环介导等温扩增检测恶性疟原虫与其他疟原虫的效果评价

doi:10.12140/j.issn.1000-7423.2020.04.004

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (4): 423-428.doi: 10.12140/j.issn.1000-7423.2020.04.004

环介导等温扩增检测恶性疟原虫与其他疟原虫的效果评价

周水茂¹(), 涂祖武², 杨燕¹, 陈芳¹, 贾西帅¹^,^*()

1 武汉市疾病预防控制中心,武汉 430015
2 湖北省疾病预防控制中心,武汉 430070

收稿日期:2020-03-07 出版日期:2020-08-30 发布日期:2020-09-09
通讯作者: 贾西帅
作者简介:周水茂（1967-）,男,本科,副主任技师,从事寄生虫病防治和诊断工作。E-mail：867494784@qq.com
基金资助:
武汉市卫生计生委医学科研项目(WG17B04);武汉市卫生计生委医学科研项目(WG18Q05);武汉市卫生健康委第七批中青年医学骨干人才培养工程

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

ZHOU Shui-mao¹(), TU Zu-wu², YANG Yan¹, CHEN Fang¹, JIA Xi-shuai¹^,^*()

1 Wuhan Centers for Disease Prevention and Control, Wuhan 430015, China
2 Hubei Provincial Center for Disease Control and Prevention, Wuhan 430070, China

Received:2020-03-07 Online:2020-08-30 Published:2020-09-09
Contact: JIA Xi-shuai
Supported by:
Wuhan Municipal Health and Family Planning Commission Medical Research Project(WG17B04);Wuhan Municipal Health and Family Planning Commission Medical Research Project(WG18Q05);Seventh Batch of Young and Middle-aged Medical Backbone Talent Training Projects of Wuhan Municipal Health and Health Commission

摘要/Abstract

摘要：

目的采用环介导等温扩增（LAMP）方法检测疟原虫DNA,并评价其检测效果。方法采集2013-2018年从疟疾流行区回国人员中确诊为疟疾患者的滤纸血样。根据疟原虫的18S rRNA序列,使用PrimerExplorer V5软件设计恶性疟原虫与非恶性疟原虫LAMP引物,建立疟原虫LAMP检测方法,进行敏感性、特异性、灵敏度和扩增效率评价,并与巢式PCR比较。检测钙黄绿素指示剂对LAMP反应的影响。结果 LAMP和巢式PCR各检测96份疟疾患者血样（47份恶性疟血样和49份其他疟疾血样）,均检出93份疟原虫阳性。LAMP检测结果显示,恶性疟血样均被检出,非恶性疟假阴性率为2.1%,10份利什曼原虫玻片血、8份日本血吸虫感染兔血和38份健康人血均为阴性,与巢式PCR检测结果具有极高的一致性（Kappa值 = 0.956）。LAMP检测恶性疟原虫、间日疟原虫、卵形疟原虫、三日疟原虫的灵敏度分别为25、3、4、2个疟原虫/μl血。扩增效率检测结果显示,4种疟原虫LAMP反应出现浊度时间（Tt值）均小于60 min,速率曲线峰值（Df）达0.14以上,其中恶性疟原虫扩增效率较高,Tt值为20.4 min,Df达0.16。可视化LAMP检测结果显示,钙黄绿素指示剂对不同种疟原虫LAMP反应延时约8~22 min,Df下降约21%~35%。结论 LAMP技术检测疟原虫有高度敏感性、特异性和灵敏度,且可视,具有在现场及基层医疗机构应用前景。

关键词: 疟原虫, LAMP, 巢式PCR, 恶性疟原虫

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

中图分类号:

R382.31

周水茂, 涂祖武, 杨燕, 陈芳, 贾西帅. 环介导等温扩增检测恶性疟原虫与其他疟原虫的效果评价[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(4): 423-428.

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

环介导等温扩增检测恶性疟原虫与其他疟原虫的效果评价

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

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方法Method	Pf	Pv	Po	Pm	Pf + Pv/Po/Pm^a	合计Total
镜检Microscopy	47	27	18	4	0	96
巢式PCR Nested-PCR	46	25	17	4	1	93
LAMP	45	25	16	4	3	93

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