检测利什曼原虫的实时荧光定量PCR的建立及应用

doi:10.12140/j.issn.1000-7423.2021.04.021

摘要/Abstract

摘要：

建立一种快速准确检测利什曼原虫的实时荧光定量PCR（qPCR）方法。以利什曼原虫动基体小环保守序列为靶基因,设计1对特异性引物和Taq-Man探针,以利什曼原虫阳性患者骨髓样品DNA为模板进行PCR扩增,扩增产物长83 bp。扩增产物连接至pESI-T载体进行克隆、测序,测序结果在GenBank上进行BLAST比对,结果显示,其序列与婴儿利什曼原虫和杜氏利什曼原虫的序列一致性为100%。取测序正确的质粒梯度稀释至10²~10⁷拷贝/μl作为标准品进行qPCR,绘制获得标准曲线方程y = 39.23-2.956 x,扩增效率为117.93%,R²为0.994;当阈值循环数为35时,最低检测限为26.98拷贝/μl,理论上可检出小于1个利什曼原虫。用所建立的qPCR检测内脏利什曼病患者骨髓样品14份、血样26份,利什曼病病犬的骨髓、血液、脾、肺、淋巴结、肝、肾组织样品各1份,利什曼原虫阳性白蛉2份,患者和病犬骨髓培养物各1份,结果均为阳性;检测利什曼原虫血清抗体阳性犬骨髓9份,结果7份阳性;检测利什曼原虫血清抗体阴性人群血样56份,沙门菌、志贺菌、蜡样芽胞杆菌DNA各1份,疟疾患者血样4份,刚地弓形虫病患者血样、卫氏并殖吸虫病患者血样各1份,结果均为阴性。所建立的qPCR具有较高灵敏度及特异度,可用于利什曼病患者、宿主动物、传播媒介利什曼原虫感染与带虫状态的快速检测,可用于患者的疗效判定。

关键词: 利什曼原虫, Taq-Man探针, 实时荧光定量PCR, 动基体DNA

Abstract:

To establish a real-time fluorescence quantitative PCR (qPCR) method for rapid and accurate detection of Leishmania. A pair of specific primers and a Taq-Man probe were designed, based on the conserved sequence of the small circle in kinetoplast of Leishmania, and the DNA of bone marrow samples from positive cases of Leishmania was used as a template for PCR amplification. The amplified product was 83 bp long, and ligated to the pESI-T vector for cloning and sequencing. The sequences were BLAST aligned on GenBank, and found 100% homologous to L. infantum and L. donovani. The plasmids with correct sequences were diluted to 10²-10⁷ copies/μl as the standard sample for qPCR. The obtained standard curve equation was y = 39.23-2.956 x, the amplification efficiency was 117.93%, R² was 0.994, and when the threshold cycle number was 35, the minimum detection limit was 26.98 copies/μl, theoretically less than 1 Leishmania parasite could be detected. The qPCR was used to detect 14 bone marrow and 26 blood samples from leishmaniasis patients, and respective, 1 sample of bone marrow, blood, spleen, lung, lymph node, liver, and kidney from dogs with leishmaniasis, 2 samples of sandfly positive for Leishmania, and 2 samples of Leishmania culture from bone marrow of patient and dog with leishmaniasis, and all had positive results; 56 blood samples from persons with negative Leishmania seroantibodies, 1 DNA sample of each of Salmonella, Shigella, and Bacillus cereus, 4 blood samples from patients with malaria, 1 blood sample from each of patients with toxoplasmosis gondii or paragonimiasis wesleyi, and all had negative results. The method has high sensitivity and specificity, and can be applied to the rapid detection of infection and carrier status of leishmaniasis patients, host animals, and vectors, and can also be used to determine the therapeutic efficacy in patients.

Key words: Leishmania, Taq-Mann probe, Real-time fluorescence quantitative PCR, Kinetoplast DNA

中图分类号:

R382.221

马琳, 张铮, 王安礼, 刘东立. 检测利什曼原虫的实时荧光定量PCR的建立及应用[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(4): 548-552.

MA Lin, ZHANG Zheng, WANG An-li, LIU Dong-li. Establishment and application of real-time fluorescence quantitative PCR for detection of Leishmania[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2021, 39(4): 548-552.

图/表 3

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