OBJECTIVE: To improve the sensitivity, specificity and stability of the Tag-primer nested/multiplex PCR for malaria diagnosis. METHODS: Filter paper blood samples were collected from 30 non-malaria fever patients and 20 infectious disease patients (common cold, influenza, typhoid, hepatitis, etc.). Four ml blood each taken from one falciparum malaria patient and one vivax malaria patient was serially diluted. Healthy blood sample was used as negative control. Improved direct heating method was used to prepare DNA template. The cytochrome oxidase gene (coxI) located in mitochondrion was selected as target gene. Relevant web resources and software (PUBMED, NCBI-BLAST, Mfold server and Primer Premier 5.0) were employed to design and optimize Tag-primer nested/multiplex PCR (UT-PCR) which was used to test all blood samples. RESULTS: A 611 bp band and a 255 bp band were seen in serially diluted infected blood samples (1,000, 100, 10 and 1 parasite/microl) from P.f and P.v patient tested by UT-PCR. The detection limit of either P. falciparum or P. vivax reached 1 parasite/microl, and the tested blood samples of non-malaria fever patients, patients with other infectious diseases and healthy persons were all negative. Consistent results of each sample in more than 3 duplicated tests were obtained. CONCLUSION: The optimized Tag-primer nested/multiplex PCR shows high sensitivity, specificity and stability in malaria diagnosis.
OBJECTIVE: To improve the sensitivity, specificity and stability of the Tag-primer nested/multiplex PCR for malaria diagnosis. METHODS: Filter paper blood samples were collected from 30 non-malaria feverpatients and 20 infectious diseasepatients (common cold, influenza, typhoid, hepatitis, etc.). Four ml blood each taken from one falciparum malariapatient and one vivax malariapatient was serially diluted. Healthy blood sample was used as negative control. Improved direct heating method was used to prepare DNA template. The cytochrome oxidase gene (coxI) located in mitochondrion was selected as target gene. Relevant web resources and software (PUBMED, NCBI-BLAST, Mfold server and Primer Premier 5.0) were employed to design and optimize Tag-primer nested/multiplex PCR (UT-PCR) which was used to test all blood samples. RESULTS: A 611 bp band and a 255 bp band were seen in serially diluted infected blood samples (1,000, 100, 10 and 1 parasite/microl) from P.f and P.v patient tested by UT-PCR. The detection limit of either P. falciparum or P. vivax reached 1 parasite/microl, and the tested blood samples of non-malaria feverpatients, patients with other infectious diseases and healthy persons were all negative. Consistent results of each sample in more than 3 duplicated tests were obtained. CONCLUSION: The optimized Tag-primer nested/multiplex PCR shows high sensitivity, specificity and stability in malaria diagnosis.