A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Malaria/*diagnosis ; Malaria/*parasitology ; Plasmodium knowlesi/*genetics ; Plasmodium knowlesi/*isolation & purification ; Polymerase Chain Reaction/*methods; Animals ; Base Sequence ; DNA Primers/genetics ; DNA, Protozoan/genetics ; DNA, Ribosomal/genetics ; Humans ; Limit of Detection ; Molecular Diagnostic Techniques/methods ; Plasmodium falciparum/genetics ; Plasmodium falciparum/isolation & purification ; Plasmodium malariae/genetics ; Plasmodium malariae/isolation & purification ; Plasmodium ovale/genetics ; Plasmodium ovale/isolation & purification ; Plasmodium vivax/genetics ; Plasmodium vivax/isolation & purification ; Species Specificity ; Time Factors

A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient's blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a "fast PCR enzyme". In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the "fast PCR enzyme", with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses.

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0 (DNA Primers)
0 (DNA, Protozoan)
0 (DNA, Ribosomal)

Date Created: 20180126 Date Completed: 20180312 Latest Revision: 20220331

20221213

PMC5785027

10.1371/journal.pone.0191886

29370297