humans.

Keywords:

• 28S rRNA gene
• Nested PCR
• Plasmodium detection
• Malaria diagnosis
• Surveillance tools

This study was partially supported by a grant from the Council of Scientific and Industrial Research (37/1176/04/EMRII). DP was supported by CSIR-SRF (09/719(0031)/2008 EMRI). We thank the clinical support staff of SPM and lab staff of BITS-Pilani for their support at various stages. The support of BITS-Pilani is duly acknowledged for providing the required lab facilities. An Indian patent IN0659DEL2009 has been filed with Council of Scientific and Industrial Research, India as the applicant.