1 Background Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as target sequence, because they contain conserved regions and are in high-copy number. However, due to sequence similarity between Leishmania species, most of the available qPCR assays had potential cross-species amplification. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographic area. An accurate diagnostic method for distinction between these two species is necessary to provide prognosis, treatment and monitor disease evolution. 2 Methods DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were used in this study. All samples were amplified by a previously developed qPCR (qPCR-ML). Selected PCR products were directly and bidirectionally sequenced using an ABI PRISM 310 Genetic Analyzer. Sequences were manually edited and aligned in MUSCLE. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. 3 Results The novel qPCR-ama assay achieved a detection limit of 0.1 pg of parasite DNA and did not show cross amplification with host DNA or Trypanosoma cruzi. The qPCR-ama showed positive results also with L. (L.) infantum strains, but the Ct values were dramatically increased compared to qPCR-ML (13-19 cycles). Therefore, combined results of qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples, provided that template DNA was diluted so as to have Ct>25 in qPCRML. 4 Conclusions A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a particular minicircle sequence rather than targeting a hypothetical species-specific minicircle sequence.

USING KDNA MINICIRCLE SUBCLASS RELATIVE ABUNDANCE TO DIFFERENTIATE LEISHMANIA (L.) INFANTUM FROM LEISHMANIA (L.) AMAZONENSIS

Marcello Ceccarelli;Luca Galluzzi
;
Aurora Diotallevi;Francesca Andreoni;Mauro Magnani
2017

Abstract

1 Background Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as target sequence, because they contain conserved regions and are in high-copy number. However, due to sequence similarity between Leishmania species, most of the available qPCR assays had potential cross-species amplification. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographic area. An accurate diagnostic method for distinction between these two species is necessary to provide prognosis, treatment and monitor disease evolution. 2 Methods DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were used in this study. All samples were amplified by a previously developed qPCR (qPCR-ML). Selected PCR products were directly and bidirectionally sequenced using an ABI PRISM 310 Genetic Analyzer. Sequences were manually edited and aligned in MUSCLE. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. 3 Results The novel qPCR-ama assay achieved a detection limit of 0.1 pg of parasite DNA and did not show cross amplification with host DNA or Trypanosoma cruzi. The qPCR-ama showed positive results also with L. (L.) infantum strains, but the Ct values were dramatically increased compared to qPCR-ML (13-19 cycles). Therefore, combined results of qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples, provided that template DNA was diluted so as to have Ct>25 in qPCRML. 4 Conclusions A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a particular minicircle sequence rather than targeting a hypothetical species-specific minicircle sequence.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11576/2656480
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