Development of a recombinase polymerase amplification-CRISPR/Cas12a assay for detection of Leishmania major

doi:10.12140/j.issn.1000-7423.2025.06.011

Abstract

Abstract:

Objective To develop a rapid and accurate assay for detection of Leishmania major based on the recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system, so as to improve the diagnostic efficiency of cutaneous leishmaniasis. Methdos Based on the internal transcribed spacer 1 (ITS1) gene sequence of L. major (GenBank accession number: KM052753.1) retrieved from NCBI as the target sequence, RPA primers, CRISPR RNA (crRNA), and single-stranded DNA (ssDNA) reporter probe were designed. An RPA-CRISPR/Cas12a assay was developed by screening RPA primers and optimizing the RPA reaction temperature. Genomic DNA was extracted from L. major, and the ITS1 gene sequence was amplified. The amplification product was cloned into the pUC18 plasmid to construct a recombinant plasmid, which was serially diluted to 8 concentrations of 2.3 × 10⁶, 2.3 × 10⁵, 2.3 × 10⁴, 2.3 × 10³, 2.3 × 10², 2.3 × 10¹, 2.3 × 10⁰, and 2.3 × 10⁻¹ copies/μl as templates for RPA-CRISPR/Cas12a assay and PCR assay to evaluate the detection sensitivity of the plasmid. L. major promastigotes were added to 200 μl of canine whole blood samples at a concentration of 1.8 × 10⁷ parasites/ml, and genomic DNA was extracted, and serially diluted into 4 concentrations of 1.8 × 10², 1.8 × 10¹, 1.8 × 10⁰, and 1.8 × 10⁻¹ parasites/ml as templates for RPA-CRISPR/Cas12a assay to evaluate the detection sensitivity of parasites. Genomic DNA from L. major, L. infantum, Toxoplasma gondii, Babesia gibsoni, Neospora caninum, and Anaplasma phagocytophilum were used as templates for the RPA-CRISPR/Cas12a assay to evaluate the detection specificity. Repeatability of the RPA-CRISPR/Cas12a assay was assessed by performing 5 independent experiments with 3 replicates each using the 2.3 × 10⁴ copies/μl recombinant plasmid as the template. To compare the detection performance of the RPA-CRISPR/Cas12a and the PCR assay, L. major promastigotes were added to 10 samples of 200 μl canine whole blood at a concentration of 1.8 × 10⁵ parasites/ml for RPA-CRISPR/Cas12a and PCR assays, with negative controls set simultaneously. Results The RPA-CRISPR/Cas12a method was established using the F7/R7 primer pair, with optimized reaction conditions set as 41 ℃ for 25 minutes. The limits of detection (LOD) of RPA-CRISPR/Cas12a and PCR assays were 2.3 copies/μl and 2.3 × 10³ copies/μl of the recombinant plasmid, and the LOD of the RPA-CRISPR/Cas12a assay was 1.8 promastigotes/ml of parasites; however, this assay showed no cross-reactivity of L. major with L. infantum, T. gondii, B. gibsoni, N. caninum, or A. phagocytophilum. Repeatability testing showed stable and consistent fluorescence intensities across different batches, with a relative standard deviation of 4.27% (< 5%). Both RPA-CRISPR/Cas12a and PCR assays accurately detected all 10 spiked canine whole blood samples containing L. major promastigotes, achieving the same detection rate and a 100% coincidence rate. Conclusion The RPA-CRISPR/Cas12a assay developed in this study is rapid, sensitive, specific, and reproducible for detection of L. major, which may provide reliable technical supports for diagnosis of L. major infections.

Key words: Leishmania major, Recombinase polymerase amplification, CRISPR/Cas12a

CLC Number:

R383.63

CHEN Xinyi, ZHANG Bingke, LANG Jiashu, XU Huiyan, LI Junqiang, WU Longfei, ZHANG Sumei, ZHANG Longxian. Development of a recombinase polymerase amplification-CRISPR/Cas12a assay for detection of Leishmania major[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2025, 43(6): 821-826.

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References 18

[1]	Alvar J, Vélez ID, Bern C, et al. Leishmaniasis worldwide and global estimates of its incidence[J]. PLoS One, 2012, 7(5): e35671.
[2]	罗卓韦, 周正斌, 公衍峰, 等. 我国内脏利什曼病的流行现状和防控挑战[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 146-152.
	Luo ZW, Zhou ZB, Gong YF, et al. Epidemic status and prevention and control challenges of visceral leishmaniasis in China[J]. Chin J Parasitol Parasit Dis, 2022, 40(2): 146-152. (in Chinese)
[3]	王奇, 师悦, 秦瑶, 等. 2017—2022年中国内脏利什曼病流行特征及时空聚集性分析[J]. 热带病与寄生虫学, 2024, 22(2): 68-75.
	Wang Q, Shi Y, Qin Y, et al. Epidemiological characteristics and spatial-temporal clustering analysis of visceral leishmaniasis in China from 2017 to 2022[J]. J Trop Dis Parasitol, 2024, 22(2): 68-75. (in Chinese)
[4]	何俊辰, 王绍瑜, 王中江, 等. DNA-病原微生物宏基因组检测输入性皮肤利什曼病1例[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(2): 271-272.
	He JC, Wang SY, Wang ZJ, et al. Detection of imported cutaneous leishmaniasis by DNA-pathogenic microorganism metagenome: A case report[J]. Chin J Parasitol Parasit Dis, 2021, 39(2): 271-272. (in Chinese)
[5]	李素华, 高丽君, 张雅兰, 等. 河南省1例输入性皮肤利什曼病的诊断与分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 339-342.
	Li SH, Gao LJ, Zhang YL, et al. Diagnosis and analysis of an imported cutaneous leishmaniasis in Henan Province[J]. Chin J Parasitol Parasit Dis, 2018, 36(4): 339-342. (in Chinese)
[6]	王萌, 高利昆, 童永清, 等. 湖北省输入性皮肤利什曼病一例并文献复习[J]. 中国麻风皮肤病杂志, 2022, 38(9): 621-625.
	Wang M, Gao LK, Tong YQ, et al. A case of imported cutaneous leishmaniasis in Hubei Province and literature review[J]. China J Lepr Skin Dis, 2022, 38(9): 621-625. (in Chinese)
[7]	Layouni S, Remadi L, Kidar A, et al. Clinical polymorphism of zoonotic cutaneous leishmaniasis: Combination of the clinical and the parasitological diagnosis[J]. Parasitol Res, 2024, 123(6): 238.
[8]	Remadi L, Haouas N, Chaara D, et al. Clinical presentation of cutaneous leishmaniasis caused by Leishmania major[J]. Dermatology, 2016, 232(6): 752-759.
[9]	Mouttaki T, Morales-Yuste M, Merino-Espinosa G, et al. Molecular diagnosis of cutaneous leishmaniasis and identification of the causative Leishmania species in Morocco by using three PCR-based assays[J]. Parasit Vectors, 2014, 7: 420.
[10]	Li X, Dang ZS, Tang WQ, et al. Detection of parasites in the field: The ever-innovating CRISPR/Cas12a[J]. Biosensors, 2024, 14(3): 145.
[11]	Yin LJ, Man SL, Ye SY, et al. CRISPR-Cas based virus detection: Recent advances and perspectives[J]. Biosens Bioelectron, 2021, 193: 113541.
[12]	Yu FC, Zhang KH, Wang YL, et al. CRISPR/Cas12a-based on-site diagnostics of Cryptosporidium parvum Ⅱd-subtype-family from human and cattle fecal samples[J]. Parasit Vectors, 2021, 14(1): 208.
[13]	Talmi-Frank D, Nasereddin A, Schnur LF, et al. Detection and identification of Old World Leishmania by high resolution melt analysis[J]. PLoS Negl Trop Dis, 2010, 4(1): e581.
[14]	de Vries HJC, Schallig HD. Cutaneous leishmaniasis: A 2022 updated narrative review into diagnosis and management developments[J]. Am J Clin Dermatol, 2022, 23(6): 823-840.
[15]	Nateghi Rostami M, Darzi F, Farahmand M, et al. Performance of a universal PCR assay to identify different Leishmania species causative of Old World cutaneous leishmaniasis[J]. Parasit Vectors, 2020, 13(1): 431.
[16]	Fotouhi-Ardakani R, Ghafari SM, Ready PD, et al. Developing, modifying, and validating a TaqMan real-time PCR technique for accurate identification of Leishmania parasites causing most leishmaniasis in Iran[J]. Front Cell Infect Microbiol, 2021, 11: 731595.
[17]	Bel Hadj Ali I, Saadi-Ben Aoun Y, Khammeri I, et al. Recombinase-based amplification coupled with lateral flow chromatography for the specific and sensitive detection and identification of Leishmania major in cutaneous leishmaniasis patients[J]. Front Microbiol, 2024, 15: 1514684.
[18]	Chaouch M, Aoun K, Ben Othman S, et al. Development and assessment of Leishmania major and Leishmania tropica specific loop-mediated isothermal amplification assays for the diagnosis of cutaneous leishmaniasis in Tunisia[J]. Am J Trop Med Hyg, 2019, 101(1): 101-107.

引物Primer	序列（5′→3′）Sequence（5′→3′）
F1	CATATACAACTCGGGGAGGCTTATTCTAT
R1	TTCTTCAACGAAATAGGAAGCCAAGTCATC
F2	ACATATACAACTCGGGGAGGCTTATTCTAT
R2	GTTCTTCAACGAAATAGGAAGCCAAGTCAT
F3	ATAGTATAGGCTTTTCCCACATACACAGC
R3	CTTCAACGAAATAGGAAGCCAAGTCATC
F4	TATAGTATAGGCTTTTCCCACATACACAGC
R4	CTTTACTGCGTTCTTCAACGAAATAGGAAG
F5	AAAACATATACAACTCGGGGAGGCTTATTC
R5	CTTTACTGCGTTCTTCAACGAAATAGGAAG
F6	TCCCACATACACAGCAAACTTTTATACTCG
R6	GCAATTGATACCACTTATCGCACTTTACTG
F7	AAACATATACAACTCGGGGAGGCTTATTC
R7	TTCTTCAACGAAATAGGAAGCCAAGTCATC
crRNA	AAUUUCUACUGUUGUAGAUUAGAACGCACCGC-CUAUACACAAA
单链DNA ssDNA	FAM/CCCCCC/BHQ1