Abstract:

Objective: This study aims to develop a multiplex PCR assay for identification of larvae of three noctuid moth species feeding on bamboo shoots in Zhejiang Province. Method: Species-specific multiplex PCR primers were designed for COI gene variation regions. The parameters influencing the multiplex PCR reaction were optimized, and the multiplex PCR assay was developed for identification of noctuids. The sensitivity and specificity of the proposed multiplex PCR assay were determined using standard templates. Species identification of larvae of noctuids collected from shoot-used bamboo forests was performed by the proposed multiplex PCR assay. Result: In the study, three species-specific multiplex PCR primers were designed for variation regions of COI gene in three noctuid species, and these primers were used in conjunction with the universal primer LCO1490 to amplify fragments of the COI genes of Kumasia kumaso, Oligia vulgaris, and Apamea apameoides with the sizes of 290, 390, and 590 bp, respectively. The optimized multiplex PCR was performed in a 20 μL reaction mixture including 10 μL 2× HotStart Taq PCR premix reagent, 0.5 μL (10 μmol·L^?1) each of primers LCO1490, JYE290, HYE390 and QTYE590, 1 μL DNA template, and added ddH₂O to 20 μL. The reaction conditions for the optimized multiplex PCR were 94 ℃ for 5 min followed by 35 cycles of 94 ℃ for 30 s, 59 ℃ for 30 s, and 72 ℃ for 30 s, followed by a final extension for 10 min at 72 ℃. The minimum detection limit of proposed multiplex PCR assay was 0.01 ng·μL^?1 for A. apameoides, and less than 0.001 ng·μL^?1 for K. kumaso and O. vulgaris. The identification results of larvae of noctuids collected from shoot-used bamboo forests showed that all 38 samples were able to amplify obvious specific bands, and the success rate of the multiplex PCR assay was 100%. Moreover, the species identified according to the technique in this study were completely consistent with the species identified by the COI gene sequence. Conclusion: A multiplex PCR assay for identification of larvae of noctuid species has been developed, which can quickly and efficiently identify noctuid species in shoot-used bamboo forests in Zhejiang Province. The proposed multiplex PCR assay has the advantages of less time consumption, high sensitivity, strong specificity and high accuracy.

Key words: Phyllostachys violascens ‘Prevernalis’, noctuid, primer, multiplex PCR, DNA barcoding