Nuclease free water

According to the specific DNA fragment generated by ISSR 827, qRT-PCR primers Qerc19F and Qerc19R were designed and synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). To verify the specificity of the primers, we used 10 sets of T. laevis DNA (100 ng/μL) and 3 sets of T. caries DNA (100 ng/μL) to perform qPCR. The reaction was carried out by using an ABI 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) machine. The reaction was performed in 20 μL, including 1 μL DNA, 0.4 μL Qerc19F (10 μM), 0.4 μL of Qerc19R (10 μM), 8.2 μL of nuclease-free water (TransGen Biotech, China) and 10 μl of 2 × TransStart Top Green qPCR SuperMix (+DyeI/+DyeII) (TransGen Biotech, China). The program settings were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 58°C for 30 s. Then, a series of 10-fold diluted plasmids (2.4 pg-0.24 fg) were used as templates. Three repeats for every concentration and 2 μL of nuclease-free water (TransGen Biotech, China) were used as controls for each repeat. The reaction system was the same as mentioned above.

The optimization of conditions for real-time qPCR method was carried out in triplicates using TransStart Probe qPCR SuperMix (TransGen Biotech, Beijing, China) on Mastercycler ep realplex (Eppendorf, Hamburg, Germany), based on the manufacturer's instructions. Different concentrations of the primers and probe were prepared into reaction tubes to optimize the assay by evaluating the highest fluorescence and lowest threshold cycle (C_T). The optimized reaction was carried out in a 20 μl reaction system containing 10.0 μl of supplied master mix, 0.4 μl of each primer (qPCMV-F, qPCMV-R and qPCMV-P, 10 μM each), 2 μl templates DNA, and 6.8 μl of Nuclease-free Water (TransGen Biotech, Beijing, China). The thermal profile for the real-time PCR was 94°C for 50s, followed by 40 cycles of 94°C for 5 sec, 60°C for 35 sec. The ten-fold dilution of plasmid from 5.19 × 10⁸ to 5.19 × 10¹ copies/μl was used as target DNA to generate the standard curve.

Neonicotinoid insecticides, i.e., imidacloprid (analytical standard 96%), and acetamiprid (analytical standard 97.5%) were obtained from Shandong Lianhe Chemical Co., Ltd., China. Technical piperonyl butoxide, PBO (analytical grade 95%) was purchased from Shanghai Macklin Biochemical Co., China.
Takara DNA Marker DL1000 and Taq^TM DNA Polymerase were obtained from Clontech Takara, China. The TransScript^® One-Step gDNA Removal and cDNA synthesis SuperMix, 6×DNA loading buffer, TransStart^® Top Green qPCR SuperMix, nuclease-free water and GelStain (10000×) were all provided by TransGen Biotech Co., Ltd. RNeasy^® Mini Kit and QIAquick^® Gel Extraction Kit was obtained from Qiagen, ON, Canada. The rest of the chemicals and reagents used in the study were of analytical grade.