A dual-tube multiplex qPCR system for detection of Plasmodium and Babesia parasites in human blood

doi:10.12140/j.issn.1000-7423.2026.04.013

Abstract

Abstract:

Objective To develop a multiplex real-time quantitative PCR (qPCR) assay for the simultaneous detection of multiple pathogens in a single sample, which can identify Plasmodium and Babesia at the genus level and determine clinically important Plasmodium species. Methods The 18S rDNA sequences of Homo sapiens, 16 18S rDNA sequences of 8 Plasmodium species and 14 18S rDNA sequences of 14 Babesia species were aligned using the bioinformatic tools DNAman 4.0 and BioEdit. The V4 region of Plasmodium 18S rDNA and the corresponding region of Babesia were selected to design universal primers to identify Plasmodium and Babesia, genus-specific probes to distinguish Plasmodium and Babesia, and species-specific probes to differentiate P. falciparum, P. vivax, and P. ovale with software Oligo 6.0. The 18S rDNA gene sequences of 3 mongkey Plasmodium species and 7 Babesia species with 400 to 500 bp in length were artificially synthesized, prepared into plasmids and diluted in a 10-fold gradient for subsequent experiments. Specific primers and species/genus probes were screened using 5 × 10⁵ copies/μL 18S rDNA plasmid as templates. The qPCR system was optimized through assigning degenerate primers and probes and introducing mismatched base into primers. The 18S rDNA plasmids of P. falciparum, P. vivax, B. microti, and B. venatorum with gradient dilution of 5 × 10⁰ to 5 × 10⁸ copies/μL were used as templates, and amplifications were performed in separate tubes using dye-based and probe-based qPCR assays to determine the lowest limit of detection. The standard curve of the system was plotted to calculate the amplification efficiency. The 18S rDNA plasmids of P. malariae, P. ovale (variant subspecies), P. knowlesi, 3 Plasmodium species of monkey, 5 species of Babesia, and genome DNA from 18 clinical samples, including 11 samples diagnosed positive for malaria (4 species), one clinical sample diagnosed positive for B. microti and 6 clinical samples diagnosed negative for malaria was collected to test the application of the system. Results A dual-tube multiplex qPCR system was developed and optimized for detecting multiple pathogens, including 3 universal primers, 3 genus- specific probes, and 5 species-specific probes. The amplification efficiency of the system was 90% to 110%, with the lowest limit of detection ranging from 5 × 10⁰ to 5 × 10¹ copies/μL for the original template concentration and the stable limit of detection of 5 × 10¹ copies/μL and higher. The 10 18S rDNA plasmid samples were tested positive with this system, and the 18S rDNA plasmid of B. bigemina, which had mismatched base with the first base of forward primer at 3′ terminal, was tested negative. The results for testing 12 positive clinical samples and 5 negative samples were consistent with previous diagnosed results, and one malaria-negative clinical sample was tested positive for P. vivax. The accordance rate was 17/18 for detection of clinical samples. Conclusion A dual-tube multiplex qPCR system has been established to detect 18 species of Plasmodium and Babesia, distinguish between Plasmodium and Babesia, and differentiate P. falciparum, P. vivax, and P. ovale, which may be used for pathogen detection of two genera in clinical blood samples.

Key words: Plasmodium, Babesia, Multiplex qPCR, Degenerate primer, Degenerate probe

CLC Number:

R382.3

LI Mei, XIA Zhigui, ZHOU Hejun, LIU Qin. A dual-tube multiplex qPCR system for detection of Plasmodium and Babesia parasites in human blood[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2026, 44(4): 543-549.

Figures/Tables 3

References 21

[1]	杨维中. 以多病同防新格局应对多重传染病[N]. 健康报, 2025-12-3(5).
	Yang WZ. Responding to multiple infectious diseases with a new paradigm of preventing multiple diseases together[N]. Health News, 2025-12-3(5). (in Chinese)
[2]	李雍龙. 人体寄生虫学[M]. 7版. 北京: 人民卫生出版社, 2008: 58-70.
	Li YL. Parasitology of humans[M]. 7th ed. Beijing: People’s Medical Publishing House, 2008: 58-70. (in Chinese)
[3]	中华人民共和国国家卫生和计划生育委员会. 巴贝虫病诊断: WS/T 564-2017[S]. 北京: 中国标准出版社, 2017: 1-11.
	National Health and Family Planning Commission of the People’s Republic of China. Diagnosis of Babesiosis: WS/T 564-2017[S]. Beijing: Standards Press of China, 2017: 1-11. (in Chinese)
[4]	张丽, 夏志贵, 李石柱. 2024年全国疟疾疫情特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(2): 162-166.
	Zhang L, Xia ZG, Li SZ. Epidemiological characteristics of malaria in China, 2024[J]. Chin J Parasitol Parasit Dis, 2025, 43(2): 162-166. (in Chinese)
[5]	国家移民管理局. 政务公开, 统计数据: 2024年6.1亿人次出入境同比上升43.9%[EB/OL]. https://www.nia.gov.cn/n741440/n741567/c1693512/content.html. [2025-01-14].
	National Immigration Administration. Government transparency, statistical data:610 million person-times of entry and exit in 2024, up 43.9% year-on-year. [EB/OL]. https://www.nia.gov.cn/n741440/n741567/c1693512/content.html. [2025-01-14]. (in Chinese)
[6]	World Health Organization. World malaria report[R]. Geneva: WHO, 2025: 6-29.
[7]	Zheng JX, Liu Y, Guan SY, et al. Global, regional, and national burden of neglected tropical diseases and malaria in the general population, 1990-2021: systematic analysis of the global burden of disease study 2021[J]. J Adv Res, 2026, 79: 769-781.
[8]	李美, 燕贺, 夏志贵, 等. 《疟原虫检测虫种核酸鉴定法》标准解读[J]. 中国寄生虫学与寄生虫病杂志, 2026, 44(1): 110-117.
	Li M, Yan H, Xia ZG, et al. Interpretation of the standard detection of Plasmodium spp. nucleic acid identification of the species[J]. Chin J Parasitol Parasit Dis, 2026, 44(1): 110-117. (in Chinese)
[9]	陈木新, 薛靖波, 艾琳, 等. 我国巴贝虫病流行现状与研究进展[J]. 热带病与寄生虫学, 2022, 20(3): 149-157.
	Chen MX, Xue JB, Ai L, et al. Epidemic status and research progress of babesiosis in China[J]. J Trop Dis Parasitol, 2022, 20(3): 149-157. (in Chinese)
[10]	周霞, 王慧, 薛靖波, 等. 国内外巴贝虫病流行现状与研究进展[J]. 中国血吸虫病防治杂志, 2019, 31(1): 63-70.
	Zhou X, Wang H, Xue JB, et al. Epidemic and research progress of babesiosis[J]. Chin J Schisto Control, 2019, 31(1): 63-70. (in Chinese)
[11]	国家疾病预防控制局. 疟原虫检测虫种核酸鉴定法: WS/T 10030-2025[S]. 北京: 中国标准出版社, 2025: 1-17.
	National Disease Control and Prevention Administration of the People’s Republic of China. Detection of Plasmodium-nucleic acid identification of species: WS/T 10030-2025[S]. Beijing: China Standards Press, 2025: 1-17.
[12]	李美, 肖宁, 夏志贵. 基于无性期18S rDNA特异性引物检测5种疟原虫qPCR的建立和应用[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(1): 36-43.
	Li M, Xiao N, Xia ZG. Establishment and application of a specific primers-based qPCR targeting asexual stage 18S rDNA for detecting five Plasmodium species[J]. Chin J Parasitol Parasit Dis, 2023, 41(1): 36-43. (in Chinese)
[13]	李金春, 李家鹏, 周彤, 等. 引物3'端不同碱基错配情况下实时荧光定量PCR非特异性扩增的发生规律[J]. 食品科学, 2017, 38(10): 277-283.
	Li JC, Li JP, Zhou T, et al. Pattern of occurrence of nonspecific amplification in real-time fluorescent PCR with different base pair mismatches at the primer 3'End[J]. Food Sci, 2017, 38(10): 277-283. (in Chinese)
[14]	Hawkins SFC, Guest PC. Multiplex analyses using real-time quantitative PCR[M]//Guest PC. Methods in molecular biology, New York: Humana Press, 2017: 125-133.
[15]	张家祺, 徐昌平, 阮卫, 等. 多重qPCR检测疟原虫方法的建立及应用评价[J]. 中国卫生检验杂志, 2023, 33(4): 393-396.
	Zhang JQ, Xu CP, Ruan W, et al. Development and application of a multiplex qPCR assay for the detection of four Plasmodium species[J]. Chin J Heal Lab Technol, 2023, 33(4): 393-396. (in Chinese)
[16]	杨亚闪. 巴贝西虫核酸检测方法的建立及无偿献血者中巴贝西虫流行情况调查[D]. 北京: 北京协和医学院, 2018: 22-23.
	Yang YS. Establishment of nucleic acid detection method for Babesia spp. and investigation of epidemic situation of Babesia spp. in blood donors[D]. Beijing: Peking Union Medical College, 2018: 22-23.
[17]	Calchi AC, Moore CO, Bartone L, et al. Development of multiplex assays for the identification of zoonotic Babesia species[J]. Pathogens, 2024, 13(12): 1094.
[18]	杨珺源. 基于通用引物与通用探针的TaqMan定量PCR法直接检测血液中的RNA: 检测疟原虫的配子体及巴贝斯虫的新方法研究[D]. 北京: 北京协和医学院, 2022: 5-9.
	Yang JY. Direct detection of RNA in blood using TaqMan quantitative PCR method based on universal primers and universal probes: a new method for detecting gametocytes of malaria parasites and Babesia parasites[D]. Beijing: Peking Union Medical College, 2022: 5-9. (in Chinese)
[19]	Patiño LH, Castañeda S, Camargo M, et al. Validation of real-time PCR assays for detecting Plasmodium and Babesia DNA species in blood samples[J]. Acta Trop, 2024, 258: 107350.
[20]	Lamien-Meda A, Fuehrer HP, Leitsch D, et al. A powerful qPCR-high resolution melting assay with taqman probe in Plasmodium species differentiation[J]. Malar J, 2021, 20(1): 121.
[21]	Cordier C, Hamane S, Ghelfenstein-Ferreira T, et al. Implementation and validation of a new qPCR assay to detect imported human Plasmodium species[J]. Microbiol Spectr, 2025, 13: e01622-e01624.

检测目标 Targets	名称 Name	序列（5'→3'） Sequences (5'→3')	5′端 5′End	3′端 3′End
通用引物 Common primers
上游引物 Forward	UP	GTTAAAACGCTCGTAGTTGA	-	-
下游引物 Reverse primer	LW	GTTATTCCATGCTGTAGTAATC	-	-
下游引物 Reverse primer	LF	GTTATTCCATGCTATAGTAATC	-	-
探针 Probe
疟原虫属 Plasmodium	PG	CTGTTTGCTTTGAACACTCTAATTTACTC	HEX	BHQ1
巴贝虫属 Babesia	BG1	CTGCTTGAAACACTCTAATTTTCTCAAAGTAA	CY5.5	BHQ3
巴贝虫属 Babesia	BG2	CTGTTTGAAACACTCTAGTTTTCTCAAAGTAA	CY5.5	BHQ3
恶性疟原虫 P. falciparum	PFA	TAGCTTTTCGCTTTAATACGCTTCCTC	CY5	BHQ3
恶性疟原虫 P. falciparum	PFS	ACTCAATCATGACTACCCGTCTGTTAT	CY5	BHQ3
间日疟原虫 P. vivax	PV	TCCAAGCCGAAGCAAAGAAAGTCCT	FAM	BHQ1
卵形疟原虫 P. ovale	POC	CTTTGCATTCCTTATGCAAAATGTGTTC	Texas RED	BHQ2
卵形疟原虫 P. ovale	POW	CTTTGCATTCCTTATCCAAAATGTGTTC	Texas RED	BHQ2