| Objective (1) To establish a method that can inactivate virus using riboflavin plus UV light, and explore the influence of inactivation effects of riboflavin work concentrates and UV light dosage. (2) In this study, the new technique of PCR was investigated for its feasibility in the estimation of virus inactivation. (3) To explore PCR was consistency of cell-based infection assays in estimation virus inactivation effect. Metheds (1) Using human cytomegalovirus (HCMV AD169) as a model virus. The virus suspension and the ratio of platelet mixed by 1:9, Adding riboflavin (0, 50, 100, 150, 200μmol/L) to HCMV (7.25±0.15logTCID50) in PVC platelet conservation bag and then the mixture was exposed to ultraviolet light for 0, 600, 900, 1200, 1500 and 1800mJ/cm2, Mixture of riboflavin and virus suspensions treated with different concentration riboflavin concentration and illuminating time was collected and inoculated in single layer cells, at the same time,cell control , virus control , single riboflavin control, single UV light control and inactivated samples do so. And the virus titer was measured. The inactivation effects of UV light plus riboflavin for HCMV was studied. In this study, human cytomegalovirus (HCMV) was used as model virus. Riboflavin (0, 50, 100, 150, 200μmol/L) were added to HCMV (7.25±0.15logTCID50) in PVC platelet conservation bag which exposed to ultraviolet light (λ: 250~350nm) for 0, 600, 900, 1200, 1500 and 1800mJ/cm2, and then the virus titer was measured by infection experiments of HF. The inactivation effects of UV light plus riboflavin for virus was studied. According to the principle of orthogonal test to select the optimal conditions for inactivation. (2) 2 pieces of primer were designed according to the conservative region of UL83 gene in HCMV, which will be used to amplify the DNA fragments 547bp and 1505bp. At same time, the treated HCMV was inoculated on the vero cell. The CPE was observed and the virus titre was counted according to Reed-Muench. After HCMV was treated by riboflavin and UV light, the non-specificity damage was amplified in different fragments (547bp, 1505bp) by PCR method, and then human cytomegalovirus (HCMV), which was a model virus, have cell pathological effect (CPE) in vitro, were used as the target viruses. After replicated in vero cell line and treated by riboflavin and UV light, HCMV nucleic acids were extracted and analyzed by PCR. Results (1)The best inactivation of this method was screened by Orthogonal assay, With a riboflavin concentration of 150μmol/L and UV light intensity of 1500mJ/cm2, the titer of HCMV in blood bag could be reduced from 7.25±0.15logTCID50 to <0.50 logTCID50. (2) After HCMV was treated by riboflavin and UV light, The PCR results showed that the amplification of 1505bp DNA fragment was negative and the short DNA fragment of 547bp was amplified positive. (3) The amplification results by PCR are well corresponding to the results of cell-based infection assays. PCR technique can be used to estimate the effects of HCMV inactivation by the method. Conclusions (1) Riboflavin plus ultraviolet light could significantly inactivate pathogens in apheresis platelet concentrates. (2)The main influences of riboflavin plus ultraviolet light were the concentration of riboflavin and the intensity of UV light. (3) PCR technique probably was treated by some of inactivation useful method for detecting the infectivity of virus methods, although it has some limitations in use. It is helpful to solve the problem that some inactivated virus can not be evaluated directly with cell infection method. Objective (1) To establish a method that can inactivate germ using riboflavin plus UV light, and explore the work concentration of riboflavin and UV radiation dosage on the effect of inactivated bacteria. (2) To study the influences on PLT cell quality of disinfected apheresis platelet concentrates by riboflavin plus UV light. Metheds (1) In this study, Escherichia coli ATCC25922 and Staphylococcus epidermidis ATCC12228 were used as G- and G+ model bacteria respective. Adding Escherichia coli and Staphylococcus epidermidis to PCs According to the ratio of 1:100. Adding riboflavin(0, 50, 100, 150, 200μmol/L) to model bacteria in PVC platelet maintain bag which exposed to ultraviolet light (λ:250~350nm) for 0, 600, 900, 1200, 1500 and 1800mJ/cm2, and then the germ concentration was measured by bacteria count. Mixture of riboflavin and bacterium suspensions treated with different riboflavin concentration and illuminating time was collected. The inactivation effects of UV light plus riboflavin for bacteria was studied. The principle of orthogonal test to select the best conditions for inactivation. (2) As the best inactivated conditions, inactivating a high concentration (6logCFU/ml) and low concentration (2logCFU/ml) model bacteria in PCs. Counting the CFU after the high concentrations test group was treated, the treated low concentration test group placed in the platelet oscillations instrument for 5d, With Bact / ALERT 3D system for monitoring. (3) Detecting physiology and biochemistry index of platelet of these groups. Results (1) Escherichi coli and Staphylococcus epidermidis in blood bag were effectively inactivated with a riboflavin concentration of 150μmol/L and UV light intensity of 1500mJ/cm2. (2) The best inactivation of this method was screened by Orthogonal assay, Escherichi coli and Staphylococcus epidermidis could be killed 99.9993 and 100% respective. After the test group of low concentration bacteria were treated, they were placed the platelet oscillations instrument for 5d, and with BacT/ ALERT 3D microbial monitoring systems continue to monitor 5d were negative. (3)PLT cell quality was adequately maintained after treatment and during storage, but no significant difference and negative control P>0.05. Conclusions (1) Riboflavin plus ultraviolet light could significantly inactivate pathogens in apheresis platelet concentrates, Bact/ALERT 3D system can evaluation inactivation effect. (2) The main influences of Riboflavin plus ultraviolet light were the concentration of riboflavin and the intensity of UV light. PLT cell quality was adequately maintained after treatment and during storage, but this was not beyond accepted levels.
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