Evaluation of the efficacy of loop-mediated isothermal amplification in detecting Plasmodium falciparum and other species of Plasmodium

doi:10.12140/j.issn.1000-7423.2020.04.004

Abstract

Abstract:

Objective To detect the DNA of malaria parasites using the loop-mediated isothermal amplification (LAMP) method and evaluate the efficacy of this method.Methods Blood samples on filter papers were collected from confirmed malaria patients returning from endemic areas of other countries during 2013-2018. According to the 18S rRNA sequences of malaria parasites, LAMP primers for Plasmodium falciparum and other species of Plasmodium were designed using the Primer Explorer V5 software. The LAMP method for detecting malaria parasites was established and its sensitivity, specificity, detection limit and amplification efficiency were assessed and compared with the nested-PCR. The effect of calcein indicator on LAMP reaction was tested.Results The LAMP and nested-PCR were performed separately on 96 blood samples, including 47 with P. falciparum and 49 with other species of Plasmodium. Either method found 93 samples as malaria-positive. The LAMP method produced positive results for all the 47 blood samples with P. falciparum, while having a false negative rate of 2.1% among the samples with other Plasmodium spp.. The LAMP method provided negative results for 10 samples with Leishmania, 8 with Schistosomiasis japonicum, and 38 blood samples from healthy participants, highly consistent with the results of nested-PCR (Kappa value, 0.956). The sensitivity of LAMP for P. falciparum, P. vivax, P. ovale and P. malariae was 25, 3, 4, and 2 parasites/μl blood, respectively. Amplification efficiency test showed that the turbity time (Tt) and peak value of rate (Df) of LAMP for the four malaria species were less than 60 min and above 0.14, respectively, of them the amplification efficacy was particularly high for P. falciparum (Tt, 20.4 min; Df, 0.16). In visualized LAMP assay for the four malaria species, using calcein indicator delayed LAMP reaction Tt about 8-22 min, and lower the Df by about 21%-35%.Conclusion The LAMP method is highly sensitive, specific, of low detection limit and has potentials for application on-site in the field and in primary medical settings.

Key words: Plasmodium, LAMP, Nested-PCR, P. falciparum

CLC Number:

R382.31

ZHOU Shui-mao, TU Zu-wu, YANG Yan, CHEN Fang, JIA Xi-shuai. Evaluation of the efficacy of loop-mediated isothermal amplification in detecting Plasmodium falciparum and other species of Plasmodium[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(4): 423-428.

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引物名称	虫种	引物序列(5′→3′)
Primer	Species	Sequence（5′→3′）
Pf-FIP	Pf	CAGTATATTGATATTGCGTGACGACCTTGCAATAAATAATATCTAGCGTGT
Pf-BIP	Pf	AACTCCAGGCGTTAACCTGTAATGATCTTTACGTTAAGGGC
Pf-LF	Pf	GTGTACAAGGCAACAATACACG
Pf-LB	Pf	GTTGAGATGGAAACAGCCGG
Pf-F3	Pf	TATTGGCACCTCCATGTCG
Pf-B3	Pf	AACATTTTTTAGTCCCATGCTAA
P-FIP^a	Pv, Po, Pm	TTAAGGCTGCGATGAGACGACGCCGGGGATAACAGGTAGT
P-BIP^a	Pv, Po, Pm	AGCGTGTATTGTTGCCTTGTACTTAACGCCTGGAGTTCTTTATCTT
P-LF^a	Pv, Po, Pm	TGGAGGTGCCAATAGTATATAAAGAT
P-LB^a	Pv, Po, Pm	TGTACACACCGCTCGTCAC
Pa-F3^a	Pv, Po	GGTGATTTTGTGTGCCGTT
Pb-F3^a	Pm	ACGGTTATTTTGTGTACCGTT
P-B3^a	Pv, Po, Pm	CGGCTGTTTCCATCTCAACT

方法Method	分类 Malaria parasite	LAMP				合计Total
方法Method	分类 Malaria parasite	Pf	其他疟原虫 Other species of Plasmodium	Pf+P^a	阴性Negative	合计Total
巢式PCR Nested-PCR	Pf	45	0	1	0	46
	其他疟原虫 Other species of Plasmodium	0	44	1	1	46
	Pf+P^a	0	0	1	0	1
	阴性Negative	0	1	0	40	41
合计Total		45	45	3	41	134

虫种Species	未加钙黄绿素指示剂 Without calcein indicator								加钙黄绿素指示剂 With addition of calcein indicator
	1		10¹		10²		10³		1		10¹		10²		10³
	Tt/min	Df	Tt/min	Df	Tt/min	Df	Tt/min	Df	Tt/min	Df	Tt/min	Df	Tt/min	Df	Tt/min	Df
Pf	20.4	0.163	21.7	0.164	23.1	0.163	25.5	0.162	29.6	0.123	31.7	0.119	34.4	0.113	36.6	0.108
Pv	34.7	0.16	36.3	0.156	39.1	0.157	42.4	0.149	42.7	0.117	44.7	0.109	47.2	0.107	50.4	0.097
Po	45.1	0.141	48.0	0.134	52.9	0.132	56.5	0.118	67.2	0.114	70.6	0.106	73.7	0.103	76.5	0.094
Pm	32.0	0.151	33.6	0.154	39.6	0.149	45.1	0.141	45.8	0.113	47.3	0.113	51.9	0.111	54.3	0.102