TaqMan‐Based Detection of Leishmania infantum DNA Using Canine Samples

Abstract

Leishmaniasis is a typical example of a worldwide diffused zoonosis. Geographic distribution depends on the presence of sand fly vectors and animal reservoirs. In Southern Europe, canines are considered the main reservoir of infection, and the phlebotomines are the vectors. In Sicily, as in all Mediterranean areas, sand flies are present almost all year around because the climate permits an uninterrupted lifecycle for the vectors. Visceral leishmaniasis is becoming a real public health concern especially in endemic areas; in fact, it is an opportunistic infection in immunocompromised patients and in HIV-positive subjects. In Italy, the visceral form of the disease is due exclusively to Leishmania infantum ZMON1, and its prevalence is growing. We have developed a highly accurate, reproducible, and sensible real-time polymerase chain reaction (PCR) assay. In a procedure that used a specific couple of primers, a 117-bp fragment was amplified from minicircle kinetoplast DNA (kDNA). The assay was able to detect even a single parasite (200 fg of DNA). In fact, a single parasite contains hundreds of kinetoplast minicircles for each class. We applied a rapid extraction method coupled with the real-time PCR assay. It was not only as sensitive as a conventional PCR assay for detection of Leishmania kDNA, but also more rapid. The assay is useful for the diagnosis of leishmaniasis in dogs and humans, and it facilitates the monitoring of parasite levels during pharmacological treatment.

Sensitivity of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood

“…This sensitivity of this assay was also higher than that reported in previous studies, owing to the target utilized (kinetoplasts) and the use of TaqMan probes [7,15,24,41,[44][45][46] . A slope of -3.3 corresponds to an efficiency of 100.0%, indicating that the number of amplified molecules doubles with each cycle of PCR [47] .…”

“…How does not show the various handling steps required after conventional PCR amplification reduces the risk of contamination of the material and allows, simultaneously, detecting, measuring and comparing the number of parasites in various samples [7,41] .…”

Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples

“…DNA include hybridization of infected tissues (17) and amplification using routine PCR, PCR-enzyme-linked immunosorbent assay (ELISA) (13,35,53,55), or quantitative PCR (40,46,62). These methods have been successfully applied for detection of individual Leishmania species in clinical samples, including peripheral blood leukocytes of patients with visceral leishmaniasis or asymptomatic infection (39,47,59).…”

SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples

“…Development of the SYBR-Green and TaqMan real-time polymerase chain reaction assay has provided sensitive, rapid, accurate, and reproducible diagnosis of VL (Mary et al 2004;Nicolas et al 2002;Bretagne et al 2001;Bossolasco et al 2003;Vitale et al 2004). SYBR Green I is an intercalating dye that binds non-specifically to the minor groove of double-stranded DNA.…”