关键词: 疟原虫, 巴贝虫, 多重qPCR, 兼并引物, 兼并探针

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