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Research article
Genetic diversity analysis of Chinese Leishmania isolates and development of L. donovani complex specific markers by RAPD
Jianping Chen
Corresponding Author
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Background: Leishmaniasis is one of the most neglected tropical diseases in the world and is still endemic in some underdeveloped regions including western of China. The phylogeny and classification of Chinese Leishmania has not yet been clarified, especially within Leishmania (L.) donovani complex, although phylogenetic analyses based on a series of gene markers were carried out. More different analytic method and data still be needed. Random amplified polymorphic DNA (RAPD) technology could identify slight intraspecific differences sensitively, and it always be a powerful tool to seek the species-specific markers. This work aimed to identify Chinese Leishmania isolates from diverse geographic regions at genomic level. Meanwhile, the specific markers of L. donovani complex were also developed by RAPD.
Methods: The RAPD was applied on 14 Chinese Leishmania isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into data matrix, based on which genetic similarity were calculated and UPGMA dendrogram were constructed to analyze genetic diversity of these Leishmania isolates. Meanwhile, the specific amplification loci of L. donovani complex were TA-cloned, sequenced and converted into sequence characterized amplified regions (SCAR) markers, which were validated preliminarily in available 17 Leishmania strains in this study and analyzed by bioinformatics.
Results: The cluster analyses showed that the three Leishmania sp. isolates SC10H2, SD and GL clustered together and apart from others, and the strains of L. donovani complex clearly divided into two clades and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of L. donovani complex, i.e. 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on available 17 Leishmania strains in this study. Through bioinformatics analyses, Marker 1-AD17 may have more specificity on PCR detection of VL and Marker 3-O13 has the potential of encoding protein.
Conclusions: The RAPD result verified that the undescribed Leishmania species causing visceral Leishmaniasis (VL) in China was a unique clade distinguished from L. donovani, and revealed that there was genetic differentiation among Chinese L. donovani. The development of L. donovani specific markers may provide the foundation for developing new specific diagnostic markers of VL and the research of specific gene function.
Keywords
Leishmania donovani, China, RAPD, SCAR
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Received 21 Oct, 2020
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On 29 Sep, 2020
Editor assigned
On 21 Sep, 2020
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On 20 Sep, 2020
Editor invited
On 20 Sep, 2020
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Editorial decision: Major revision
On 30 Aug, 2020
Review #1 received
Received 25 Aug, 2020
Review #2 received
Received 25 Aug, 2020
Reviewer #3 agreed
On 01 Aug, 2020
Reviewer #2 agreed
On 25 Jul, 2020
Reviewer #1 agreed
On 22 Jul, 2020
Reviewers invited
Invitations sent on 19 Jul, 2020
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On 06 Jul, 2020
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On 05 Jul, 2020
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This preprint is under consideration at BMC Infectious Diseases. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Genetic diversity analysis of Chinese Leishmania isolates and development of L. donovani complex specific markers by RAPD
Jianping Chen
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 2
Posted 23 Sep, 2020
No community comments so far
Review #1 received
Received 21 Oct, 2020
Reviewers invited
Invitations sent on 29 Sep, 2020
Reviewer #1 agreed
On 29 Sep, 2020
Editor assigned
On 21 Sep, 2020
Submission checks complete
On 20 Sep, 2020
Editor invited
On 20 Sep, 2020
No comments provided
Editorial decision: Major revision
On 30 Aug, 2020
Review #1 received
Received 25 Aug, 2020
Review #2 received
Received 25 Aug, 2020
Reviewer #3 agreed
On 01 Aug, 2020
Reviewer #2 agreed
On 25 Jul, 2020
Reviewer #1 agreed
On 22 Jul, 2020
Reviewers invited
Invitations sent on 19 Jul, 2020
Editor assigned
On 06 Jul, 2020
Submission checks complete
On 05 Jul, 2020
Editor invited
On 05 Jul, 2020
First submitted
On 03 Jul, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Infectious Diseases
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Leishmaniasis is one of the most neglected tropical diseases in the world and is still endemic in some underdeveloped regions including western of China. The phylogeny and classification of Chinese Leishmania has not yet been clarified, especially within Leishmania (L.) donovani complex, although phylogenetic analyses based on a series of gene markers were carried out. More different analytic method and data still be needed. Random amplified polymorphic DNA (RAPD) technology could identify slight intraspecific differences sensitively, and it always be a powerful tool to seek the species-specific markers. This work aimed to identify Chinese Leishmania isolates from diverse geographic regions at genomic level. Meanwhile, the specific markers of L. donovani complex were also developed by RAPD.
Methods: The RAPD was applied on 14 Chinese Leishmania isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into data matrix, based on which genetic similarity were calculated and UPGMA dendrogram were constructed to analyze genetic diversity of these Leishmania isolates. Meanwhile, the specific amplification loci of L. donovani complex were TA-cloned, sequenced and converted into sequence characterized amplified regions (SCAR) markers, which were validated preliminarily in available 17 Leishmania strains in this study and analyzed by bioinformatics.
Results: The cluster analyses showed that the three Leishmania sp. isolates SC10H2, SD and GL clustered together and apart from others, and the strains of L. donovani complex clearly divided into two clades and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of L. donovani complex, i.e. 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on available 17 Leishmania strains in this study. Through bioinformatics analyses, Marker 1-AD17 may have more specificity on PCR detection of VL and Marker 3-O13 has the potential of encoding protein.
Conclusions: The RAPD result verified that the undescribed Leishmania species causing visceral Leishmaniasis (VL) in China was a unique clade distinguished from L. donovani, and revealed that there was genetic differentiation among Chinese L. donovani. The development of L. donovani specific markers may provide the foundation for developing new specific diagnostic markers of VL and the research of specific gene function.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5