Abstract:

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 5.58×102， 1.56×103， 1.66×103， and 1.80×102 copies/μl. The minimum detection limit of blood DNA from falciparum malaria patients by the multiplex PCR system was 1.43×102-8.84×103 copies/μl or 5.10×10-4.92×102 parasites/μl， higher than that of P. vivax（17.4-69.1 copies/μl or 13.5-83.2 parasites/μl）. 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.

Key words: Multiple PCR, Nested PCR, Plasmodium ovale wallikeri