Establishment and application of real-time fluorescence quantitative PCR detection technology for cherry virus A

doi:10.14088/j.cnki.issn0439-8114.2023.07.028

Abstract

Abstract: Three pairs of detection primers were designed in the conserved region of cherry virus A （CVA） mp gene. After specific screening, primers were obtained that could be used for virus quantitative research. Preparation of plasmid standards, and establishment of standard curves were conducted the sensitivity and specificity of this method, were verified and it is applied to the quantitative detection of CVA in field fruit tree samples. One pair of primers with high detection efficiency and strong specificity （CVA-dF2, CVA-dR2） was successfully screened, and a real-time reverse transcription fluorescence quantitative PCR method for detecting CVA was established based on SYBR Green I fluorescent dye. This method had good repeatability and high sensitivity. It could accurately detect the target viral load without the help of internal reference genes. The slope of the absolute quantitative standard curve was -3.574 6, the coefficient of determination R² was 0.998 6, and the amplification efficiency was 0.904 4, which was 10 times higher than the sensitivity of conventional RT-PCR detection.The establishment of this method provided a powerful tool for quantitative research on CVA, which could be used for batch detection of CVA in fruit trees or detection of low abundance virus samples.

Key words: cherry virus A （CVA）, reverse transcription real-time fluorescence quantitative PCR, quick detection

CLC Number:

S432

LIU Huan, LIU Ge, LI Rui. Establishment and application of real-time fluorescence quantitative PCR detection technology for cherry virus A[J]. HUBEI AGRICULTURAL SCIENCES, 2023, 62(7): 163-169.

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