[Establishment and Application of Multiplex PCR System for Detecting Four Human Plasmodium Species].

OBJECTIVE: To establish a multiplex PCR detection system for identifying 4 human Plasmodium species and evaluate its applicability. METHODS: The sequences of 18S rDNA gene of the 4 human Plasmodium species were compared using DNAman software, and 4 downstream primers were designed using Oligo 6.0 software, which targeted the region of variability between conserved regions 5 and 6 of the sequences. Using these primers, the specificity and sensitivity of the multiplex PCR system were evaluated, with plasmids containing the 18S rDNA gene sequence as a template. Further, a new nest PCR system (M-Nest) was established by combining the multiplex PCR system with the first-cycle genus-specific primer of the NP-1993 system. The sensitivities of the multiplex PCR system and the M-nest system were evaluated in serial dilutions of blood DNA samples from patients infected by P. falciparum and P. vivax. In addition, the NP-1993 and M-Nest systems were applied to screen the Plasmodium species in 307 blood samples from people returning to Guangxi from Ghana, a malaria epidemic area. And the NP-2002 and M-Nest systems were applied to re-check Plasmodium species in 66 blood samples collected in Guangxi from 2014 January to May, which were identified by microscopy to be infected mainly by P. ovale. RESULTS: The sizes of multiplex PCR products for P. falciparum, P. vivax, P. ovale, and P. malariae were 268 bp, 323 bp, 394 bp, and 446 bp, respectively, located in-between 50-bp DNA ladders. However, their melting curves had similar Tm values, thus could not be used to identify the 4 species. The minimum detection limits of P. falciparum, P. malariae, P. ovale, and P. vivax 18S rDNA gene by the multiplex PCR system were 5.58x102, 1.56x103, 1.66x103, and 1.80 x 10(2) copies/[A. The minimum detection limit of blood DNA from falciparum malaria patients by the multiplex PCR system was 1.43 x 10(2)-8.84 x 10(3) copies/p.1 or 5.10 x 10-4.92 x 10(2) parasites/µl, higher than that of P. vivax (17.4-69.1 copies/L or 13.5-83.2 parasites/p). Compared with this multiples PCR system, The M-Nest system further reduced the minimum detection limit of Plasmodium by 10-100 folds. Further, the M-Nest and NP-1993 systems reached inconsistent detection results in 307 blood samples from people returned to from Ghana; the former detected 2 cases of P. ovale infection while the latter failed. In addition, the NP-2002 and M-Nest systems came to the same results in re-checking Plasmodium species in the 66 blood samples. CONCLUSION: The established multiplex PCR system can identify 4 human Plasmodium species simultaneously and has good applicability in practice.