Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis
Objective:Leishmania RNA virus was detected the first time in the New World Leishmania species. Recent studies were also showed the presence of Leishmania RNA virus 2 (LRV2) in Old Word Leishmania species including Turkish L. major and L. tropica isolates. This study aimed to increase the sensitivit...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Galenos Yayinevi
2022-06-01
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| Series: | Türkiye Parazitoloji Dergisi |
| Subjects: | |
| Online Access: |
http://www.turkiyeparazitolderg.org/archives/archive-detail/article-preview/ncreasing-the-sensitivity-of-ileishmania-i-rna-vir/51972
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| _version_ | 1828024315706081280 |
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| author | Muhammed Nalçacı Mehmet Karakuş Yusuf Özbel Ahmet Özbilgin Seray Töz |
| author_facet | Muhammed Nalçacı Mehmet Karakuş Yusuf Özbel Ahmet Özbilgin Seray Töz |
| author_sort | Muhammed Nalçacı |
| collection | DOAJ |
| description | Objective:Leishmania RNA virus was detected the first time in the New World Leishmania species. Recent studies were also showed the presence of Leishmania RNA virus 2 (LRV2) in Old Word Leishmania species including Turkish L. major and L. tropica isolates. This study aimed to increase the sensitivity of qPCR with a modification in the denaturation step of cDNA preparation protocol.Methods:In this study, LRV2+ three L. major, two L. tropica strains and L. major control strain (MHOM/SU/73/5-ASKH) were included. Total RNA isolation was done using different numbers of Leishmania promastigotes (108, 105 and 103). Before cDNA synthesis, samples were denatured at 95 °C for 2 min, as a modification of the kit procedure. qPCR was undertaken using 0.5 mM primers (LRV F-HR/LRV R-HR) diluted in SYBR Green Master mix.Results:We observed lower Ct values in amplicons with the modified version than with the classical kit protocol for cDNA synthesis, in all of the strains used in the study. The addition of pre-denaturation step at 95 °C showed lower Ct values meaning the sensitivity increased. Different parasite dilutions showed similar results.Conclusion:It is important to increase the sensitivity especially with the aim for detecting LRV in clinical samples obtained from patients probably have less number of parasites. The presence and burden of the virus can help to understand the relationship between the clinical findings and the pathogenicity of the parasite which may lead to changes in the course of treatment. |
| first_indexed | 2024-04-10T12:43:08Z |
| format | Article |
| id | doaj.art-82592f8c570a4c11aacb450496f05d5a |
| institution | Directory Open Access Journal |
| issn | 1300-6320 2146-3077 |
| language | English |
| last_indexed | 2024-04-10T12:43:08Z |
| publishDate | 2022-06-01 |
| publisher | Galenos Yayinevi |
| record_format | Article |
| series | Türkiye Parazitoloji Dergisi |
| spelling | doaj.art-82592f8c570a4c11aacb450496f05d5a2023-02-15T16:14:14ZengGalenos YayineviTürkiye Parazitoloji Dergisi1300-63202146-30772022-06-01462869010.4274/tpd.galenos.2022.3007413049054Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA SynthesisMuhammed Nalçacı0Mehmet Karakuş1Yusuf Özbel2Ahmet Özbilgin3Seray Töz4 Ege University Graduate School of Natural and Applied Sciences, Department of Biology, İzmir, Turkey University of Health Sciences Turkey Hamidiye Faculty of Medicine, Department of Medical Microbiology, İstanbul, Turkey Ege University Faculty of Medicine, Department of Parasitology, İzmir, Turkey Celal Bayar University Faculty of Medicine, Department of Parasitology, Manisa, Turkey Ege University Faculty of Medicine, Department of Parasitology, İzmir, Turkey Objective:Leishmania RNA virus was detected the first time in the New World Leishmania species. Recent studies were also showed the presence of Leishmania RNA virus 2 (LRV2) in Old Word Leishmania species including Turkish L. major and L. tropica isolates. This study aimed to increase the sensitivity of qPCR with a modification in the denaturation step of cDNA preparation protocol.Methods:In this study, LRV2+ three L. major, two L. tropica strains and L. major control strain (MHOM/SU/73/5-ASKH) were included. Total RNA isolation was done using different numbers of Leishmania promastigotes (108, 105 and 103). Before cDNA synthesis, samples were denatured at 95 °C for 2 min, as a modification of the kit procedure. qPCR was undertaken using 0.5 mM primers (LRV F-HR/LRV R-HR) diluted in SYBR Green Master mix.Results:We observed lower Ct values in amplicons with the modified version than with the classical kit protocol for cDNA synthesis, in all of the strains used in the study. The addition of pre-denaturation step at 95 °C showed lower Ct values meaning the sensitivity increased. Different parasite dilutions showed similar results.Conclusion:It is important to increase the sensitivity especially with the aim for detecting LRV in clinical samples obtained from patients probably have less number of parasites. The presence and burden of the virus can help to understand the relationship between the clinical findings and the pathogenicity of the parasite which may lead to changes in the course of treatment. http://www.turkiyeparazitolderg.org/archives/archive-detail/article-preview/ncreasing-the-sensitivity-of-ileishmania-i-rna-vir/51972 leishmanialeishmania rna virus 2lrv2cdnaqpcr |
| spellingShingle | Muhammed Nalçacı Mehmet Karakuş Yusuf Özbel Ahmet Özbilgin Seray Töz Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis Türkiye Parazitoloji Dergisi leishmania leishmania rna virus 2 lrv2 cdna qpcr |
| title | Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis |
| title_full | Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis |
| title_fullStr | Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis |
| title_full_unstemmed | Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis |
| title_short | Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis |
| title_sort | increasing the sensitivity of leishmania rna virus 2 lrv2 detection with a modification in cdna synthesis |
| topic | leishmania leishmania rna virus 2 lrv2 cdna qpcr |
| url |
http://www.turkiyeparazitolderg.org/archives/archive-detail/article-preview/ncreasing-the-sensitivity-of-ileishmania-i-rna-vir/51972
|
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