常用消毒剂对Sabin株脊髓灰质炎病毒的杀灭效果

目的  评价杀孢子剂、“84”消毒液、酸性苯酚盐、碱性苯酚盐对Sabin株脊髓灰质炎病毒的杀灭效果。方法  首先采用中和剂鉴定试验鉴定消毒剂的中和剂,然后通过悬液定量病毒杀灭试验比较4种消毒剂对Sabin株脊髓灰质炎病毒Ⅱ型的杀灭效果。杀灭对数值>4.0 lg半数细胞培养感染量（50% cell culture infective dose,CCID50）/ml,表示达到消毒效果。结果  中和剂鉴定结果表明,0.5% 硫代硫酸钠可有效中和杀孢子剂和“84”消毒液对病毒的杀灭作用,10%胰蛋白胨大豆肉汤培养基可有效中和酸性苯酚盐和碱性苯酚盐对病毒的杀灭作用。杀孢子剂和1.0%“84”消毒液分别与病毒作用30 min,平均杀灭对数值均>4.0 lgCCID50/ml,达到消毒效果。0.5%“84”消毒液、酸性苯酚盐、碱性苯酚盐分别与病毒作用45 min,平均杀灭对数值仍<4.0 lgCCID50/ml,没有达到预期的消毒效果。结论  杀孢子剂和氯含量较高的“84”消毒液对Sabin株脊髓灰质炎病毒的杀灭效果远大于氯含量较低的“84”消毒液以及酸性和碱性苯酚盐。     

清洁消毒新方法的建立及在腮腺炎疫苗车间的应用

 目的  以酸性/碱性苯酚盐作为常规消毒剂,在腮腺炎疫苗生产车间建立新的清洁消毒方法。方法  每月轮换使用酸性/碱性苯酚盐溶液进行擦拭,且每周使用杀孢子剂进行熏蒸。检测新消毒方法的消毒效果,并与原消毒方法进行比较。 结果  新方法的消毒效果好于原方法,使用新方法消毒后,腮腺炎疫苗车间的沉降菌检出率下降,而且腮腺炎疫苗单收液和原液的污染率分别降至0.1%和0.0%。  结论  新消毒方法可用腮腺炎疫苗生产车间的日常清洁消毒。     

疫苗生产洁净室消毒剂的验证

目的  根据消毒剂的实际使用方式,验证疫苗生产洁净室4种消毒剂的使用效果,为无菌生产控制提供依据和保障。方法  对于洁净室的日常表面消毒,采用2%84消毒液、0.1%新洁尔灭消毒液、1% Minncare杀孢子剂和75%乙醇进行实验室悬液定量杀菌试验、环境获得菌不锈钢载体杀菌试验以及材料耐受性确认。另外,用75%乙醇进行人员手部冲淋消毒现场确认,用84消毒液、新洁尔灭消毒液和杀孢子剂各连续3次消毒后进行洁净室静态环境微生物检测。结果  在悬液定量杀菌试验中,新洁尔灭消毒液和乙醇对枯草杆菌黑色变种芽孢菌悬液作用10和20 min、84消毒液作用10 min,杀灭对数值均<5.00,未达到标准;其余实验室试验结果均符合规定。在乙醇手部冲淋消毒现场确认中,所有人员五手指印微生物检测均为阴性。洁净室经连续3次84消毒液、新洁尔灭消毒液和Minncare杀孢子剂表面消毒后,静态环境微生物检测均为阴性。 结论  经过验证,以上4种消毒剂在预定使用条件下均有效,能够较好地控制洁净室的洁净度。     

制药企业常用消毒剂消毒效果验证

对酸性苯酚、碱性苯酚、75%乙醇、70%异丙醇、0.1%新洁尔灭、1.5%双氧水、杀孢子剂、1.5%雷克特合能8种消毒剂的消毒效果进行验证.结果表明,酸性苯酚、碱性苯酚、1.5%双氧水、杀孢子剂对微生物的杀灭能力较强.75%乙醇、70%异丙醇、0.1%新洁尔灭对白色念珠菌和枯草芽孢杆菌杀灭能力较弱,需和其它消毒剂配合使用.1.5%雷克特合能消毒剂对微生物的杀灭能力较弱.     

疫苗生产中使用的酸性和碱性苯酚对MRC-5细胞生长的影响

 目的   观察疫苗生产中使用的消毒剂酸性苯酚和碱性苯酚对MRC-5细胞生长的影响。 方法   将酸性苯酚（0.37%）和碱性苯酚（0.74%）分别加入MRC-5细胞培养瓶中,观察细胞生长形态,绘制细胞生长曲线,测定细胞贴壁率。分别采用t检验和x²检验对结果进行比较。 结果  加入酸性和碱性苯酚后,第7天的细胞数分别为6.65×10⁵和7.12×10⁵,与对照组（6.93×10⁵）相比,差异无统计学意义（t=0.476,P=0.654;t=0.334,P=0.752）;第8小时的细胞贴壁率分别为88.3%和96.0%,与对照组（90.3%）相比,差异亦无统计学意义（x²=0.765,P=0.659;x²=0.376,P=0.585）。 结论  微量酸性和碱性苯酚对MRC-5细胞的生长没有显著影响。     

水痘减毒活疫苗的无菌生产工艺验证

 目的  通过培养基模拟灌装试验,验证水痘减毒活疫苗（水痘疫苗）无菌生产工艺的有效性。方法  模拟水痘疫苗的无菌灌装工艺,对胰酪胨大豆肉汤培养基（TSB）按0.6 ml/瓶进行模拟灌装,对灌装后小瓶进行培养,根据小瓶内培养基的污染情况,评估现行水痘疫苗无菌生产工艺的有效性。结果  3次TSB模拟灌装试验的灌装批量分别为10 160、10 181和10 175瓶,经20～25 ℃和30～35 ℃先后培养后,均未出现TSB污染。在水痘疫苗生产区域的各监测点,≥0.5 μm悬浮粒子均≤70粒/m3,≥5.0 μm悬浮粒子均为0;沉降菌和表面微生物的菌落计数均为0;培养基灵敏度检查和微生物挑战试验均合格。结论  水痘疫苗的无菌生产工艺符合药品生产质量管理规范的要求,可确保产品的无菌性。     

冻干Vero细胞狂犬病纯化疫苗在120例门诊病人中的临床效果

目的:观察国产冻干非洲绿猴肾传代细胞(Vero细胞)为基质制备的狂犬病纯化疫苗临床应用的免疫效果和安全性。方法:选择门诊部就诊的犬致伤者240例,分为试验组和对照组。试验组接种冻干Vero细胞狂犬病纯化疫苗,对照组接种地鼠肾狂犬病纯化疫苗。2组人群均按照狂犬病疫苗暴露后常规免疫程序进行接种,于全程接种后15d测定中和抗体,并观察不良反应。结果:试验组抗体阳性率为96.7%,明显高于对照组的90.0%,P<0.05;不良反应发生率为10.8%,明显低于对照组的21.7%,P<0.05。结论:Vero细胞纯化疫苗抗体阳转率高、不良反应发生率低,临床应用优于地鼠肾纯化疫苗,适宜推广使用。     

免疫细胞化学灶试验检测麻疹-流行性腮腺炎-风疹三价疫苗的效力

作者应用免疫细胞化学染色灶试验分别测定麻疹、流行性腮腺炎和风疹三价疫苗(MMR)中每个疫苗的效力。用该法测定MMR中一种疫苗的效力不受其他两种疫苗影响,其结果与常规的蚀斑法和终点稀释法一致。本法避免了常规方法中在试验前必须用高效价特异抗血清中和另两利病毒的程序。作者用Vero细胞株测定麻疹和流行性腮腺炎疫苗病毒效价;用RK13细胞测风疹病毒效价。按10倍或3.16倍连续稀释疫苗病毒,取0.1ml病毒接种到生长于孔经为3.5cm的6孔细胞培养板中的单层细胞内,吸附1小时后,复盖MEM琼脂培养基。Vero细     

双价肾综合征出血热纯化疫苗(Vero细胞)在儿童和老年人中的免疫原性观察

目的    评价双价肾综合征出血热纯化疫苗(Vero细胞)在6～15岁儿童和>60岁老年人中的免疫原性。方法   对知情同意的500名6～15岁(儿童组)、16～60岁(成年人组)和>60岁(老年人组)的健康人群按2:1:2的人数比例非随机接种双价肾综合征出血热纯化疫苗(Vero细胞),其中以成年人组作为对照。用ELlSA和空斑减少中和试验测定儿童和老年人接种疫苗后的抗体阳转率和几何平均滴度(GMT)。结果 经ELISA测定,试验组(儿童和老年人)免疫后的抗体阳转率与成年人对照组的差异无统计学意义;老年人组与成年人组的抗体GMT差异无统计学意义,儿童组的GMT水平低于成年人组,差异有统计学意义,但GMT达到有效保护水平。经空斑减少中和试验测定,试验组的抗体阳转率和GMT与对照组的差异均无统计学意义。结论   疫苗对≥6岁的所有健康人群接种(禁忌证除外)均具有良好的免疫效果。     

不同培养基和胰蛋白酶对Vero细胞培养及消化效果的比较

目的 比较不同公司生产的无血清培养基对病毒性疫苗生产用Vero细胞培养效果,及基因工程胰蛋白酶的细胞消化效果,以判断是否适用。方法 实验组用VirusPro Vero-A培养基进行Vero细胞的培养,用Trpzyme消化细胞。对照组用VP-SFM培养基进行细胞培养,用TrypLE Select消化细胞。两组Vero细胞以相同密度接种在T175培养瓶中,以相同的培养条件和传代方法在T175瓶和细胞工厂中各培养3代。培养期间观察细胞的上清液、形态以及汇合度,消化后检测细胞活率并计算细胞收获量。采用配对t检验比较两组消化液的pH值、总消化时长、37 ℃消化孵育时长、细胞活率以及细胞收获量。结果 两组细胞生长状态均良好。配对t检验显示,实验组消化液的pH值（6.99）、总消化时长（17.28 min）和37 ℃消化孵育时长（6.93 min）均值均大于对照组消化液的（分别为6.75、12.34 min、3.30 min）,细胞活率（94.79%）及细胞收获量（T175瓶：3.91×10⁷个;细胞工厂：1.90×10⁹个）均值均高于对照组的（分别为90.20%、3.33×10⁷个、1.26×10⁹个）,且差异有统计学意义（t值分别为9.17、3.46、2.98、2.31和4.38,P值均<0.05）。结论 实验组无血清培养基培养效果较好,基因工程胰蛋白酶细胞消化液温和、细胞损伤小,均适用于Vero细胞。     

Method for testing aseptic technique of intravenous admixture personnel

A method of checking the aseptic technique of personnel that prepare i.v. admixtures is described. The following sterile dosage forms of trypticase soy broth (TSB) were prepared: (1) single-strength ampuls 5 ml, (2) six-times (6X) concentrated broth ampuls 10 ml, (3) lyophilized broth sufficient to make 5 ml single-strength broth in 10-ml vials, and lyophilized broth sufficient to make 10.6 ml 6X concentrated broth in 20-ml vials. Admixture personnel were told these were investigational products, and they were given simulated orders to prepare syringes and 50-ml piggyback admixtures of the products. The specimens containing the broth were intercepted by an investigator, incubated for seven days at 37 degree C, and observed for turbidity on days 2 and 7. To verify that the broth supported the growth of bacteria, two common contaminants were inoculated into the broth in the syringes and admixtures to serve as positive controls. At least 100 samples from each of four procedures were processed during the three-month study period. A total of 405 samples prepared by 13 individuals were tested. None of the samples became turbid; hence, the bacterial contamination rate was 0%. Among the positive controls, 98% inoculated with one species and 100% inoculated with the other became turbid. This method is now used for monitoring the aseptic technique of new admixture personnel. This unique method of checking the aseptic technique of personnel is accurate and cost effective, and it avoids the risk of adventitious contamination.     

洋葱伯克霍尔德菌群(Bcc)及干扰菌在不同增菌培养基中的生长研究

目的：考察Bcc以及Bcc与干扰菌的混合物在不同的增菌培养基中能否生长和增殖;探讨不同的 Bcc菌种对增菌培养基的营养需求和抗生素的耐受是否存在差异。方法：将Bcc的代表菌株、干扰菌以及 Bcc与干扰菌的混合物分别接种至TSB、10倍稀释的TSB、添加10 mg·L-1庆大霉素的TSB和添加250000 U·L-1多粘菌素B的TSB中培养,划线分离至选择性培养基上观察结果,并对可疑菌落进行分离和鉴定。结果：只接种Bcc的4种增菌培养基均能使之良好地增殖。低量的Bcc(约102 cfu)与高量的干扰菌 (104 cfu)的混合接种,除了神秘伯克霍尔德菌CMCC(B)23010外,4种增菌培养基能使Bcc增殖并被检出。结论：添加抗生素的TSB与未添加抗生素的相比没有表现出明显的优势。TSB可以作为Bcc检验时的增菌培养基使用。生长缓慢且活力弱的Bcc菌株在特定条件下有可能会被漏检。     

Effect of technetium Tc 99m pertechnetate on bacterial survival in solution

Survival of Staphylococcus epidermidis (10(2) organisms/ml) in solutions containing various levels of radioactivity was assessed. Six test preparations contained nonbacteriostatic 0.9% sodium chloride solution; four of these contained technetium Tc 99m pertechnetate (99mTcO-4) in various quantities (80, 250, 500, and 750 mCi). A fifth contained technetium that had decayed to an essentially nonradioactive form, and a sixth contained 0.9% sodium chloride solution only. Each of the six 20-ml solutions was inoculated with 2 ml of single-strength trypticase soy broth (TSB) containing 10(3) organisms/ml. At various times up to 12 hours after inoculation, 1-ml aliquots of each test solution were withdrawn and passed through 0.22-micron filters, thereby preventing further irradiation of the filtered organisms. The filters were incubated in single-strength TSB at 37 degrees C, and samples were examined for turbidity at 24, 48, and 72 hours. After 24 hours, 25 of the 36 sample tubes showed turbidity; after 48 hours, the turbid samples totaled 28. Bacteria in the two nonradioactive solutions remained viable throughout the 12-hour sampling period. Accumulated doses of radiation obtained in the 250-, 500-, and 750-mCi samples inhibited bacterial growth. To be a valid quality-control measure, sterility monitoring of prepared radiopharmaceutical dosage forms may need to be performed concurrently with their preparation.     

有限稀释法筛选轮状病毒LD9株纯化方法的建立及应用

目的:建立有限稀释法克隆纯化轮状病毒的试验方法,筛选出感染性强、遗传特性稳定的轮状病毒基因重配株LD9。方法:梯度稀释LD9毒种,用5个稀释度(10-4～10-8)的病毒感染铺满单层Vero细胞的96孔板细胞,培养6 d,-20℃冻融,培养物传代至24孔板继续培养6 d,显微镜下观察细胞病变(CPE),-20℃冻融后检测。以相同方法重复克隆纯化3次,筛选获得病毒滴度稳定、具有稳定遗传特性的纯化病毒,由T25、T75到2,4,10层细胞工厂逐级放大培养。结果:筛选出适用于疫苗生产用的遗传特性稳定、感染性强的LD9疫苗候选株。结论:建立了适用于轮状病毒克隆纯化的筛选方法。     

Degradation of organochlorine pesticides by meat starter in liquid media and fermented sausage.

The effect of meat starter on the degradation of DDT and lindane was investigated. The insignificant role of Lactobacillus plantarum in degrading p,p'-DDT and lindane presented in tryptone soya broth (TSB) and mineral salt medium (MSM) with or without 120 ppm nitrite was observed.The degradation of DDT and lindane by Micrococcus varians in TSB and MSM with or without nitrite were studied. The results indicated that DDT or lindane were degraded during the incubation period. The reduction in DDT at the end of the incubation period (15 days) was about 24.1 and 32.5% in TSB and MSM without nitrite, respectively. Corresponding values in the same media with nitrite were 37.5 and 46.4%. Regarding the reduction in lindane, it was recorded as 27.9 and 40.0% in TSB and MSM without nitrite, respectively and 38.4 and 48.4% in the same media with nitrite. The results indicated that culture media M. varians metabolized DDT mainly to DDD and lindane mainly to 2,4-, 2,5-, 2,6- and 3,4-dichlorophenol; 2,3,4- and 2,3,5-trichlorophenol; hexachlorobenzene; and pentachlorophenol. The effect of pesticides on the growth rate of meat starter was also investigated. The addition of DDT or lindane resulted in a slight decrease in counts of the strains during the initial incubation in TSB or MSM. Then the microorganisms recovered and began to grow logarithmically, but not as well as in a normal situation. The effect of fermentation stage by meat starter on DDT and lindane in fermented sausage was recorded. The results indicated that during the 72 h of fermentation, the reduction was 10 and 18% of DDT and lindane, respectively. These results confirmed that the fermentation process in meat products reduced pesticide residues and these reductions were due to the activity of meat starter.     

Development and characterization of a stable luciferase dengue virus for high-throughput screening

To facilitate dengue virus (DENV) drug discovery, we developed a stable luciferase reporter DENV-2. A renilla luciferase gene was engineered into the capsid-coding region of an infectious cDNA clone of DENV-2. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated high titers (>10⁶ PFU/ml) of luciferase reporter DENV-2. The reporter virus was infectious to a variety of cells, producing robust luciferase signals. Compared with wild-type virus, the reporter virus replicated slower in both mammalian Vero and mosquito C6/36 cells. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. All passaged viruses stably maintained the luciferase gene, demonstrating the stability of the reporter virus. Furthermore, we found that the passaged virus accumulated a mutation (T108M) in viral NS4B gene that could enhance viral RNA replication in a cell-type specific manner. Using the reporter virus, we developed a HTS assay in a 384-well format. The HTS assay was validated with known DENV inhibitors and showed a robust Z′ factor of 0.79. The Luc-DENV-2 HTS assay allows screening for inhibitors of all steps of the viral life cycle. The reporter virus will also be a useful tool for studying DENV replication and pathogenesis.     

国产与进口牛血清促进Vero细胞生长的比较

 目的  通过对国产与进口牛血清促进Vero细胞生长的比较,筛选可替代进口血清用于轮状病毒疫苗生产中Vero细胞培养的国产牛血清。方法  以国产的3批胎牛血清和1批新生牛血清为待评血清,以进口胎牛血清为对照血清,制备细胞培养液。在T175细胞培养瓶（T瓶）和2层细胞工厂中连续传代培养Vero细胞各3代,观察每一代次的培养上清液、细胞形态和细胞汇合度,计算细胞收获量和与对照血清相比较的相对增长率。采用单样本t检验对细胞收获量与生产要求的细胞产量（T瓶：>1.50×10⁷ 个/ml;细胞工厂：>3.00×10⁸ 个/ml）进行比较。根据细胞相对增长率判断国产血清与进口对照血清促细胞生长活性的相近程度。结果  国产血清培养的Vero细胞生长状态均良好。T瓶培养时,国产血清组的细胞收获量为（1.56～9.30）×10⁷ 个/ml,与生产要求细胞产量的差异有统计学意义（t=2.44～3.76,P<0.05）,且t值均>0,说明符合生产要求。细胞相对增长率接近或超过100%。细胞工厂培养时,国产血清组的细胞收获量为（1.80～4.92）×10⁸ 个/ml,与生产要求产量的差异无统计学意义（t=－0.23～1.16,P>0.05）,但是只有1批胎牛血清和新生牛血清的细胞收获量均值>3.00×10⁸ 个/ml,且细胞相对增长率>90%。 结论  经与进口牛血清比较,国产的1批胎牛血清和1批新生牛血清可替代进口血清,用于轮状病毒疫苗生产中的Vero细胞培养。