| Title |
Real-time PCR using FRET technology for Old World cutaneous leishmaniasis species differentiation
|
|---|---|
| Published in |
Parasites & Vectors, May 2016
|
| DOI | 10.1186/s13071-016-1531-4 |
| Pubmed ID | |
| Authors |
Milli Nath-Chowdhury, Mugundhine Sangaralingam, Patrick Bastien, Christophe Ravel, Francine Pratlong, Juan Mendez, Michael Libman, Momar Ndao |
| Abstract |
Recently, there has been a re-emergence of cutaneous leishmaniasis in endemic countries and an increase in imported cases in non-endemic countries by travelers, workers, expatriates, immigrants, and military force personnel. Old World cutaneous leishmaniasis is caused primarily by Leishmania major, L. tropica and L. aethiopica. Despite their low sensitivity, diagnosis traditionally includes microscopic and histopathological examinations, and in vitro cultivation. Several conventional PCR techniques have been developed for species identification, which are time-consuming and labour-intensive. Real-time PCR using SYBR green dye, although provides rapid detection, may generate false positive signals. Therefore, a rapid and easy method such as a FRET-based real-time PCR would improve not only the turn-around time of diagnosing Old World cutaneous Leishmania species but will also increase its specificity and sensitivity. A FRET-based real-time PCR assay which amplifies the cathepsin L-like cysteine protease B gene encoding a major Leishmania antigen was developed to differentiate L. major, L. tropica, and L. aethiopica in one single step using one set of primers and probes. Assay performance was tested on cutaneous and visceral strains of Leishmania parasite cultures and isolates of other protozoan parasites as well as human biopsy specimen. The assay readily differentiates between the three Old World cutaneous leishmaniasis species based on their melting curve characteristics. A single Tm at 55.2 ± 0.5 °C for L. aethiopica strains was distinguished from a single Tm at 57.4 ± 0.2 °C for L. major strains. A double curve with melting peaks at 66.6 ± 0.1 °C and 48.1 ± 0.5 °C or 55.8 ± 0.6 °C was observed for all L. tropica strains. The assay was further tested on biopsy specimens, which showed 100 % agreement with results obtained from isoenzyme electrophoresis and Sanger sequencing. Currently, there are no published data on real-time PCR using FRET technology to differentiate between Old World cutaneous Leishmania species. In summary, our assay based on specific hybridization addresses the limitations of previous PCR technology and provides a single step, reliable method of species identification and rapid diagnostic applications. |
X Demographics
The data shown below were collected from the profiles of 7 X users who shared this research output. Click here to find out more about how the information was compiled.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United States | 3 | 43% |
| Italy | 1 | 14% |
| Unknown | 3 | 43% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 5 | 71% |
| Science communicators (journalists, bloggers, editors) | 1 | 14% |
| Practitioners (doctors, other healthcare professionals) | 1 | 14% |
Mendeley readers
The data shown below were compiled from readership statistics for 70 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Spain | 1 | 1% |
| Unknown | 69 | 99% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 12 | 17% |
| Student > Ph. D. Student | 10 | 14% |
| Student > Doctoral Student | 7 | 10% |
| Other | 6 | 9% |
| Student > Bachelor | 4 | 6% |
| Other | 14 | 20% |
| Unknown | 17 | 24% |
| Readers by discipline | Count | As % |
|---|---|---|
| Agricultural and Biological Sciences | 12 | 17% |
| Biochemistry, Genetics and Molecular Biology | 11 | 16% |
| Medicine and Dentistry | 11 | 16% |
| Immunology and Microbiology | 4 | 6% |
| Veterinary Science and Veterinary Medicine | 2 | 3% |
| Other | 9 | 13% |
| Unknown | 21 | 30% |
Attention Score in Context
This research output has an Altmetric Attention Score of 4. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 20 September 2017.
All research outputs
#6,453,639
of 23,577,761 outputs
Outputs from Parasites & Vectors
#1,407
of 5,582 outputs
Outputs of similar age
#89,472
of 300,439 outputs
Outputs of similar age from Parasites & Vectors
#38
of 180 outputs
Altmetric has tracked 23,577,761 research outputs across all sources so far. This one has received more attention than most of these and is in the 72nd percentile.
So far Altmetric has tracked 5,582 research outputs from this source. They typically receive a little more attention than average, with a mean Attention Score of 5.8. This one has gotten more attention than average, scoring higher than 74% of its peers.
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We're also able to compare this research output to 180 others from the same source and published within six weeks on either side of this one. This one has done well, scoring higher than 78% of its contemporaries.