Supriya Sharma1, Kamlesh Kaitholia1, Ram Suresh Bharti1, Mrigendra Pal Singh2, Neelima Mishra3. 1. ICMR-National Institute of Malaria Research, New Delhi 110077, India. 2. ICMR-NIMR, Field Unit, NIRTH Campus, Jabalpur 482003, India. 3. ICMR-National Institute of Malaria Research, New Delhi 110077, India. Electronic address: neelima.nimr@gmail.com.
Abstract
BACKGROUND: Sensitive diagnostic techniques are needed for timely detection of malaria parasite and disease control. Molecular diagnostic techniques involving Polymerase chain reaction (PCR) with 18 s rRNA as a known diagnostic target with an overall sensitivity of 10 parasites per microliter is used as a gold standard. Till date, no attempt has been undertaken to develop a technique for the identification of four Plasmodium species in a single step PCR combined with restriction digestion with enzymes. METHOD: Plasmodium species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays have been developed, based on RFLP of amplified PCR product of mitochondrial gene as a target. This approach identifies Plasmodium species in two steps involving amplification of mitochondrial (Mt) gene by PCR followed by digestion with restriction enzymes. RESULT: A total of 36 clinical samples were subjected to PCR-RFLP for the diagnosis and detection of malaria parasites targeting mitochondrial gene (Mt). The findings of the method were compared with gold standard methods (Microscopy, RDTs and Nested PCR) and was able to detect mixed infection with a sensitivity of 100% and specificity of 93.8% with respect to nested PCR. The results obtained by PCR-RFLP were validated with Sanger sequencing (n = 32) and were found to be consistent with the method. CONCLUSION: This method identifies and distinguishes four species of human malaria parasite namely P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm) and P. ovale (Po) in approximately 4 h. To overcome and address PCR difficulties, continuous efforts are needed for the development of newer diagnostic techniques.
BACKGROUND: Sensitive diagnostic techniques are needed for timely detection of malaria parasite and disease control. Molecular diagnostic techniques involving Polymerase chain reaction (PCR) with 18 s rRNA as a known diagnostic target with an overall sensitivity of 10 parasites per microliter is used as a gold standard. Till date, no attempt has been undertaken to develop a technique for the identification of four Plasmodium species in a single step PCR combined with restriction digestion with enzymes. METHOD: Plasmodium species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays have been developed, based on RFLP of amplified PCR product of mitochondrial gene as a target. This approach identifies Plasmodium species in two steps involving amplification of mitochondrial (Mt) gene by PCR followed by digestion with restriction enzymes. RESULT: A total of 36 clinical samples were subjected to PCR-RFLP for the diagnosis and detection of malaria parasites targeting mitochondrial gene (Mt). The findings of the method were compared with gold standard methods (Microscopy, RDTs and Nested PCR) and was able to detect mixed infection with a sensitivity of 100% and specificity of 93.8% with respect to nested PCR. The results obtained by PCR-RFLP were validated with Sanger sequencing (n = 32) and were found to be consistent with the method. CONCLUSION: This method identifies and distinguishes four species of humanmalaria parasite namely P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm) and P. ovale (Po) in approximately 4 h. To overcome and address PCR difficulties, continuous efforts are needed for the development of newer diagnostic techniques.