Abstract
A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 (TCID50) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.
| Original language | English |
|---|---|
| Pages (from-to) | 100-108 |
| Number of pages | 9 |
| Journal | Journal of Microbiology and Biotechnology |
| Volume | 21 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2011 Jan 1 |
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Keywords
- Method comparison
- Method validation
- Newcastle disease virus (NDV)
- Real-time RT-PCR (RRT-PCR)
- Virus quantification
ASJC Scopus subject areas
- Biotechnology
- Applied Microbiology and Biotechnology
Cite this
Validation of a real-time RT-PCR method to quantify newcastle disease virus (NDV) titer and comparison with other quantifiable methods. / Jang, Juno; Hong, Sung Hwan; Kim, Ik Hwan.
In: Journal of Microbiology and Biotechnology, Vol. 21, No. 1, 01.01.2011, p. 100-108.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Validation of a real-time RT-PCR method to quantify newcastle disease virus (NDV) titer and comparison with other quantifiable methods
AU - Jang, Juno
AU - Hong, Sung Hwan
AU - Kim, Ik Hwan
PY - 2011/1/1
Y1 - 2011/1/1
N2 - A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 (TCID50) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.
AB - A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 (TCID50) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.
KW - Method comparison
KW - Method validation
KW - Newcastle disease virus (NDV)
KW - Real-time RT-PCR (RRT-PCR)
KW - Virus quantification
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U2 - 10.4014/jmb.1006.06006
DO - 10.4014/jmb.1006.06006
M3 - Article
VL - 21
SP - 100
EP - 108
JO - Journal of Microbiology and Biotechnology
JF - Journal of Microbiology and Biotechnology
SN - 1017-7825
IS - 1
ER -