Development of a real-time polymerase chain reaction assay for the quantification of Leishmania species and the monitoring of systemic distribution of the pathogen.
We have developed a highly accurate and sensitive real-time polymerase chain reaction (PCR) assay to detect and quantify Leishmania parasites. The assay targets GP63, a highly conserved gene in Leishmania. We demonstrate that, with a single assay, we are able to detect and quantify several species of Leishmania. Our assay system detects Leishmania donovani and Leishmania major down to 0.1 parasite. The dynamic range of the assay extends over 6 log cycles of target, with an average correlation coefficient >0.988. In addition, we utilize a simple approach to distinguish between Leishmania species causing diverse spectra of disease. We have also used this assay to follow the course of cutaneous disease in CBA/CaJ mice, known to be resistant to L. major. The assay is sensitive enough to quantify parasite load in the absence of overt lesions and reveals a systemic distribution of Leishmania, which has implications for our understanding of the disease.
Tupperwar
Nitin
N
Centre for Cellular and Molecular Biology, Hyderabad 500007, India.
Vineeth
Varanasi
V
Rath
Satyajit
S
Vaidya
Tushar
T
D000818:Animals; D001483:Base Sequence; D005528:Foot / Q000469:parasitology; D007893:Leishmania donovani / Q000235:genetics / Q000302:isolation & purification*; D018320:Leishmania major / Q000235:genetics / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000469:parasitology*; D008198:Lymph Nodes / Q000469:parasitology; D008666:Metalloendopeptidases / Q000235:genetics; D051379:Mice; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013154:Spleen / Q000469:parasitology
D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania