Real-time RT-RAA assay and primer selection

Fluorescent RT-RAA nucleic acid amplification kit (Jiangsu Qitian Gene Biotechnology Co., Ltd.) was used, including reverse transcription and DNA amplification enzymes. The total volume of RT-RAA reaction system is 50μL (Table ​(Table3).3). After the system was configured, it was immediately transferred to a fluorescence quantitative PCR instrument preheated to 39 °C. The denaturation temperature, annealing temperature and extension temperature were all set at 39 °C, and each cycle was 1 min and 30 cycles were set. Nuclease–free water was used as negative control.

RT-RAA reaction system.

Three forward primers and three reverse primers of RT-RAA were combined. From 1 to 9, they were F1/R1, F1/R2, F1/R3, F2/R1, F2/R2, F2/R3, F3/R1, F3/R2, F3/R3, F3/R3. And the combined primers were used for RT-PCR detection. The amplified strips were connected with pEASY-Blunt Zero Cloning Kit after glue recovery, and the ligation products were sent to Sangon Bioengineering (Shanghai) Co., Ltd for sequencing. Whether the alignment and sequencing results are GETV sequences. The combined 9 pairs of primers were used to detect GETV viral RNA, and the primers were screened by fluorescence value and peak time.