Acta Entomologica Sinica ›› 2020, Vol. 63 ›› Issue (2): 149-158.doi: 10.16380/j.kcxb.2020.02.004

• RESEARCH PAPERS • Previous Articles     Next Articles

Primer screening and amplification protocol optimization of rapid detection technique for Cucurbit chlorotic yellows virus

WANG Ke, HE Yan-Yan, ZHANG You-Jun, WU Qing-Jun, WANG Shao-Li*    

  1. (Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China)
  • Online:2020-02-20 Published:2020-02-25

Abstract: 【Aim】 Cucurbit chlorotic yellows virus (CCYV) is an emerging plant virus, in semi-persistent manner, transmitted by the sweetpotato whitefly, Bemisia tabaci. Induced virus disease by CCYV has caused severe economic losses to cucurbit crops. CCYV monitoring based on PCR method is a common technique for detecting this virus. However, some commonly used primers have the problems of unstable amplification results and insufficient reproducibility. This study aims to screen and obtain the more suitable primers for stable detection of CCYV under optimized conditions. 【Methods】 The bacterial liquid of Agrobacterium tumefaciens strain GV3101 carrying CCYV and the cDNA from its infected cucumber leaves were amplified by PCR with 14 pairs of primers known currently to screen primers for stable detection of CCYV. The annealing temperatures were also optimized. Using the cDNA from cucumber leaves infected by A. tumefaciens carrying CCYV, the stability and sensitivity of the selected four primer pairs in PCR amplification were determined. Then, the CCYV infection of 13 samples including B. tabaci adults and host plant leaves collected from the field was detected and validated by the selected primers and the optimized amplification procedure. 【Results】 Four pairs of primers were screened out from the 14 known primer pairs, which could stably amplify cDNA from A. tumefaciens carrying CCYV and its infected cucumber leaves, and the optimized amplification procedure was obtained. Sensitivity test revealed that the minimum cDNA concentration of CCYV-infected cucumber leaves in PCR assay with the selected four pairs of primers was 0.25 ng/μL. Detection results of CCYV infection in 13 samples including B. tabaci adults and host plant leaves collected from the field by PCR with the selected four pairs of primers showed that 69.23% samples were CCYV positive. 【Conclusion】 The selected four pairs of primers and the corresponding optimized amplication procedure could be applied for accurate detection of CCYV in A. tumefaciens carrying CCYV, CCYV-infected plant leaves and field samples.

Key words: Cucurbit chlorotic yellows virusBemisia tabaci, rapid detection, primer screening, detection sensitivity, viruliferous rate