Semi-quantitative, duplexed qPCR assay for the detection of leishmania spp. using bisulphite conversion technology

Gow, I; Millar, D; Ellis, J; Melki, J; Stark, D

Semi-quantitative, duplexed qPCR assay for the detection of leishmania spp. using bisulphite conversion technology

Gow, I Millar, D Ellis, J

Melki, J Stark, D

Permalink

Publication Type:: Journal Article
Citation:: Tropical Medicine and Infectious Disease, 2019, 4 (4)
Issue Date:: 2019-01-01

Open Access

Copyright Clearance Process

Recently Added
In Progress
Open Access

This item is open access.

Adobe PDF

Download Published VersionAdobe PDF (731.81 kB)

View on publisher's site

View statistics

Full metadata record

Field	Value	Language
dc.contributor.author	Gow, I	en_US
dc.contributor.author	Millar, D	en_US
dc.contributor.author	Ellis, J https://orcid.org/0000-0001-7328-4831	en_US
dc.contributor.author	Melki, J	en_US
dc.contributor.author	Stark, D	en_US
dc.date.available	2019-10-28	en_US
dc.date.issued	2019-01-01	en_US
dc.identifier.citation	Tropical Medicine and Infectious Disease, 2019, 4 (4)	en_US
dc.identifier.uri	http://hdl.handle.net/10453/138180
dc.description.abstract	© 2019 by the authors. Leishmaniasis is caused by the flagellated protozoan Leishmania, and is a neglected tropical disease (NTD), as defined by theWorld Health Organisation (WHO). Bisulphite conversion technology converts all genomic material to a simplified form during the lysis step of the nucleic acid extraction process, and increases the efficiency of multiplex quantitative polymerase chain reaction (qPCR) reactions. Through utilization of qPCR real-time probes, in conjunction with bisulphite conversion, a new duplex assay targeting the 18S rDNA gene region was designed to detect all Leishmania species. The assay was validated against previously extracted DNA, from seven quantitated DNA and cell standards for pan-Leishmania analytical sensitivity data, and 67 cutaneous clinical samples for cutaneous clinical sensitivity data. Specificity was evaluated by testing 76 negative clinical samples and 43 bacterial, viral, protozoan and fungal species. The assay was also trialed in a side-by-side experiment against a conventional PCR (cPCR), based on the Internal transcribed spacer region 1 (ITS1 region). Ninety-seven percent of specimens from patients that previously tested positive for Leishmania were positive for Leishmania spp. with the bisulphite conversion assay, and a limit of detection (LOD) of 10 copies per PCR was achieved, while the LOD of the ITS1 methodology was 10 cells/1000 genomic copies per PCR. This method of rapid, accurate and simple detection of Leishmania can lead to improved diagnosis, treatment and public health outcomes.	en_US
dc.relation.ispartof	Tropical Medicine and Infectious Disease	en_US
dc.relation.isbasedon	10.3390/tropicalmed4040135	en_US
dc.title	Semi-quantitative, duplexed qPCR assay for the detection of leishmania spp. using bisulphite conversion technology	en_US
dc.type	Journal Article
utslib.citation.volume	4	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
utslib.for	1103 Clinical Sciences	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
pubs.organisational-group	/University of Technology Sydney/Students
utslib.copyright.status	open_access
pubs.issue	4	en_US
pubs.publication-status	Published	en_US
pubs.volume	4	en_US

Abstract:

© 2019 by the authors. Leishmaniasis is caused by the flagellated protozoan Leishmania, and is a neglected tropical disease (NTD), as defined by theWorld Health Organisation (WHO). Bisulphite conversion technology converts all genomic material to a simplified form during the lysis step of the nucleic acid extraction process, and increases the efficiency of multiplex quantitative polymerase chain reaction (qPCR) reactions. Through utilization of qPCR real-time probes, in conjunction with bisulphite conversion, a new duplex assay targeting the 18S rDNA gene region was designed to detect all Leishmania species. The assay was validated against previously extracted DNA, from seven quantitated DNA and cell standards for pan-Leishmania analytical sensitivity data, and 67 cutaneous clinical samples for cutaneous clinical sensitivity data. Specificity was evaluated by testing 76 negative clinical samples and 43 bacterial, viral, protozoan and fungal species. The assay was also trialed in a side-by-side experiment against a conventional PCR (cPCR), based on the Internal transcribed spacer region 1 (ITS1 region). Ninety-seven percent of specimens from patients that previously tested positive for Leishmania were positive for Leishmania spp. with the bisulphite conversion assay, and a limit of detection (LOD) of 10 copies per PCR was achieved, while the LOD of the ITS1 methodology was 10 cells/1000 genomic copies per PCR. This method of rapid, accurate and simple detection of Leishmania can lead to improved diagnosis, treatment and public health outcomes.

Please use this identifier to cite or link to this item:

http://hdl.handle.net/10453/138180