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Dry- down probe free qPCR for detection of KFD in resource limited settings

Kyasanur Forest Disease is a tick-borne flavivirus is endemic in the Southern India. The recent expansion and resurgence of sporadic outbreaks in southern parts of country is the most important concern. Although only formalin inactivated vaccine is available for treatment with limited efficacy the e...

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Autores principales: Yadav, Pooja, Sharma, Shashi, Dash, Paban Kumar, Dhankher, Suman, V. K., Sandhya, Kiran, S. K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10171661/
https://www.ncbi.nlm.nih.gov/pubmed/37163557
http://dx.doi.org/10.1371/journal.pone.0284559
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author Yadav, Pooja
Sharma, Shashi
Dash, Paban Kumar
Dhankher, Suman
V. K., Sandhya
Kiran, S. K.
author_facet Yadav, Pooja
Sharma, Shashi
Dash, Paban Kumar
Dhankher, Suman
V. K., Sandhya
Kiran, S. K.
author_sort Yadav, Pooja
collection PubMed
description Kyasanur Forest Disease is a tick-borne flavivirus is endemic in the Southern India. The recent expansion and resurgence of sporadic outbreaks in southern parts of country is the most important concern. Although only formalin inactivated vaccine is available for treatment with limited efficacy the early detection and timely identification is a only way to prevent spread of cases. If the disease can be identified prior to infection in humans like in forest areas from ticks and vectors the disease spread supposed to be managed quickly. Here we have standardized a single tube ready to use dry-down probe free real time RT-PCR targeted against virus envelope gene for detection of KFDV infection. The assay was standardized in liquid format first, later it was converted into dry-down format with addition of stabilizers with a similar sensitivity and specificity (10RNA Copies/rxn). The sensitivity was comparable to the most widely used and accepted diagnostic platform i.e. TaqMan qRT-PCR. However as the reported assay here omit the need of probes makes it cost effective and dry-down reagents makes more stability to the developed assay in this study if compare to TaqMan qPCR. The assay was evaluated with KFD positive samples and healthy sample panel which revealed high concordance with TaqMan qRT-PCR. Stability was unaffected by temperature fluctuations during transportation even in cold chain free conditions, thus reduce the maintenance of strict cold storage. These findings demonstrated that the reported assay is convenient with 100% sensitivity and specificity to TaqMan qPCR. Thus this assay has the potential usefulness for diagnosis KFDV for routine surveillance in resource limited laboratory settings omitting the use costly and heat sensitive TaqMan qRT-PCR reagents without compromising the sensitivity and specificity of the diagnosis assay.
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spelling pubmed-101716612023-05-11 Dry- down probe free qPCR for detection of KFD in resource limited settings Yadav, Pooja Sharma, Shashi Dash, Paban Kumar Dhankher, Suman V. K., Sandhya Kiran, S. K. PLoS One Research Article Kyasanur Forest Disease is a tick-borne flavivirus is endemic in the Southern India. The recent expansion and resurgence of sporadic outbreaks in southern parts of country is the most important concern. Although only formalin inactivated vaccine is available for treatment with limited efficacy the early detection and timely identification is a only way to prevent spread of cases. If the disease can be identified prior to infection in humans like in forest areas from ticks and vectors the disease spread supposed to be managed quickly. Here we have standardized a single tube ready to use dry-down probe free real time RT-PCR targeted against virus envelope gene for detection of KFDV infection. The assay was standardized in liquid format first, later it was converted into dry-down format with addition of stabilizers with a similar sensitivity and specificity (10RNA Copies/rxn). The sensitivity was comparable to the most widely used and accepted diagnostic platform i.e. TaqMan qRT-PCR. However as the reported assay here omit the need of probes makes it cost effective and dry-down reagents makes more stability to the developed assay in this study if compare to TaqMan qPCR. The assay was evaluated with KFD positive samples and healthy sample panel which revealed high concordance with TaqMan qRT-PCR. Stability was unaffected by temperature fluctuations during transportation even in cold chain free conditions, thus reduce the maintenance of strict cold storage. These findings demonstrated that the reported assay is convenient with 100% sensitivity and specificity to TaqMan qPCR. Thus this assay has the potential usefulness for diagnosis KFDV for routine surveillance in resource limited laboratory settings omitting the use costly and heat sensitive TaqMan qRT-PCR reagents without compromising the sensitivity and specificity of the diagnosis assay. Public Library of Science 2023-05-10 /pmc/articles/PMC10171661/ /pubmed/37163557 http://dx.doi.org/10.1371/journal.pone.0284559 Text en © 2023 Yadav et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Yadav, Pooja
Sharma, Shashi
Dash, Paban Kumar
Dhankher, Suman
V. K., Sandhya
Kiran, S. K.
Dry- down probe free qPCR for detection of KFD in resource limited settings
title Dry- down probe free qPCR for detection of KFD in resource limited settings
title_full Dry- down probe free qPCR for detection of KFD in resource limited settings
title_fullStr Dry- down probe free qPCR for detection of KFD in resource limited settings
title_full_unstemmed Dry- down probe free qPCR for detection of KFD in resource limited settings
title_short Dry- down probe free qPCR for detection of KFD in resource limited settings
title_sort dry- down probe free qpcr for detection of kfd in resource limited settings
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10171661/
https://www.ncbi.nlm.nih.gov/pubmed/37163557
http://dx.doi.org/10.1371/journal.pone.0284559
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