Establishment and application of real-time fluorescence quantitative PCR for detection of Leishmania

doi:10.12140/j.issn.1000-7423.2021.04.021

Abstract

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

CLC Number:

R382.221

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.

Figures/Tables 3

References 21

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