To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes.
Amino Acid Sequence ; Base Sequence ; DNA ; genetics ; DNA Restriction Enzymes ; genetics ; DNA-Directed DNA Polymerase ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; methods ; Plasmids ; Receptors, Polymeric Immunoglobulin ; genetics

Objective:To summarize the scientific research management response strategies and practices of designated hospitals under the prevention and control of new coronary pneumonia, provide reference for the scientific and technological management of public health emergencies in domestic hospitals.Methods:Take the hospital scientific research management response under the prevention and control of the new coronavirus pneumonia epidemic in 2020 as an example, discuss and summarize the response strategies and mechanism establishment, work practice and existing problems of designated hospitals in the research management of prevention and control of public health emergencies represented by COVID-19 from the aspects of management system, resource integration, project operation, and scientific and technological service.Results:As a designated hospital of COVID-19 in Beijing, Youan hospital has actively carried out scientific and technological research on epidemic prevention and control while successfully completing patient treatment, established a scientific and efficient scientific and technological research management system, and integrated resources to form an efficient project operation mechanism, Providing a full range of scientific management services, providing policy and material guarantee for the rapid and high-quality completion of scientific and technological research on epidemic prevention and control.Conclusions:The scientific and technological research on public health emergencies has the characteristics of demand-driven, focused on key points, collaborative research, scientific decision-making, In order to carry out the research work smoothly, it is particularly important to form a benign interaction, straighten out the system, do a good job in long-term planning and layout, and establish a scientific and efficient research and management mechanism.

OBJECTIVE： To systematically evaluate the mode of personnel training in pharmacy intravenous admixture services （PIVAS） in China， and to provide reference for the comprehensive training of pharmacist in PIVAS in China. METHODS： PubMed， Embase， Cochrane Library， CBM， CJFD， VIP and Wanfang database were searched from the establishment of database to Sept. 2018. Studies which evaluated the training mode of PIVAS in China were included， and the results were presented by descriptive analysis in respects of training objects， training objectives， contents and evaluation indicators. RESULTS： A total of 5 literatures were included. The research types were 2 before-after control studies， 2 experience sharing studies and 1 review. 3 subjects were pharmacists， 1 subject was clinical pharmacists， and 1 subject was nurses. The training objectives were comprehensive quality training， clinical rational drug use level， pharmacy personnel training path and professional service ability. The specific training content of the training mode varied greatly， including professional theoretical knowledge， practical operation ability， pre-job training， professional psychological quality， professional ethics and laws and regulations， continuing education learning ability， career development planning and teaching ability. There were great differences in the evaluation indicators of training effectiveness， which were mainly reflected in team execution motivation and creativity， discoveny rate of unreasonable doctor’s advice， work efficiency， service quality， drug treatment level and satisfaction of PIVAS， etc. CONCLUSIONS： There are certain differences in the training objectives， training targets， specific contents and evaluation indicators of the PIVAS pharmacist training model in China. It is necessary to use the evidence- based method to construct the training mode for PIVAS pharmacist to provide support for clinical intravenous drug use.

OBJECTIVE： To systematically evaluate current status of charges in pharmacy intravenous admixture services （PIVAS）， and to provide reference for the formulation of China’s pharmacy intravenous admixture services （PIVAS） charging standards. METHODS： Retrieved from PubMed， Embase， Cochrane library， CBM， CNKI， VIP， Wanfang database and related goverment websets， the literatures about current status evaluation of charges in PIVAS of China were collected during the establishment of database to Jan. 2019. Cost estimation， charge standard， influential factors and other indicators were collected， and the results were presented by descriptive analysis. RESULTS： A total of 5 literatures were included， all of which were reviewed. According to the existing literatures， except for Shandong， Guangdong and Yunnan provinces， there were no regional charge standards in other provinces （districts and cities）. The cost estimation methods of PIVAS in these three provinces were basically the same. The cost could be obtained by adding up the business fees， labor fees， fees of medical instruments purchase and use， indirect fees etc. Dispensing charges in PIVAS were 3-5 yuan per piece for general drug， 5 yuan per piece for antibiotics and 8-12 yuan per piece for cancer chemotherapeutics， 20-35 yuan per piece for TPN. The charging level was mainly affected by local prices， PIVAS scale， hardware investment， management and other factors. CONCLUSIONS： There is no unified charging standard for PIVAS in most provinces （districts， cities） of China. The cost estimation methods of the hospitals from the included literatures are basically the same. It is necessary to construct national PIVAS charging standard and cost estimation method， which could provide a basis for formulating the price of medical and health services.

OBJECTIVE： To systematically evaluate the status quo of cost estimation in pharmacy intravenous admixture services （PIVAS）， and to provide cost basis for the construction of PIVAS in China. METHODS： Retrieved from PubMed， Embase， Cochrane library， CBM， CNKI， CSJD and Wanfang database from database establishment to Jan. 2019， the studies about the status quo of cost estimation in PIVAS of China were included. The descriptive analysis was conducted for content and method of cost estimation， infection to hospital. RESULTS： A total of 17 literatures were included， involving 8 before and after control studies， 6 experience sharing studies and 3 reviews. Existing reports showed that the estimation contents and methods of PIVAS cost were roughly the same. The cost included manpower， medical and health materials， fixed asset purchase， depreciation， repair costs， medicine cost and indirect costs. At the same time， the infection to hospital were reported， such as in manpower adopting， formulating detailed management measures and systems， concurrent allocation of the same kind of drugs， shortening infusion preparation and replacement time， in order to save manpower cost. CONCLUSIONS： PIVAS cost calculation method is roughly the same in some hospitals， but there is no uniform standard. It is necessary to further improve the PIVAS cost measurement standard and provide a basis for the construction and development of PIVAS in China.