噻唑橙光化学法对红细胞中病毒及细菌灭活作用的研究

Inactivation of virus and bacteria in red blood cell suspension by Thiazole orange photo-chemistry

目的探讨噻唑橙(TO)光化学法对红细胞中病毒及细菌灭活效果。方法以伪狂犬病毒(PRV)和辛德毕斯(Sindbis)病毒为指示病毒,分别加入红细胞比积为20%的红细胞悬液中,TO孵育1 h后做476 nm光照射处理;研究病毒灭活动力学特征;做TO浓度(C)、光照强度(I)、光照时间(T)三因素三水平正交试验确定最优灭活条件;最优条件下检测裸照、密闭血袋中、密闭血袋充氧后的病毒灭活效果;最优条件下检测血液污染常见细菌小肠结肠炎耶尔森氏菌、金黄色葡萄球菌及表皮葡萄球菌灭活效果。结果病毒灭活动力学研究显示:在60μmol/LTO、1.06E-01 w·m-2·nm-1光强条件下,照射5 min可将大部分PRV和Sindbis病毒灭活,20 min灭活作用达峰值,分别为5.88和5.12 LogTCID50;正交试验显示:裸照时PRV及Sindbis病毒灭活最优条件均为I=1.33E-01 w·m-2·nm-1,T=20 min,C=80μmol/L;在此条件下,可灭活红细胞中PRV≥6.13LogTCID50(裸照,n=4)、(4.75±0.62)LogTCID50(密闭血袋,n=4)、(6.06±0.16)LogTCID50(密闭血袋充氧,n=4);可灭活红细胞中Sindbis病毒(5.41±0.12)LogTCID50(裸照,n=4)、(3.72±0.77)LogTCID50(密闭血袋,n=4)、(5.76±0.25)LogTCID50(密闭血袋充氧,n=4)。在此条件下细菌灭活效果:小肠结肠炎耶尔森氏菌(5.91±0.13)Log10、金黄色葡萄球菌>6.8Log10、表皮葡萄球菌>6.0 Log10。结论 TO光化学法可有效灭活红细胞中病毒,有氧情况下(裸照或充氧)病毒灭活效果好于密闭无氧。TO光化学法可有效灭活红细胞中细菌。

Objective To evaluate the inactivation effect of thiazole orange（TO） photo-chemistry on virus and bacteria in red blood cell（RBC） suspension.Methods Pseudorabies virus（PRV） and Sindbis virus were used as indicator virus,and virus infected samples were prepared by adding PRV or Sindbis virus to RBC suspension（RBC）.Incubated the samples with TO 1 h and exposed them to 476 nm light.Viral inactivation kinetics was first studied.Then the optimal condition for virus inactivation was determined by orthogonal test,in which 3 factors were studied namely TO concentration（C）,light intensity（I）,irradiation time（T）.Finally,the inactivation effects of TO on virus were tested on naked samples,samples in bags,and oxygenated samples in bags under the optimal inactivation condition.Moreover,the inactivation effects of TO on bacteria were also tested,including Staphylococcus aureus,Staphylococcus epidermidis,Yersinia enterocolitica.Results Samples with 60 μmol/L TO exposed to 1.06E-01 w·m-2·nm-1 light for 5 min resulted in inactivation of most PRV and Sindbis virus in RBC suspension,and for 20 min,5.88 Log TCID50 PRV and 5.12 LogTCID50 Sindbis virus were inactivated.The optimal inactivation condition for both PRV and Sindbis virus was,I=1.33E-01w·m-2·nm-1,T=20 min,C=80 μmol/L.Under this condition,≥6.13 Log TCID50（naked samples,n=4）,（4.75±0.62）LogTCID50（samples in bags,n=4） and（6.06±0.16） LogTCID50（oxygenated samples in bags,n=4） PRV were inactivated;（5.41±0.12）LogTCID50（naked samples,n=4）,（3.72±0.77）LogTCID50（samples in bags,n=4） and（5.76±0.25）LogTCID50（oxygenated samples in bags,n=4） Sindbis virus were inactivated,respectively.Moreover,under this optimal condition,（5.91±0.13）Log10 Yersinia enterocolitica,〉6.8 Log10 Staphylococcus aureus,and 〉6.0 Log10 Staphylococcus epidermidis were inactivated.Conclusion TO photo-chemistry is an effective method for virus inactivation in RBC,and its effect is enhanced under oxygen condition compared with low oxygen closed bag;TO photo-chemistry is also an effective method for bacteria inactivation in RBC.