RESUMO
【Objective】 To evaluate the ability of different disinfection methods to remove nucleic acid pollution in 2019-nCoV so as to obtain the best removal scheme. 【Methods】 2019-nCoV positive quality control nucleic acid of 50 μL was applied to plastic， metal and glass with medical cotton swabs， respectively. After drying， we dropped 50 μL of 750 mL/L alcohol （ethanol）， chlorine-containing disinfectant （2 000 mg/L and 5 500 mg/L）， and PCR Cleaner， respectively. After 1 min， the contaminated area was wiped with medical cotton swabs and soaked in 300 μL of pure water. After shaking and mixing， 5 μL was taken as a template. The Ct values of ORF1ab and N genes and IC genes of internal standard fragment in the amplified target area of 2019-nCoV after wiping with different disinfection methods were compared to evaluate the effect of eliminating nucleic acid pollution， and each experiment was repeated for three times. Similarly， the effects of ultraviolet irradiation for 0， 1， 2， 3， 4， and 5 hours on the removal of nucleic acid pollution were compared. 【Results】 After 2 000 mg/L and 5 500 mg/L chlorine-containing disinfectant wiped the contaminated area， the Ct values of ORF1ab and N genes and IC genes of internal standard fragment in the amplified target area in 2019-nCoV were all 0， and the Ct values of all genes in the contaminated area in groups 3， 4 and 5 h after UV irradiation were all 0， which completely cleared the pollution and had a strong effect. The effect of PCR Cleaner was second， and 750 mL/L ethanol was the worst. 【Conclusion】 2 000 mg/L and 5 500 mg/L chlorine-containing disinfectant and ultraviolet irradiation for 3 hours have the best effect of eliminating nucleic acid pollution， which is worth popularizing under appropriate conditions.
RESUMO
Agrobacterium tumefaciens-mediated transformation system has been widely applied. However, the function of target gene is affected by multiple factors. With this system, we obtained a transgenic rice line CX8621 carrying the bacterial blight resistance gene Xa21. In previous work, we have confirmed that it was selectable maker-free and vector backbone-free. And after 16 generations of breeding, it still maintained perfect resistance to bacterial blight disease. On this basis, we analyzed the integration and expression of Xa21 in CX8621 at the present study. First, based on the border sequences of plasmid pBXa21 and Xa21, we designed nested primers and assured the integrity of Xa21 in CX8621. Second, we cloned the flanking sequences and located Xa21 on chromosome 2 using improved Tail-PCR. Then we analyzed the expression pattern of Xa21 in several tissues and at different developmental stages by RT-PCR. The results show that Xa21 can be stably expressed in CX8621, agreeing well with the disease resistance response as reported previously. In addition, we detected the protein levels of XA21 in CX8621 with antibody of natural XA21 protein. Surprisingly, no XA21 protein was detected in the seeds of CX8621. Thus, the integration and expression analysis of Xa21 in CX8621 provided a part of scientific evidences for the safety assessment of genetically modified rice.