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Published on: June 10, 2015
[Establishment and evaluation of loop-mediated isothermal amplification based on Plasmodium falciparum unique genes
Objective:
To establish a novel convenient loop-mediated isothermal amplification (LAMP) method with the unique genes coding Plasmodium helical interspersed sub-telomeric superfamily (PHIST) for the rapid molecular diagnosis of P. falciparum.
Methods:
The unique genes coding PHIST with high expression mRNA profile during the ring form or schizont period of P. falciparum were screened and selected from the PlasmoDB database. The LAMP primers of targeted genes were designed by the online software (PrimerExplorer V4). The LAMP assay was executed by the color-displaying method with SYBR Green. The dried blood spots of P. falciparum from clinical isolates were collected and the genomic DNA (gDNA) was extracted. For evaluation of sensitivity, the gDNA was diluted to four gradients (10⁻¹, 10⁻², 10⁻³, and 10⁻⁴). For assessment of specificity, the gDNA (s) of P. vivax, P. yoelii, Taenia saginata, and Schistosoma japonicum were also extracted.
Results:
Totally, 61 P. falciparum unique genes coding PHIST were found. The PF3D7_1372300 with high expression value during the ring form and PF3D7_1401600 with high expression value during the schizont period were selected for LAMP assay. The lowest detectable limits of PF3D7_1372300 and PF3D7_1401600 were 130.5 parasite/µl and 1305.3 parasite/µL, respectively. Specific tests showed the amplified products of P. falciparum was positive and all the others including P. vivax, P. yoelii, T. saginata, and S. japonicum were negative.
Conclusions:
The established LAMP method with PF3D7_1372300 gene is sensitive, specific, simple and useful. It can be applied to the field investigation and clinical diagnosis for falciparum malaria.
