Establishment and preliminary evaluation of a visualized detection technique for Schistosoma mansoni nucleic acid based on recombinase polymerase amplification

doi:10.12140/j.issn.1000-7423.2022.03.009

Abstract

Abstract:

Objective To establish and preliminarily evaluate a fast, convenient and visualized method for detection of Schistosoma mansoni nucleic acid by combining the recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD). Methods Selecting the cytochrome c oxidase subunit 1 (COX1) gene of S. mansoni as the target sequence, specific primers and probes were designed using Primer Primer5 with manual assistance and screened to establish a LFD-RPA method for detection of S. mansoni. The sensitivity of the established method was evaluated by detecting the genomic DNA of adult S. mansoni at different concentrations (1 ng/μl, 100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, 10 fg/μl, 1 fg/μl, 0.1 fg/μl) and the recombinant SmCox1 plasmids with different copies (10⁵, 10⁴, 10³, 10², 10¹, 10⁰, 10^-1 copies/μl). The specificity of LFD-RPA was evaluated by detecting the genomic DNA of S. japonicum adult worms, S. haematobium eggs, S. mansoni infected and non-infected Biomphalaria spp., S. japonicum infected and non-infected Oncomelania hupensis and other trematodes. Thirty female BALB/c mice were randomly divided into low infection group (40 cercariae per mouse), high infection group (80 cercariae per mouse) and control group (no infection) to establish the mouse infection model. The DNA was extracted from the feces and serum samples from the mice at different time points post infection (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks) and detected by LFD-RPA to evaluate its effectiveness in early detection of S. mansoni infection. Results The specific band of S. mansoni could be detected by the established SmCox1-LFD-RPA method after 20 min of reaction at 39 ℃. The detectable limits of LFD-RPA method for S. mansoni were 10 fg/μl for the genomic DNA of adult worms and 10 copies/μl for SmCox1 recombinant plasmid, respectively. The established SmCox1-LFD-RPA method was specific to the DNA of S. mansoni and S. mansoni infected Biomphalaria spp., no cross-reaction with other flukes were observed. When detecting DNA samples extracted from mouse blood and feces from the 40 cercariae group 1 week to 8 weeks post infection, SmCox1-LFD-RPA presented positive reaction since 3 weeks post infection. While for the mice of the 80 cercariae group, positive band began to appear from 1-week post infection. The color of the band was gradually deepened with the infection time. Conclusion A LFD-RPA based visualized rapid detection method for S. mansoni nucleic acid was developed, with higher sensitivity, specificity, rapidity and easy-to-use.

Key words: Schistosoma mansoni, Recombinase polymerase, Isothermal amplification, Visualization, Lateral flow dipstick

CLC Number:

R383.24

WANG Li-ping, LV Chao, QIN Zhi-qiang, XU Jing, DENG Wang-ping. Establishment and preliminary evaluation of a visualized detection technique for Schistosoma mansoni nucleic acid based on recombinase polymerase amplification[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(3): 337-343.

Figures/Tables 4

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