Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance
The major arboviruses mainly belong to the <i>Bunyaviridae</i>, <i>Togaviridae</i>, and <i>Flaviviridae</i> families, among which the chikungunya virus and dengue virus have emerged as global public health problems. The main objective of this study was to develop...
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MDPI AG
2024-03-01
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| Series: | Current Issues in Molecular Biology |
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| Online Access: | https://www.mdpi.com/1467-3045/46/3/134 |
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| author | Louis Robert W. Belem Sylvester Agha Ibemgbo Michel Kiréopori Gomgnimbou Dileep Kumar Verma Antoinette Kaboré Ankit Kumar Ibrahim Sangaré Sujatha Sunil |
| author_facet | Louis Robert W. Belem Sylvester Agha Ibemgbo Michel Kiréopori Gomgnimbou Dileep Kumar Verma Antoinette Kaboré Ankit Kumar Ibrahim Sangaré Sujatha Sunil |
| author_sort | Louis Robert W. Belem |
| collection | DOAJ |
| description | The major arboviruses mainly belong to the <i>Bunyaviridae</i>, <i>Togaviridae</i>, and <i>Flaviviridae</i> families, among which the chikungunya virus and dengue virus have emerged as global public health problems. The main objective of this study was to develop specific, sensitive, and cost-effective molecular multiplex RT-PCR and RT-qPCR assays for the rapid and simultaneous detection of CHIKV and the four serotypes of DENV for arbovirus surveillance. Specific primers for all viruses were designed, and one-step multiplex RT-PCR (mRT-PCR) and RT-qPCR (mRT-qPCR) were developed using reference strains of the CHIKV and DENV serotypes. The specificity of the test for all the viruses was confirmed through sequencing. The standard curves showed a high correlation coefficient, R<sup>2</sup> = 0.99, for DENV-2 and DENV-3; R<sup>2</sup> = 0.98, for DENV-4; and CHIKV; R<sup>2</sup> = 0.93, for DENV-1. The limits of detection were calculated to be 4.1 × 10<sup>−1</sup> copies/reaction for DENV-1, DENV-3, and CHIKV and 4.1 × 10<sup>1</sup> for DENV-2 and DENV-4. The specificity and sensitivity of the newly developed mRT-PCR and mRT-qPCR were validated using positive serum samples collected from India and Burkina Faso. The sensitivity of mRT-PCR and mRT-qPCR are 91%, and 100%, respectively. The specificity of both assays was 100%. mRT-PCR and mRT-qPCR assays are low-cost, and a combination of both will be a useful tool for arbovirus surveillance. |
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| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1467-3037 1467-3045 |
| language | English |
| last_indexed | 2024-04-24T18:25:26Z |
| publishDate | 2024-03-01 |
| publisher | MDPI AG |
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| series | Current Issues in Molecular Biology |
| spelling | doaj.art-3c11c3ab55724249912494436d16bafb2024-03-27T13:31:24ZengMDPI AGCurrent Issues in Molecular Biology1467-30371467-30452024-03-014632093210410.3390/cimb46030134Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus SurveillanceLouis Robert W. Belem0Sylvester Agha Ibemgbo1Michel Kiréopori Gomgnimbou2Dileep Kumar Verma3Antoinette Kaboré4Ankit Kumar5Ibrahim Sangaré6Sujatha Sunil7Vector Borne Diseases Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, IndiaVector Borne Diseases Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, IndiaCentre d’Excellence Africain en Innovations Biotechnologiques pour l’Elimination des Maladies à Transmission Vectorielle (CEA/ITECH-MTV), Université Nazi Boni, Bobo-Dioulasso 01 BP 1091, Burkina FasoVector Borne Diseases Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, IndiaLaboratoire National de Référence, Institut National de Santé Publique, Ouagadougou BP 10278, Burkina FasoVector Borne Diseases Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, IndiaCentre d’Excellence Africain en Innovations Biotechnologiques pour l’Elimination des Maladies à Transmission Vectorielle (CEA/ITECH-MTV), Université Nazi Boni, Bobo-Dioulasso 01 BP 1091, Burkina FasoVector Borne Diseases Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, IndiaThe major arboviruses mainly belong to the <i>Bunyaviridae</i>, <i>Togaviridae</i>, and <i>Flaviviridae</i> families, among which the chikungunya virus and dengue virus have emerged as global public health problems. The main objective of this study was to develop specific, sensitive, and cost-effective molecular multiplex RT-PCR and RT-qPCR assays for the rapid and simultaneous detection of CHIKV and the four serotypes of DENV for arbovirus surveillance. Specific primers for all viruses were designed, and one-step multiplex RT-PCR (mRT-PCR) and RT-qPCR (mRT-qPCR) were developed using reference strains of the CHIKV and DENV serotypes. The specificity of the test for all the viruses was confirmed through sequencing. The standard curves showed a high correlation coefficient, R<sup>2</sup> = 0.99, for DENV-2 and DENV-3; R<sup>2</sup> = 0.98, for DENV-4; and CHIKV; R<sup>2</sup> = 0.93, for DENV-1. The limits of detection were calculated to be 4.1 × 10<sup>−1</sup> copies/reaction for DENV-1, DENV-3, and CHIKV and 4.1 × 10<sup>1</sup> for DENV-2 and DENV-4. The specificity and sensitivity of the newly developed mRT-PCR and mRT-qPCR were validated using positive serum samples collected from India and Burkina Faso. The sensitivity of mRT-PCR and mRT-qPCR are 91%, and 100%, respectively. The specificity of both assays was 100%. mRT-PCR and mRT-qPCR assays are low-cost, and a combination of both will be a useful tool for arbovirus surveillance.https://www.mdpi.com/1467-3045/46/3/134multiplexmoleculardetectiondengue viruschikungunya virusarbovirus |
| spellingShingle | Louis Robert W. Belem Sylvester Agha Ibemgbo Michel Kiréopori Gomgnimbou Dileep Kumar Verma Antoinette Kaboré Ankit Kumar Ibrahim Sangaré Sujatha Sunil Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance Current Issues in Molecular Biology multiplex molecular detection dengue virus chikungunya virus arbovirus |
| title | Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance |
| title_full | Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance |
| title_fullStr | Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance |
| title_full_unstemmed | Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance |
| title_short | Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance |
| title_sort | development of multiplex molecular assays for simultaneous detection of dengue serotypes and chikungunya virus for arbovirus surveillance |
| topic | multiplex molecular detection dengue virus chikungunya virus arbovirus |
| url | https://www.mdpi.com/1467-3045/46/3/134 |
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