流感裂解疫苗生产中沙门菌和微生物限度的监控研究

通过对2008年流感裂解疫苗生产中的监控,结果显示,流感病毒接种鸡胚尿囊液中未检出沙门菌,除菌过滤前微生物限度均小于10CFU/ml、细菌内毒素均小于25EU/ml,纯化过程去除了99%的杂蛋白。表明生产中使用的健康鸡胚尿囊液是符合生物制品规程的要求,现行生产工艺对微生物限度和细菌内毒素的控制是有效的,现行纯化过程对病毒液的纯化是有效的。     

硅藻土、珍珠岩微生物限度检查方法的探讨

文章尝试对助滤剂微生物计数方法和控制菌大肠埃希菌检查法进行适用性试验,建立助滤剂微生物限度检测方法,对血液制品生产过程中添加的助滤剂进行微生物控制。具体通过采用涂布法、倾注法、薄膜过滤法、涂布（分解求和）法对珍珠岩和硅藻土进行微生物回收试验,计算各试验菌回收率;用控制菌检查法对控大肠埃希菌检查法进行确认。结果发现涂布法各试验组菌落数减去供试品对照组菌落数的值与菌液对照组菌落数的比值均在0.5～2.0范围内,微生物计数方法通过;控大肠埃希菌检查法通过。     

表达绿色荧光蛋白重组新城疫病毒 LaSota疫苗株的构建

新城疫病毒是理想的新型活病毒疫苗载体,具有巨大的优势和应用前景。采用生产实践中广泛应用、免疫效果良好的NDV LaSota弱毒疫苗株,建立了反向遗传操作系统。在此基础上,进一步构建了表达绿色荧光蛋白(GFP)的重组NDV基因组cDNA克隆,成功救获了重组病毒rLaSota-EGFP,病毒F1代尿囊病毒液按1×104EID50接种9~10日龄SPF鸡胚尿囊腔,接种后分别于24h、48h、72h及96h收获尿囊液,检测平均HA滴度分别为28、210.3、211.3和211,每mL尿囊液病毒量EID50分别为108.64、109.22、109.21和109.64,重组病毒与亲本株生长滴度在相近时间达到峰值,生长动力学特性与亲本株无明显差异。各代次重组病毒按1×106EID50病毒量接种9~10日龄SPF鸡胚,96h内完全不致死鸡胚。救获重组病毒保持了LaSota弱毒疫苗亲本毒株对鸡胚良好的高滴度生长适应和低致病特性,并且鸡胚连续传9代次仍保持GFP的稳定表达及生物学特性不变。重组病毒rLaSota-EGFP的成功救获为开展新城疫病毒活载体疫苗研制提供了可行的技术平台。     

血吸附法浓缩NDV病毒及与其他几种方法的比较

用人O型红细胞吸附－释放病毒的方法纯化被感染的鸡胚尿囊液中的新城疫病毒（NDV）,然后分别对纯化的病毒液及弃去的上清进行血凝检测。结果表明,使用1％的红细胞悬液只能吸附部分病毒,损失较大,只有将红细胞悬液的浓度增加到20％时,才基本将病毒全部吸附住。同时还比较了差速离心,PEG沉淀,PEG包埋等方法。     

表达绿色荧光蛋白重组新城疫病毒 LaSota疫苗株的构建

新城疫病毒是理想的新型活病毒疫苗载体,具有巨大的优势和应用前景。采用生产实践中广泛应用、免疫效果良好的NDV LaSota弱毒疫苗株,建立了反向遗传操作系统。在此基础上,进一步构建了表达绿色荧光蛋白（GFP）的重组NDV基因组cDNA克隆,成功救获了重组病毒rLaSota_EGFP,病毒F1代尿囊病毒液按1×10.4EID50接种9～10日龄SPF鸡胚尿囊腔,接种后分别于24h、48h、72h及96h收获尿囊液,检测平均HA滴度分别为2.8、2 10.3、2 11.3和2 11,每mL尿囊液病毒量EID50分别为108.64、109.22、109.21和109.64,重组病毒与亲本株生长滴度在相近时间达到峰值,生长动力学特性与亲本株无明显差异。各代次重组病毒按1×10.6EID50病毒量接种9～10日龄SPF鸡胚,96h内完全不致死鸡胚。救获重组病毒保持了LaSota弱毒疫苗亲本毒株对鸡胚良好的高滴度生长适应和低致病特性,并且鸡胚连续传9代次仍保持GFP的稳定表达及生物学特性不变。重组病毒rLaSota_EGFP的成功救获为开展新城疫病毒活载体疫苗研制提供了可行的技术平台。     

止痛透骨贴微生物限度检查方法的验证

目的:建立适合于止痛透骨贴微生物限度检查的方法。方法:参照《中国药典》2010版一部微生物限度检查法验证试验的要求,采用中和-离心薄膜过滤法进行细菌数检查,采用平皿法进行霉菌和酵母菌数检查,采用直接接种法进行控制菌检查。结果:5种验证菌株的回收率均高于70%,控制菌检查经方法验证,可用直接接种法对金黄色葡萄球、铜绿假单胞菌、大肠菌群进行检查。结论:建立的方法准确可靠,适用于止痛透骨贴的微生物检查。     

花蛇解痒胶囊微生物限度检查方法学验证试验研究

目的:建立花蛇解痒胶囊微生物限度检查方法。方法:采用培养基稀释法和常规法。结果:采用常规法验证,大肠埃希菌、金黄色葡萄球菌、白色念珠菌、黑曲霉回收率均能达到70%以上,采用培养基稀释法验证枯草芽孢杆菌回收率能达到70%以上,控制菌可检出大肠埃希菌、大肠菌群、沙门菌。结论:花蛇解痒胶囊微生物限度检查,可采用培养基稀释法检查细菌数、常规法检查霉菌和酵母菌数;控制菌采用常规法和稀释法。     

从SARS患者血清发现双相型深部真菌的报告

目的从临床诊断的SARS病人血清中分离病原微生物。方法采用SARS患者血清通过NIH小鼠腹腔、脑和鸡胚卵黄囊和尿囊接种、人胚肺细胞呼吸道病毒培养和系列细菌培养分离病原体,所获微生物经形态学、血清学、生化学和法国梅里埃(Vitek)全自动微生物系统鉴定,复制动物模型和病理学检查。结果从SARS病人血清分离的微生物,检定结果为一种双相型深部嗜肺性真菌,属流行性肺组织胞浆菌(球状酵母菌)。结论分离的肺组织胞浆菌(球状酵母菌)是呼吸系统深部真菌。     

重配技术构建高产H1N1型流感疫苗病毒株

目的以经典重配技术制备高产H1N1流感疫苗病毒株。方法以野生型A1/云南昆明/03/2009(H1N1)作为HA及NA基因的供体株,以WHO疫苗株A/Perth/16/2009(H3N2)作为高产基因供体株,共同感染SPF鸡胚,经抗H3及抗N2血清中和筛选法及终末稀释法筛选高产重配H1N1病毒。结果获得一株重配H1N1流感病毒株,病毒血凝滴度为1∶4 096,病毒滴度为7.8 lg EID50/mL,显示为鸡胚高产病毒株;血凝抑制结果为1∶1 024,单向免疫扩散试验结果为阳性,证明抗原性与野生株一致;基因测序结果表明重配株的HA及NA基因序列与野生株序列一致。结论构建了高产重配H1H1流感疫苗病毒株,并应用经典重配技术建立了制备高产流感疫苗病毒株的技术平台。     

喘咳安丸微生物限度检查方法的验证

目的建立咳喘安丸的微生物限度检查方法。方法按中国药典2010年版一部微生物限度检查方法,用常规法、培养基稀释法进行方法验证。结果常规法试验时,大肠埃希菌、白色念珠菌和黑曲霉的回收率均高于85%,而枯草芽孢杆菌与金黄色葡萄球菌采用培养基稀释法试验时,使其回收率均高于80%。控制菌采用常规法检出。结论细菌数测定采用培养基稀释法(0.2mL/皿),霉菌、酵母菌及控制菌可采用常规法进行咳喘安丸的微生物限度检验。     

添加有扩增内标的沙门氏菌荧光定量PCR 检测体系的建立与评价

【目的】建立添加有扩增内标(IAC,Internal amplification control)的沙门氏菌EvaGreen荧光定量PCR检测体系,提高PCR检测可靠性。【方法】通过比较已有沙门氏菌属细菌的基因组序列,筛选沙门氏菌属特异检测靶点,设计特异引物;再用复合引物法构建扩增内标,优化参数,建立沙门氏菌内标PCR检测体系,利用特异性和灵敏度实验评价体系的检测性能。【结果】筛选得到的新特异靶点基因编码III型分泌系统蛋白(ssaQ)。针对该基因设计特异引物(SsaQ6),建立了添加有扩增内标的常规PCR和EvaGreen荧光定量PCR检测体系;二者对151株沙门氏菌和34株非沙门氏菌的检测符合率均达100%,对基因组DNA的检测下限达14.9拷贝/PCR和2.76拷贝/PCR;人工污染牛奶样品(初始染菌量:4-6 cfu/10 mL),増菌10 h和8 h后分别可检出沙门氏菌。【结论】本研究发掘的新靶点基因ssaQ特异性强,基于这一新靶点建立的添加有扩增内标的EvaGreen荧光定量PCR比常规内标PCR的检测限更低,重复性更好,快速方便,在12 h内即可得出检测结果,并且定量准确,有利于推进沙门氏菌PCR检测方法的标准化应用。     

Loop-mediated isothermal amplification for the rapid detection of Salmonella

Loop-mediated isothermal amplification (LAMP) assay detected Salmonella within 60 min. The 220 strains of 39 serotypes of Salmonella subsp. enterica and 7 strains of Salmonella enterica subsp. arizonae were amplified, but not 62 strains of 23 bacterial species other than Salmonella. The sensitivity of the LAMP assay was found to be >2.2 cfu/test tube using nine serotypes. The specificity was similar to that of a PCR assay, but the sensitivity of LAMP was greater. Both fluorescence and turbidity were able to detect the products in the LAMP assay. S. enteritidis in a liquid egg sample artificially inoculated with the organism was detected by the LAMP assay at 2.8 cfu/test tube, although negative by PCR assay. These results indicate that the LAMP assay is a rapid, specific and sensitive detection method for Salmonella.     

Development of calcium reabsorption by the allantoic epithelium in chick embryos grown in shell-less culture

The allantoic sac of the chick embryo functions as a primitive urinary bladder, storing and modifying the excretory fluid produced by the embryo. We have used chick embryos grown in shell-less culture to study the in situ handling of Ca2+ by the allantoic epithelium. Between Days 8 and 13 of incubation (38 degrees C, 5% CO2), the [Ca2+] of the allantoic sac fluid declines from about 1.5 mM to less than 0.3 mM, with most of this Ca2+ reabsorption occurring between Days 10 and 11. In 13-day-old embryos, the allantoic epithelium reabsorbs within 24 hr 85-92% of 45Ca2+ injected into the allantoic sac, while in 9-day-old embryos 45Ca2+ reabsorption is less than 40% by 24 hr. This is evidence for the developmental onset of a Ca2+ reabsorption process in the allantoic epithelium. The allantoic fluid Ca2+ is reabsorbed into the embryo's blood in which the serum [Ca2+] is about 1.5 mM. Also, electrical potential profiles reveal that the serosal (mesenchymal) side of the allantoic epithelium is 15-30 mV positive compared to the mucosal (luminal) side. Thus, by electrochemical criteria this reabsorption process appears to be active.     

Survival of Salmonella in peanut butter and peanut butter spread

In 1996, the first documented outbreak of salmonellosis associated with the consumption of peanut butter was reported. This study was undertaken to determine survival characteristics of high (5.68 log10 cfu g(-1)) and low (1.51 log10 cfu g(-1)) inocula of a five-serotype mixture of Salmonella in five commercial peanut butters and two commercial peanut butter spreads. Populations in samples inoculated with 5.68 log10 cfu g(-1) and stored for 24 weeks at 21 or 5 degrees C decreased 4.14-4.50 log10 cfu g(-1) and 2.86-4.28 log10 cfu g(-1), respectively, depending on the formulation. The order of retention of viability was: peanut butter spreads > traditional (regular) and reduced sugar, low-sodium peanut butters > natural peanut butter. Differences in rates of inactivation are attributed to variation in product composition as well as size and stability of water droplets in the colloidal matrix, which may influence nutrient availability. With the exception of natural peanut butter, products initially inoculated with 1.51 log10 cfu of Salmonella g(-1) (32 cfu g(-1)) were positive for the pathogen after storage for 24 weeks at 5 degrees C. At 21 degrees C, however, with the exception of one peanut butter spread, all products were negative for Salmonella after storage for 24 weeks. Post-process contamination of peanut butter and spreads with Salmonella may to result in survival in these products for the duration of their shelf life at 5 degrees C and possibly 21 degrees C, depending on the formulation.     

Evaluation of RapidChek Select for the screening of Salmonella in meat and meat products

The efficacy of the RapidChek Select, an alternative rapid method based on lateral flow technology, for the screening of Salmonella in meat and meat products, was compared with the current ISO reference culture-based method. Of the 265 routine samples examined, 61 were found to be positive for Salmonella by both methods. The percentage of agreement between the results of two methods was determined as 98%. All presumptive positive results obtained by the RapidCheck Select were confirmed to be positive by ISO method. For five samples ISO method gave positive result, while RapidChek Select gave negative result. The limit of detection (LOD(50)) of RapidChek Select and ISO methods for minced beef meat samples were 1.00 cfu/25 g and 0.63 cfu/25 g, respectively. For sausage samples, LOD(50) of both methods were 2.00 cfu/25 g. As a result, the high agreement between two methods and the comparable detection limits of two methods showed that the RapidChek Select is an efficient alternative method for the screening of Salmonella in meat and meat products.     

多重PCR检测无公害畜禽肉和水产品中4种致病菌

建立无公害畜禽肉和水产品中肠出血性大肠杆菌(EHEC)、沙门氏菌、副溶血性弧菌(VP)和单核细胞增生性李斯特氏菌(LM)的多重PCR检测方法,为这些致病菌的快速诊断提供实验依据。选择分别针对EHEC溶血素基因hlyAB、副溶血性弧菌属保守序列toxR基因、沙门氏菌侵袭基因invA和LM的iap基因特异的4对引物,先分别进行单重PCR扩增,再同时加入4对引物进行多重PCR扩增,扩增产物经测序验证。建立的多重PCR方法可简便、快速、灵敏地实现对EHEC、LM、沙门氏菌和VP的同时检测,在畜禽肉和水产品中的检测灵敏度达到10^3cfu/mL。     

人母乳细菌Streptococcus salivarius F286和S. parasanguinis F278对HT29细胞的细胞毒性

为了筛选到具有抗炎特性的有益菌,研究者通常将待测细菌的发酵液上清和热致死菌体与TNF-α刺激下的人类结肠腺癌细胞HT29共孵育,并测量细菌是否能够减少HT29细胞分泌的炎症因子。该测试的前提之一是待测细菌的发酵液上清或菌体不杀死或杀死<10%的HT29细胞。在前期的工作中,我们从人母乳中分离得到Streptococcus salivarius F286和S.parasanguinis F278两株菌。在研究这两株菌的抗炎能力之前,我们利用MTT法摸索不同浓度的S.salivarius F286和S.parasanguinis F278的发酵液上清和热致死菌体对HT29细胞的细胞毒性。实验表明,两株菌的发酵液上清的原液和稀释液对HT29细胞均没有细胞毒性;浓度5×10^5~7.5×10^6cfu/mL的F286热致死菌体、浓度5×10^5~2.5×10^6cfu/mL的F278热致死菌体对HT29细胞的细胞毒性低于10%,而浓度1×10^8cfu/mL的热致死F286和F278菌体分别杀死(23±5.3)%和(22±5.3)%的HT29细胞。因此,S.salivarius F286和S.parasanguinis F278的发酵液上清原液、以及浓度5×10^5~7.5×10^6cfu/mL的F286热致死菌体和5×10^5-2.5×10^6cfu/mL的F278热致死菌体可在HT29细胞模型中进行抗炎能力测试。本研究的方法可用于确定其他细菌在HT29细胞模型中进行抗炎能力测试的合理浓度范围。     

Simple method for the concentration of influenza virus from allantoic fluid on microporous filters.

Membrane filter adsorption-elution is an efficient method for concentration and partial purification of several types of viruses from various aqueous solutions. For efficient virus adsorption to negatively charged filters, the sample is adjusted to pH 3.5 and trivalent salts are added before filtration. Since influenza virus is sensitive to extremes in pH, it cannot be concentrated by ordinary filters. Zeta Plus filters, which have a net positive charge of up to 5 or 6, were evaluated for the concentration of influenza virus from infectious allantoic fluids. Influenza virus efficiently adsorbed to Zeta Plus filters at pH 6, and addition of salts was not necessary. Adsorbed virus was eluted in a small volume of 2% bovine serum albumin plus 1 M NaCl at pH 10. By this procedure, viruses in 100 ml of allantoic fluid were concentrated to a final volume of 8 ml, with an average recovery efficiency of 71.0%.