关键词: 疟原虫, 嵌合特异性引物, 种属同检, 多重PCR

Abstract:

Objective To establish an efficient malaria parasite detection method with genus- and species-specificity. Methods Specific primers were designed, including 4 pairs for 18S rRNA ribosomes which were species specific and 1 pair for mitochondrial gene which was genus specific. They were connected to the universal primer (5′-CGAGTCCTGCGGTCTCAAATT-3′) to generate 5 pairs of chimeric primers. PCR reaction was performed to determine the optimal annealing temperature and primer concentrations. Multiple PCR was performed to optimize the reaction condition of the 5 primer pair mix to establish the new method named P5-mPCR. Patient blood samples, which contained different densities of parasites of Plasmodium vivax (Pv), P. malariae (Pm), P ovale (Po), and P falciparum (Pf), and simulated mixed infection samples were examined by P5-mPCR to determine the sensitivity of P5-mPCR. A variety of blood samples infected by other parasites or worm DNA were examined to evaluate the specificity of the method. The 212 malaria blood samples were tested to evaluate the application value of P5-mPCR in comparison to the nested PCR. Results The optimized reaction system of P5-mPCR consisted of (volume ratio): 10% of DNA template, 5% of primers mix, 35% of distilled water and 50% of Taq polymerase pre-mixed solution. The optimal condition for P5-mPCR system was as follows: 95 ℃ for 5 min, 5 cycles of 94 ℃ for 15 s, 58 ℃ for 20 s, and 72 ℃ for 20 s; 10 cycles of 94 ℃ for 15 s, 62 ℃ for 20 s, and 72 ℃ for 20 s; 25 cycles for 94 ℃ for 15 s, 68 ℃for 20 s, and 72 ℃ for 20 s; 72 ℃ for 3 min; 10 ℃ for 5 min. The amplified products were 778, 582, 400, 256 and 334 bp for Pv, Pm, Po, Pf, and genus Plasmodium, respectively, with a detection sensitivity of 4.49, 5.45, 6.39, 4.07 parsites/μl blood (average of 4.85 parsites/μl blood for species-specific detection), and 0.10-1.07 parasites/μl blood (mean value, 0.41 for genus-specific detection). Specific amplification bands were clearly seen in testing mixed blood sample containing 50-200 parsites/μl blood. No specific bands were seen for Schistosoma japonicum, Toxoplasma gondii, Leishmania donovani, Cysticercus cellulosae, Paragonimus westermani and Cryptosporidium. For the 212 clinically-suspected malaria blood samples, the consistency of the testing results was higher between the nested PCR and P5-mPCR(Kappa = 0.866, P < 0.01), and the positive rate by nested PCR and P5-mPCR was 75.0% (159/212) and 79.7% (169/212), respectively, with a significant difference (χ² = 8.100,P < 0.01). From the point of species-specificity diagnosis, the differences were mainly derived from the Pf (P < 0.01) and Po (P < 0.05) samples. While for samples with Pv, Pm, and mixed infections, there was no statistical difference between the two methods (P > 0.05). Conclusion The P5-mPCR detection system has a high sensitivity and good specificity, is simple to perform, fast and efficient, and can simultaneously identify 4 species of malaria parasites.

Key words: Plasmodium, Chimeric primers, Genus-and species-specific co-detection, Multiplex PCR