Establishment of real-time PCR methods to identify Anopheles sinensis and Anopheles lesteri
Author of the article:SHU Huang-Fang, WANG Ke-Yi, WU De, LIN Rong-Xing, LU Wen-Cheng, RUAN Cai-Wen, DENG Zhuo-Hui, ZHANG M
Author's Workplace：Guangdong Provincial Center for Disease Control and Prevention, Guangzhou 511430, China
Key Words：Anopheles sinensis; Anopheles lesteri; TaqMan probe; Real-time fluorescence quantitative PCR
Abstract：
[Objectives] To develop a real-time PCR technique to identify Anopheles sinensis and Anopheles lesteri. [Methods] Samples of each species were obtained and identified using both morphological methods and gene sequencing. Positive plasmid quality control products were synthesized and cloned, and the rDNA-ITS2 sequences of A. sinensis and A. lesteri were downloaded from the NCBI database. Prime 3 software was used to design primers and probes for each species online, and the minimum detection limit, sensitivity, specificity and repeatability of the PCR method were verified. [Results] Three hundred and eighty-three mosquitoes were collected and identified to 4 species; 159 A. sinensis, 104 A. lesteri, 60 Culex pipiens quinquefasciatus and 60 Aedes albopictus. The minimum detection limit of qPCR for A. sinensis and A. lesteri was 31.5 copy/response and the linear correlation coefficient (R2) of the standard curve was 0.99. The results of a repeatability test showed that intra and inter-group coefficients of variation for A. sinensis and A. lesteri were < 2%. [Conclusion] A TaqMan probe, real-time, fluorescent, quantitative PCR method with good sensitivity and specificity was developed to identify A. sinensis and A. lesteri.