TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth disease
Effective control and monitoring the spread of foot-and-mouth disease (FMD) relies upon rapid and accurate laboratory detection of FMD virus (FMDV). Therefore, in this report, a multiplex TaqMan probe-based one-step RT-qPCR assay simultaneously targeting FMDV 5′UTR and 3Dpol regions, and 18S rRNA ho...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-11-01
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| Series: | Acta Virologica |
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| Online Access: | https://www.frontierspartnerships.org/articles/10.3389/av.2023.12075/full |
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| author | Jitendra K. Biswal Jajati K. Mohapatra Rajeev Ranjan Manoranjan Rout Shyam Singh Dahiya Rabindra Prasad Singh |
| author_facet | Jitendra K. Biswal Jajati K. Mohapatra Rajeev Ranjan Manoranjan Rout Shyam Singh Dahiya Rabindra Prasad Singh |
| author_sort | Jitendra K. Biswal |
| collection | DOAJ |
| description | Effective control and monitoring the spread of foot-and-mouth disease (FMD) relies upon rapid and accurate laboratory detection of FMD virus (FMDV). Therefore, in this report, a multiplex TaqMan probe-based one-step RT-qPCR assay simultaneously targeting FMDV 5′UTR and 3Dpol regions, and 18S rRNA housekeeping gene (as an internal control) in a single reaction tube was developed and evaluated. The multiplex one-step RT-qPCR assay specifically detected viral genome in both FMDV-infected cell culture suspensions and clinical samples collected from known-FMD infected animals. The assay could detect FMDV RNA in the archived FMDV cell culture isolates (n = 120) collected during the last two decades in India. In addition, the new assay could also detect viral RNA in the FMD suspected clinical samples (n = 740) collected from various field outbreaks. At a cut-off Ct-value of <38, the assay could detect at least 20 and 10 copies of FMDV 3Dpol and 5′UTR genes, respectively. Further, the multiplex RT-qPCR assay proved to be robust, showing an inter-assay co-efficient of variations ranging from 1.3% to 3.03% for FMDV-3Dpol gene target, and from 1.44% to 4.69% for 5′UTR gene target. In addition, it was found that the new assay could be used to detect viral genome in a variety of samples (epithelium, saliva, OPF, milk and blood) without any significance difference in the detection limit of the assay. Hence, the multiplex one-step RT-qPCR assay could be considered a valuable tool for the detection of FMDV in India. |
| first_indexed | 2024-03-10T13:39:08Z |
| format | Article |
| id | doaj.art-da24553f67214b088a10268345c9a0fb |
| institution | Directory Open Access Journal |
| issn | 1336-2305 |
| language | English |
| last_indexed | 2024-03-10T13:39:08Z |
| publishDate | 2023-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Acta Virologica |
| spelling | doaj.art-da24553f67214b088a10268345c9a0fb2023-11-21T04:11:13ZengFrontiers Media S.A.Acta Virologica1336-23052023-11-016710.3389/av.2023.1207512075TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth diseaseJitendra K. BiswalJajati K. MohapatraRajeev RanjanManoranjan RoutShyam Singh DahiyaRabindra Prasad SinghEffective control and monitoring the spread of foot-and-mouth disease (FMD) relies upon rapid and accurate laboratory detection of FMD virus (FMDV). Therefore, in this report, a multiplex TaqMan probe-based one-step RT-qPCR assay simultaneously targeting FMDV 5′UTR and 3Dpol regions, and 18S rRNA housekeeping gene (as an internal control) in a single reaction tube was developed and evaluated. The multiplex one-step RT-qPCR assay specifically detected viral genome in both FMDV-infected cell culture suspensions and clinical samples collected from known-FMD infected animals. The assay could detect FMDV RNA in the archived FMDV cell culture isolates (n = 120) collected during the last two decades in India. In addition, the new assay could also detect viral RNA in the FMD suspected clinical samples (n = 740) collected from various field outbreaks. At a cut-off Ct-value of <38, the assay could detect at least 20 and 10 copies of FMDV 3Dpol and 5′UTR genes, respectively. Further, the multiplex RT-qPCR assay proved to be robust, showing an inter-assay co-efficient of variations ranging from 1.3% to 3.03% for FMDV-3Dpol gene target, and from 1.44% to 4.69% for 5′UTR gene target. In addition, it was found that the new assay could be used to detect viral genome in a variety of samples (epithelium, saliva, OPF, milk and blood) without any significance difference in the detection limit of the assay. Hence, the multiplex one-step RT-qPCR assay could be considered a valuable tool for the detection of FMDV in India.https://www.frontierspartnerships.org/articles/10.3389/av.2023.12075/fullFMDVTaqMan probemultiplex RT-qPCR3D polymerase5′UTR |
| spellingShingle | Jitendra K. Biswal Jajati K. Mohapatra Rajeev Ranjan Manoranjan Rout Shyam Singh Dahiya Rabindra Prasad Singh TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth disease Acta Virologica FMDV TaqMan probe multiplex RT-qPCR 3D polymerase 5′UTR |
| title | TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth disease |
| title_full | TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth disease |
| title_fullStr | TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth disease |
| title_full_unstemmed | TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth disease |
| title_short | TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth disease |
| title_sort | taqman probe based one step multiplex real time rt pcr assay for the diagnosis of foot and mouth disease |
| topic | FMDV TaqMan probe multiplex RT-qPCR 3D polymerase 5′UTR |
| url | https://www.frontierspartnerships.org/articles/10.3389/av.2023.12075/full |
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