Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry
Quantitative real-time RT-PCR (qRT-PCR) assay was developed for the detection and quantification of bovine viral diarrhea virus (BVDV) in clinical samples from persistently infected cattle. qRT-PCR was optimized to quantify the number of BVD virus copies using Light Cycler® detection system and inte...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Czech Academy of Agricultural Sciences
2007-06-01
|
| Series: | Veterinární Medicína |
| Subjects: | |
| Online Access: | https://vetmed.agriculturejournals.cz/artkey/vet-200706-0004_real-time-pcr-for-quantitation-of-bovine-viral-diarrhea-virus-rna-using-sybr-green-i-fluorimetry.php |
| _version_ | 1828006841875955712 |
|---|---|
| author | E. Kosinova I. Psikal B. Robesova K. Kovarcik |
| author_facet | E. Kosinova I. Psikal B. Robesova K. Kovarcik |
| author_sort | E. Kosinova |
| collection | DOAJ |
| description | Quantitative real-time RT-PCR (qRT-PCR) assay was developed for the detection and quantification of bovine viral diarrhea virus (BVDV) in clinical samples from persistently infected cattle. qRT-PCR was optimized to quantify the number of BVD virus copies using Light Cycler® detection system and intercalation fluorogenic dye SYBR Green I. A universal set of primers was selected from a highly conserved 5' untranslated region (5'UTR) to detect BVDV type I and II simultaneously. Quantification of BVDV cDNA was accomplished using a calibration curve generated from 10-fold serial dilutions of standard plasmid DNA in the range 1-108 copies/μl. Analysis of 290 bp amplicons enabled monitoring of the viral RNA/BVDV level in a total of five BVDV strains (BVD-NADL, A03/3004, DB03/2943, KA04/3124, KV05/3412) and sixteen bulk milk samples, and in bovine sera of persistent carriers originating from Czech farms, as well as in a batch of calf serum for cell culture. Melting temperatures of amplicons (Tm) of BVDV strains of the same genotype group I as the NADL reference strain showed variability of the thermal points, however significant differences were observed in Tm values between the representatives of genotype group I and II. Low concentrations of BVD virus in bulk milk samples were also qualitatively identified by conventional RT-PCR. Highly reproducible data were obtained as the coefficients of variation of threshold cycles values in intra-assay and inter-assay were less than 0.85% and 2.76%, respectively. The results give enough evidence of suitability of qRT-PCR assay for quantitative analysis of BVDV in clinical samples. |
| first_indexed | 2024-04-10T08:01:36Z |
| format | Article |
| id | doaj.art-215e6043075e4228b7ef64b96b46db5e |
| institution | Directory Open Access Journal |
| issn | 0375-8427 1805-9392 |
| language | English |
| last_indexed | 2024-04-10T08:01:36Z |
| publishDate | 2007-06-01 |
| publisher | Czech Academy of Agricultural Sciences |
| record_format | Article |
| series | Veterinární Medicína |
| spelling | doaj.art-215e6043075e4228b7ef64b96b46db5e2023-02-23T03:49:27ZengCzech Academy of Agricultural SciencesVeterinární Medicína0375-84271805-93922007-06-0152625326110.17221/1882-VETMEDvet-200706-0004Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetryE. Kosinova0I. Psikal1B. Robesova2K. Kovarcik3Veterinary Research Institute, Brno, Czech RepublicVeterinary Research Institute, Brno, Czech RepublicVeterinary Research Institute, Brno, Czech RepublicVeterinary Research Institute, Brno, Czech RepublicQuantitative real-time RT-PCR (qRT-PCR) assay was developed for the detection and quantification of bovine viral diarrhea virus (BVDV) in clinical samples from persistently infected cattle. qRT-PCR was optimized to quantify the number of BVD virus copies using Light Cycler® detection system and intercalation fluorogenic dye SYBR Green I. A universal set of primers was selected from a highly conserved 5' untranslated region (5'UTR) to detect BVDV type I and II simultaneously. Quantification of BVDV cDNA was accomplished using a calibration curve generated from 10-fold serial dilutions of standard plasmid DNA in the range 1-108 copies/μl. Analysis of 290 bp amplicons enabled monitoring of the viral RNA/BVDV level in a total of five BVDV strains (BVD-NADL, A03/3004, DB03/2943, KA04/3124, KV05/3412) and sixteen bulk milk samples, and in bovine sera of persistent carriers originating from Czech farms, as well as in a batch of calf serum for cell culture. Melting temperatures of amplicons (Tm) of BVDV strains of the same genotype group I as the NADL reference strain showed variability of the thermal points, however significant differences were observed in Tm values between the representatives of genotype group I and II. Low concentrations of BVD virus in bulk milk samples were also qualitatively identified by conventional RT-PCR. Highly reproducible data were obtained as the coefficients of variation of threshold cycles values in intra-assay and inter-assay were less than 0.85% and 2.76%, respectively. The results give enough evidence of suitability of qRT-PCR assay for quantitative analysis of BVDV in clinical samples.https://vetmed.agriculturejournals.cz/artkey/vet-200706-0004_real-time-pcr-for-quantitation-of-bovine-viral-diarrhea-virus-rna-using-sybr-green-i-fluorimetry.phpbovine viral diarrhea virusrnareal-time rt-pcrsybr green i |
| spellingShingle | E. Kosinova I. Psikal B. Robesova K. Kovarcik Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry Veterinární Medicína bovine viral diarrhea virus rna real-time rt-pcr sybr green i |
| title | Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry |
| title_full | Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry |
| title_fullStr | Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry |
| title_full_unstemmed | Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry |
| title_short | Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry |
| title_sort | real time pcr for quantitation of bovine viral diarrhea virus rna using sybr green i fluorimetry |
| topic | bovine viral diarrhea virus rna real-time rt-pcr sybr green i |
| url | https://vetmed.agriculturejournals.cz/artkey/vet-200706-0004_real-time-pcr-for-quantitation-of-bovine-viral-diarrhea-virus-rna-using-sybr-green-i-fluorimetry.php |
| work_keys_str_mv | AT ekosinova realtimepcrforquantitationofbovineviraldiarrheavirusrnausingsybrgreenifluorimetry AT ipsikal realtimepcrforquantitationofbovineviraldiarrheavirusrnausingsybrgreenifluorimetry AT brobesova realtimepcrforquantitationofbovineviraldiarrheavirusrnausingsybrgreenifluorimetry AT kkovarcik realtimepcrforquantitationofbovineviraldiarrheavirusrnausingsybrgreenifluorimetry |