Application of fluorescence quantitative PCR in laboratory diagnosis of malaria

doi:10.12140/j.issn.1000-7423.2019.02.021

Abstract

Abstract:

Seventy-nine malaria samples were examined by microscopy, nested PCR and fluorescence quantitative PCR, and the detection rate was analyzed among the three methods. After combining clinical symptoms of the patients and results of the three methods, 74 of the 79 samples were confirmed to be malaria infection, with a positive rate of 93.7%（74/79）. Specifically, the detection rate of microscopy, nested PCR and fluorescence quantitative PCR were 82.3%（65/79）, 82.3%65/79） and 93.7%（74/79）, respectively. The concordance rate between microscopy and nested PCR was 78.4%（58/74）, and that between microscopy and fluorescence quantitative PCR was 63.5%（47/74）, between nested PCR and fluorescence quantitative PCR was 83.8%（62/74）. In addition, the detection rate of fluorescence quantitative PCR was significantly higher than that of the nested PCR（P < 0.05）. Compared with microscopy and nested PCR, the fluorescence quantitative PCR has higher sensitivity and positive detection rate, and is less time-consuming.

Key words: Malaria, Microscopy, Nested PCR, Fluorescence quantitative PCR

CLC Number:

R513.3

Su-hua LI, Jing LI, Li-jun GAO, Ya-lan ZHANG, Rui-min ZHOU, Dan QIAN, Cheng-yun YANG, Ying LIU, Yu-ling ZHAO, Hong-wei ZHANG. Application of fluorescence quantitative PCR in laboratory diagnosis of malaria[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2019, 37(2): 232-234.

Figures/Tables 3

References 13

[1]	Lee PC, Chong ET, Anderios F, et al. Molecular detection of human Plasmodium species in Sabah using PlasmoNexTM multiplex PCR and hydrolysis probes real-time PCR[J]. Malar J, 2015, 14: 28.
[2]	Koukouikila-Koussounda F, Malonga V, Mayengue PI, et al. Genetic polymorphism of merozoite surface protein 2 and prevalence of K76T pfcrt mutation in Plasmodium falciparum field isolates from Congolese children with asymptomatic infections[J]. Malar J, 2012, 11: 105.
[3]	孙菲, 万向阳, 李欢, 等. 多重巢式PCR检测恶性疟和间日疟原虫[J]. 临床检验杂志, 2014, 32(8): 565-567.
[4]	李美, 夏志贵, 汤林华. 检测4种人体疟原虫多重PCR体系的建立和应用[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(2):91-95.
[5]	黄雨婷, 佘丹娅, 卢丽丹, 等. 单管单轮多重PCR检测4种疟原虫混合血样[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(3): 200-205.
[6]	李欢. SYBR Green实时PCR检测恶性疟原虫与间日疟原虫方法的建立[D]. 长沙: 中南大学, 2014.
[7]	张金飞, 周燕珍. 三种不同检测方法在疟疾诊断中的应用比较[J]. 中国地方病防治杂志, 2016, 31(2): 201-202.
[8]	雷露, 刘学升, 毛玲玲, 等. 3种实验室检测方法在疟疾诊断中的应用比较[J]. 中国媒介生物学及控制杂志, 2015, 26(2): 206-207.
[9]	Barker RH Jr, Banchongaksorn T, Courval JM, et al. A simple method to detect Plasmodium falciparum directly from blood samples using the polymerase chain reaction[J]. Am J Trop Med Hyg, 1992, 46(4): 416-426.
[10]	Win TT, Jalloh A, Tantular IS, et al. Molecular analysis of Plasmodium ovale variants[J]. Emerging Infect Dis, 2004, 10(7): 1235-1240.
[11]	周瑞敏, 刘颖, 钱丹, 等. 2012年河南省疟疾标本的实验室检测分析[J]. 中国病原生物学杂志, 2014, 9(2): 177-179.
[12]	鲁少平, 邓中平, 龙妍娇, 等. 运用实时荧光RT-PCR方法检测疟原虫的研究[J]. 实用预防医学, 2015, 22(10): 1171-1173.
[13]	焦炳欣, 华文浩, 陈志海, 等. 三种检测方法在疟疾诊断和疗效中的应用评估[J]. 中国医药导报, 2012, 9(270): 98-99.

引物名称		引物序列（5′→3′）	产物长度/bp
第1轮	rPLU5	CCTGTTGTTGCCTTAAACTTC	1 600
	rPLU6	TCAAAGATTAAGCCATGCAAG
恶性疟原虫	rFAL1	TTAAACTGGTTTGGGAAAACC-AAATATATT	205
	rFAL2	ACACAATGAACTCAATCATGA-CTACCCGTC
间日疟原虫	rVIV1	CGCTTCTAGCTTAATCCACAT-AACTGATAC	120
	rVIV2	ACTTCCAAGCCGAAGCAAAG-AAAGTCCTTA
三日疟原虫	rMAL1	ATAACATAGTTGTACGTTAAG-AATAACCGC	144
	rMAL2	AAAATTCCCATGCATAAAAAA-TTATACAAA
卵形疟原虫	rOVA1	ATCTCTTTTGCTATTTTTTAGT-ATTGGAGA	800
	rOVA2	GGAAAGGACACATTAATTGT-ATCCTAGTG

样品编号	镜检	巢式PCR	荧光定量PCR
3	阴性	三日疟原虫	三日疟原虫
4	阴性	恶性疟原虫	恶性疟原虫
15	卵形疟原虫	阴性	卵形疟原虫
18	阴性	恶性疟原虫	恶性疟原虫
20	卵形疟原虫	阴性	卵形疟原虫
30	卵形疟原虫	阴性	卵形疟原虫
33	卵形疟原虫	阴性	卵形疟原虫
56	卵形疟原虫	阴性	卵形疟原虫
58	阴性	恶性疟原虫	恶性疟原虫
60	阴性	阴性	恶性疟原虫
62	阴性	阴性	恶性疟原虫
63	阴性	恶性疟原虫	恶性疟原虫
66	阴性	阴性	恶性疟原虫
74	阴性	恶性疟原虫	恶性疟原虫
75	卵形疟原虫	恶性疟原虫	卵形疟/恶性疟原虫
76	三日疟原虫	三日疟原虫	三日疟/恶性疟原虫
78	卵形疟原虫	阴性	卵形疟/恶性疟原虫
79	卵形疟原虫	卵形疟原虫	卵形疟/恶性疟原虫

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