Reaction Systems of Real-Time Qpcr and RT-PCR

Supplementary Information

Reaction systems of Real-time qPCR and RT-PCR

Real-time qPCR was carried out using Premix Ex Taq (Probe qPCR) by following the manufacturer’s instructions (TaKaRa Biotech.; Dalian, China). The reaction volume (25 µl) contained 2 µl gDNA extracted from the genetically modified maize seeds, 12.5 µl of 2× Premix Ex Taq Mix, 400 nM sense primer, 400 nM antisense primer, 160 nM TaqMan probe, and 0.5 µl of ROX Reference Dye (50×). The program of qPCR detection was set as follows: 95°C for 1 min, and 40 cycles of 95°C for 5 s and 60°C for 30 s. The fluorescent signal was recorded at 60°C at the end of each cycle. The threshold cycle (Ct) was calculated automatically using SDS 2.4 software (Applied Biosystems; Foster City, CA, USA).

Real-time RT-qPCR was carried out using the One-step PrimeScript RT-PCR kit (Perfect Real-time; TaKaRa Biotech.; Dalian, China). The reaction volume (25 µl) contained 1× One Step RT-PCR Buffer III, 2.5 U of TaKaRaEx TaqHS, 0.5 µl of PrimeScript RT Enzyme Mix II, 200 nM sense primer, 200 nM antisense primer, 120 nM TaqMan probe, and 2 µl of RNA extracted from the non-genetically-modified soybean seeds infected with BPMV and TRSV. The thermal cycling was programed as follows: 42°C for 5 min, 95°C for 10 s, and 40 cycles of 95°C for 5 s and 60°C for 30 s. The fluorescence was recorded at the end of each amplification cycle. The Ct value was calculated automatically by the SDS 2.4 software.

Supplementary Table 1 Primers and probes used in this study

Namea / Sequence (5’-3’) / Target gene
CaMV35S FP / CGACAGTGGTCCCAAAGA / CaMV35S
CaMV35S RP / AAGACGTGGTTGGAACGTCTTC
CaMV35S Probe / TGGACCCCCACCCACGAGGAGCATC
NOS FP / ATCGTTCAAACATTTGGCA / NOS
NOS RP / ATTGCGGGACTCTAATCATA
NOS Probe / CATCGCAAGACCGGCAACAGG
BPMV FP / CTCTCCTTACATTAAATGTACAGTGTCTT / BPMV
BPMV RP / AACTTATTCCTGAATTTCTCCAAACT
BPMV Probe / TTCATAGCATTTGGAAATTTGGCTGATGA
TRSV FP / GAGTTTATTGTACATATAGATACCTGGCG / TRSV
TRSV RP / CGAAGTCATGAATGTATCCAGG
TRSV Probe / TGACTCTCAGGTGCATCCTCCCATGTT

a: TaqMan probes, CaMV35S probe, NOS probe, BPMV probe, and TRSV probe, were labeled with FAM and BHQ1 at both terminals.

PCR amplification of 18S rDNA from nucleic acid extracted from soybean seed

PCR was performed in a 20-µl volume containing 2 µl of template, 10 µl of Premix Taq (TaKaRa Taq™ Version 2.0 plus dye; TaKaRa Biotech., Dalian, China), 1.0 µl of N-NS1 (10 mM; GTA GTC ATA TGC TTG TCT C), 1.0 µl of C-18H (10 Mm; GCC CTT CCG TCA ATT CCT TTA AGT TTC AGC). PCR was carried out by using the ABI 2720 PCR system involving the following steps: initial denaturation at 95oCfor 5 min; 35 cycles of 95 oC for 30 s, 49 oC for 30 s, 72 oC for 2 min; final extension at 72◦C for 10 min. Ten microliter of product was determined by 1% agarose gel electrophoresis.