Comparison of conventional RT-PCR, reverse-transcription loop-mediated isothermal amplification, and SYBR green I-based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches

Shu-Qin Zhang; Bin Tan; Peng Li; Feng-Xue Wang; Li Guo; Yong Yang; Na Sun; Hong-Wei Zhu; Yong-Jun Wen; Shi-Peng Cheng

doi:10.1016/j.jviromet.2014.05.020

Comparison of conventional RT-PCR, reverse-transcription loop-mediated isothermal amplification, and SYBR green I-based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches

J Virol Methods. 2014 Oct:207:204-9. doi: 10.1016/j.jviromet.2014.05.020. Epub 2014 Jul 12.

Authors

Shu-Qin Zhang¹, Bin Tan², Peng Li², Feng-Xue Wang², Li Guo², Yong Yang², Na Sun², Hong-Wei Zhu², Yong-Jun Wen³, Shi-Peng Cheng⁴

Affiliations

¹ State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences CAAS, No. 4899 Juye Street, Chuangchun 130112, China. Electronic address: [email protected].
² State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences CAAS, No. 4899 Juye Street, Chuangchun 130112, China.
³ State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences CAAS, No. 4899 Juye Street, Chuangchun 130112, China. Electronic address: [email protected].
⁴ State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences CAAS, No. 4899 Juye Street, Chuangchun 130112, China. Electronic address: [email protected].

PMID: 25019170
DOI: 10.1016/j.jviromet.2014.05.020

Abstract

Bovine viral diarrhea virus (BVDV) can contaminate biological products produced in bovine or porcine cells or manufactured using bovine sera. A rapid, specific, sensitive, and practical method of detecting BVDV in bio-products is needed. The purpose of this study was to compare three assays with respect to their ability to accurately detect BVDV in biological samples, namely reverse-transcription loop-mediated isothermal amplification (RT-LAMP), SYBR green I-based real-time RT-PCR, and conventional RT-PCR. All assays detected BVDV nucleotide and differentiated between BVDV-free and -contaminated bovine sera successfully. In addition, the results were specific to BVDV: the amplification of samples containing the closely related classical swine fever virus or other pathogenic bovine viruses yielded negative results. The lowest detection threshold, 10(1) copies, was displayed by the SYBR green I-based real-time RT-PCR and RT-LAMP assay. This assay was also the most effective in the detection of BVDV contamination in a set of commercially available bovine sera. The field conditions suggest that RT-LAMP is specific and sensitive to detecting BVDV in biological samples and may be used for quality control of biomaterials.

Keywords: Bovine viral diarrhea virus; RT-PCR; Reverse-transcription loop-mediated isothermal amplification; SYBR green I-based real-time RT-PCR.

Publication types

Comparative Study
Evaluation Study
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Benzothiazoles
Cattle
Classical Swine Fever Virus
Diamines
Molecular Sequence Data
Nucleic Acid Amplification Techniques / methods*
Organic Chemicals / metabolism
Pestivirus / genetics
Pestivirus / isolation & purification*
Quinolines
RNA, Viral / genetics
RNA, Viral / isolation & purification*
Sequence Analysis, DNA
Serum / virology*
Staining and Labeling / methods
Temperature
Time Factors

Substances

Benzothiazoles
Diamines
Organic Chemicals
Quinolines
RNA, Viral
SYBR Green I

Associated data

GENBANK/KC176777