Establishment and application of multiplex PCR for co-detection of genus- and species-specific malaria parasites

Abstract

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

CLC Number:

R382.31

Li JIANG, Yao-guang ZHANG, Li CAI, Zhen-yu WANG, min ZHU, Xiao-jiang MA, Huan-yu WU. Establishment and application of multiplex PCR for co-detection of genus- and species-specific malaria parasites[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2018, 36(4): 380-387.

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种属名 Species	特异性引物序列（5′→3′） specific primer sequences（5′→3′）	产物长度/bp Product length/bp	参考序列 References
间日疟原虫 P. vivax	F: TAAGGACTTTCTTTGCTTCGGC R: AAATCAACCGAATTCAGTCCCAC	778	U07367
三日疟原虫 P. malariae	F: GAATTTCAAGGAATCAATATTTTAAGT R: GAATGTCTCTTTTATTTTTTACTCTATC	582	AF145336
卵形疟原虫 P. ovale	F: GCTAATACTTGCTTTAATGCGTTTG R: CCAATTACAAAACCATGAAATGGCC	400	KF696365
恶性疟原虫 P. falciparum	F: GATGTTTCATTTAAACTGGTTTGGGA R: ACAATGAACTCAATCATGACTACCCG	256	M19173
疟原虫属 Plasmodium	F: GAATAGAAACAGATGCCAGGCCA R: ATGTTAGAAGCAAACACTAGCGGTG	334	NC007243

种属名 Species	原虫计数/个虫·（μl血)^-1 Parasite count/No·（μl blood)^-1	最高稀释度 Highest dilution	最低原虫数/个虫·（μl血)^-1 Lowest parasite count/No·（μl blood)^-1	平均检出限及范围/个虫·（μl血)^-1 Average detection limit and its range/No·（μl blood)^-1
间日疟原虫 P. vivax	6 857	10^-3	6.86	4.49/（1.49~6.86）
	5 124	10^-3	5.12
	1 493	10^-3	1.49
三日疟原虫 P. malariae	4 085	10^-3	4.08	5.45/（1.60~10.66）
	1 600	10^-3	1.60
	10 666	10^-3	10.66
卵形疟原虫 P. ovale	9 627	10^-3	9.63	6.39/（2.38~9.63）
	2 378	10^-3	2.38
	716	10^-2	7.16
恶性疟原虫 P. falciparum	200 000	10^-5	2.00	4.07/（2.00~4.50）
	4 500	10^-3	4.50
	27 200	10^-4	2.72
疟原虫属 Plasmodium	6 857	10^-4	0.68	0.41/（0.10~1.07）
	5 124	10^-4	0.51
	1 493	10^-4	0.15
	4 085	10^-4	0.41
	1 600	10^-4	0.16
	10 666	10^-4	1.07
	9 627	10^-5	0.10
	2 378	10^-4	0.24
	716	10^-3	0.72
	200 000	10^-6	0.20
	4 500	10^-4	0.45
	27 200	10^-5	0.27

检测方法 Methods	样品数 No. samples	阳性数 No. positives	阳性率/% Positive rate/%	分型诊断阳性数 No. positives for classification diagnosis
检测方法 Methods	样品数 No. samples	阳性数 No. positives	阳性率/% Positive rate/%	恶性疟原虫 P. falciparum	间日疟原虫 P. vivax	卵形疟原虫 P. ovale	三日疟原虫 P. malariae	混合感染 Mixed infection	未分型 Unclassified
巢式PCR Nested PCR	212	159	75.0	112	22	10	6	2	7
多重PCR P5-mPCR	212	169	79.9	121	22	17	7	2	0
Kappa值 P值 χ²值		0.866 < 0.01 8.100		0.914 < 0.01 7.111	0 1 0	0.724 < 0.01 5.143	0.921 < 0.01 0	0 1 0	- - 5.143
P值		< 0.01		< 0.01	1	< 0.01	1	1	< 0.05