目的:建立可重复使用超声刀手术剪(简称超声刀)的清洗工艺确认方案,并对清洗有效性进行评价,为使用单位进行清洁方法确认和确保安全使用提供依据.方法:选择模拟血液污染物,按照确定的清洗工艺,选择最严苛的清洁方法挑战条件,通过对超声刀的可复用部件刀杆进行污染物负载-清洗数次后,提取残留污染物,并检测残留污染物中总蛋白和血红蛋白残留量.根据美国食品药品监督管理局(FDA)相关指导原则、美国医疗器械促进协会(AAMI)及美国材料和试验协会(ASTM)相关标准,设定可复用超声刀满足清洁要求的残留物限量指标(总蛋白残留量<6.4μg/cm2,血红蛋白残留量<2.2μg/cm2).结果:所建立的确认方案体系中,残留污染物的提取回收率均>79％;彻底提取验证研究中,单次提取回收率达到4次彻底提取回收的90％以上,满足实验要求.负载-清洗1次、4次、6次和8次清洗后,可重复使用超声刀总蛋白残留量均未检出,低于方法检测限(0.43μg/cm2);血红蛋白残留量均<0.34μg/cm2,低于确认方案体系的残留污染物中血红蛋白检测下限(1.10μg/cm2).结论:本研究建立的可复用超声刀手术剪清洁方法确认方案及清洁有效性评价方法;经验证,可重复使用超声刀8次,污染物负载-清洗后仍能满足所规定的清洁要求.

柯萨奇病毒A16(CV-A16)是小RNA 病毒科肠道病毒属的成员,是引起婴幼儿手足口病(H FM D )的常见病原体之一[1].CV-A16是含有约7 .4 kb基因组的正义单链RN A病毒,其基因组包含5'端非翻译区(5'U T R )、结构蛋白编码区P1、非结构蛋白编码区P2/P3及3'非翻译区(3'U T R ).P1编码4种结构蛋白(V P1、V P2、V P3、V P4 ) ,P2和P3分别编码7种非结构蛋白(2A、2B、2C、3A、3B、3C、3D )[2].随着研究的不断深入,在分子流行病学方面,基于V P1基因的完整序列,CV-A16可以在系统发育上分为A、B、C和D 4个基因组[3-6 ].基因组B可以进一步分为B1～B4这4 个基因亚型,其中B1 基因亚型还可以细分为B1a～B1d[3 ,7].在基础研究方面,利用反向遗传学技术构建感染性克隆[8 ] ,能够在体外对病毒基因组进行突变、缺失、嵌合等操作后,获得表型、性状发生变化的拯救病毒,为研究其基因的结构与功能、病毒复制机制、致病机制及筛选候选疫苗提供了崭新的途径.在疾病预防控制方面,单价肠道病毒71 型(EV-A71)疫苗无法预防其他病原体引起的 HFMD及其他相关疾病,应研发多价HFMD 疫苗[9] ,而EV-A71和CV-A16交替或共同流行可引发世界多个国家或地区HFMD暴发.本研究从CV-A16的分离、鉴定、系统进化分析、检测方法建立和全基因组克隆构建等方面进行了一系列研究,以期为C V-A 16 病毒的分子流行病学和基础研究提供有利的工具.

Objective:To establish and validate a crystal violet staining method for poliovirus (PV) titer detection.Methods:Based on the 50% cell culture infective dose (CCID 50) of PV, the cytopathic effect (CPE) of PV was determined by crystal violet staining and microplate reader.The accuracy, precision, linearity and robustness of the method were verified. Sixty-six lots of oral type Ⅰ type Ⅲ polio live vaccine were titrated by established crystal violet staining method and traditional microscope observation method, respectively. The correlation between the two methods was statistically analyzed by the Pearson’s correlation coefficient. The cell plate soaking solution was passed for 3 generations blindly, and the virus inactivation was determined by observing CPE of the culture and reverse transcription PCR (RT-PCR). Results:The recovery rates of 8.00, 6.00 and 4.00 lgCCID 50/ml PV samples were 98%-104% using the established method. The coefficients of variation (CVs) of intra-plate and inter-plate detection results were 0%-7.85%.The CVs of intraday and interday detection results were ≤6.44% and ≤5.27%, respectively. The CVs of detection results from different workers were≤5.10%.The linearity range of the method was 2.00-8.00 lgCCID 50/ml. The method had good robustness, and there was no effect on detection results of PV titer with different cell concentrations ( F=1.624, P=0.215) or incubation time ( F=2.731, P=0.055). The Pearson’s correlation coefficient 0.918 indicated that the detection results of the established method correlated with those of the microscope observation method. After the cell culture plate soaking solution was blindly passaged for 3 generations in Hep-2 cells, no CPE was observed in the culture, and RT-PCR result was negative. Conclusion:The crystal violet staining method has good safety, accuracy, precision, linearity and robustness, and is suitable for the virus titer detection of oral polio live vaccine.

目的 考察口服Ⅰ型Ⅲ型脊髓灰质炎减毒活疫苗在不同温度和pH环境下的稳定性.方法 将6批疫苗分别于2～8℃放置6个月、22～25℃放置7d、37℃放置7d、-20℃放置27、30、33个月,或反复冻融5次;将疫苗pH调至3.0～10.0.采用细胞培养半数感染量(cell culture infective dose 50％,CCID50)法检测病毒总滴度及分型滴度.结果 口服二价脊髓灰质炎减毒活疫苗在2～8℃保存6个月,22～25℃保存5d,37℃保存2d,反复冻融5次,-20℃保存27个月,pH3.0～10.0环境下,疫苗效力均符合质量标准.结论 疫苗的保存温度及时间对其稳定性均有影响.

Objective To establish the national reference for detecting infective titer of coxsackievirus A16 (CA16),in order to provide the national reference for viral titer control during CA16 vaccine production and immune effect evaluation of CA16 vaccine.Methods The qualified CA16 culture was divided into 0.5 ml aliquots as candidate reference.Candidate reference was collaboratively calibrated by 3 independent laboratories.The infective titer of CA16 was detected by cell culture method,and 50％ cell culture infective does (CCID50) of CA16 was calculated by Behrens-K(a)rber method.Meanwhile,CA16 candidate reference was stored at-60 ℃ for 12 months,at-20 ℃ for 6 months,at 4 ℃ for 28 days,at 22-25 ℃ (room temperature) for 7 days and at 37 ℃ for 7 days or frozen and thawed repeatedly,and the stability under different circumstances was evaluated.Results The viral titer of CA16 candidate reference was (6.80 ± 0.95) lgCCID50/ml.The viral titers of CA16 candidate reference had no significant reduction after keeping in storage at-60 ℃ for 12 months,-20 ℃ for 6 months,and 4 ℃ for 28 days,indicating high stability at these conditions.The viral titer loss rate of CA16 candidate reference at 22-25 ℃ (room temperature) and 37 ℃ were 3.4％ and 4.4％ per day respectively,and respective titer loss after 27 and 21 d storage was not significant.The viral titer loss rate of CA16 candidate reference was 4.8％ per time under repeated freeze-thaw condition.Conclusion CA16 candidate reference meets the requirement as a national reference,and its valuation was (6.80 ± 0.95) lgCCID50/ml.