目的:将牡蛎与富血小板—白细胞凝胶进行结合,并对该材料的生物安全性进行初步评估;方法:将牡蛎的形状进行重塑并使用相应的化学试剂进行脱脂脱蛋白处理,使用时采取进一步的灭菌处理。选取成年的新西兰大耳兔5只,采用抗凝血管抽取血液约...目的:将牡蛎与富血小板—白细胞凝胶进行结合,并对该材料的生物安全性进行初步评估;方法:将牡蛎的形状进行重塑并使用相应的化学试剂进行脱脂脱蛋白处理,使用时采取进一步的灭菌处理。选取成年的新西兰大耳兔5只,采用抗凝血管抽取血液约3 m L,使用3200r/min转速离心处理15min,取上清液约1 m L与含10%氯化钙的凝血酶溶液约0. 2 m L同时注入牡蛎小孔处,完成富血小板—白细胞凝胶/牡蛎复合型人工骨替代物的制作。按照国家医疗器械与有机材料的生物安全性评估方法制成该材料的浸提液。使用浸提液通过热原实验、溶血实验对富血小板—白细胞凝胶/牡蛎复合型人工骨替代物的生物安全性进行初步评估。结果:热原实验中实验兔的温度升高值分别为0.3℃、0.4℃、0.7℃、0.2℃、0.2℃;溶血实验中该材料的溶血率约为1%。结论:富血小板—白细胞凝胶/牡蛎复合型人工骨替代物无溶血作用及无致热源的存在,说明具有较为良好的生物安全性。展开更多
AIM To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus(HCV).METHODS In patients with HCV infection, resistance-associated amino acid...AIM To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus(HCV).METHODS In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80 K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. Light Cycler methods, based on real-time PCR with sequencespecific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest. RESULTS Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10%(mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80 K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80 K was detected in 14.6% of G1 a patients and 0% of G1 b in our setting. CONCLUSION A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single po展开更多
文摘目的:将牡蛎与富血小板—白细胞凝胶进行结合,并对该材料的生物安全性进行初步评估;方法:将牡蛎的形状进行重塑并使用相应的化学试剂进行脱脂脱蛋白处理,使用时采取进一步的灭菌处理。选取成年的新西兰大耳兔5只,采用抗凝血管抽取血液约3 m L,使用3200r/min转速离心处理15min,取上清液约1 m L与含10%氯化钙的凝血酶溶液约0. 2 m L同时注入牡蛎小孔处,完成富血小板—白细胞凝胶/牡蛎复合型人工骨替代物的制作。按照国家医疗器械与有机材料的生物安全性评估方法制成该材料的浸提液。使用浸提液通过热原实验、溶血实验对富血小板—白细胞凝胶/牡蛎复合型人工骨替代物的生物安全性进行初步评估。结果:热原实验中实验兔的温度升高值分别为0.3℃、0.4℃、0.7℃、0.2℃、0.2℃;溶血实验中该材料的溶血率约为1%。结论:富血小板—白细胞凝胶/牡蛎复合型人工骨替代物无溶血作用及无致热源的存在,说明具有较为良好的生物安全性。
基金Supported by Instituto de Salud Carlos III,No.PI13/00456,No.PI15/00829,No.PI15/00856,and No.PI12/01893 cofinanced by the European Regional Development Fund(ERDF)the Miguel Servet program of the Instituto de Salud Carlos III,No.CP14/00121 cofinanced by the ERDF+1 种基金Gilead,No.GLD14/00296Instituto de Salud Carlos III,CIBERehd(Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas)
文摘AIM To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus(HCV).METHODS In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80 K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. Light Cycler methods, based on real-time PCR with sequencespecific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest. RESULTS Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10%(mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80 K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80 K was detected in 14.6% of G1 a patients and 0% of G1 b in our setting. CONCLUSION A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single po
