一种甲型肝炎灭活疫苗灭活验证试验方法的建立

目的探讨细胞培养/链特异性RT-PCR方法可否作为甲型肝炎(甲肝)灭活疫苗(L-A-1减毒株)的病毒灭活验证试验方法。方法根据甲肝病毒(HAV)L-A-1株基因组序列,设计5条基因特异性引物,提取HAV基因组RNA,应用设计的正向引物进行反转录,再进行两轮PCR扩增,通过检测HAV复制过程中的负链中间体,对甲肝灭活疫苗灭活验证试验方法进行探讨,并与《中华人民共和国药典》甲肝灭活疫苗HAV灭活验证试验方法进行比较。结果细胞培养/链特异性RT-PCR方法对HAV负链RNA特异、敏感。通过方法学验证试验表明,该方法的特异性、敏感性和重复性均良好。利用此方法对5批甲肝灭活疫苗(L-A-1减毒株)进行检测,结果全为阴性,与《中华人民共和国药典》甲肝灭活疫苗HAV灭活验证试验测定结果相同。结论细胞培养/链特异性RT-PCR方法快速、灵敏可靠,可作为检测甲肝灭活疫苗(L-A-1减毒株)HAV灭活验证试验的方法。     

微生物能力验证样品均匀性试验的研究

目的：对人工制备的微生物能力验证样品进行均匀性试验,验证是否符合能力验证的要求。方法：随机抽取能力验证样品,采用AOAC990.12测试方法,菌落总数的结果经单因素方差分析评价其均匀性。结果：样品D均匀性试验结果为样品间变异较小,样品足够均匀;样品C均匀性试验统计学结果显示样品间差异存在显著性,通过借鉴方法的重复性和再现性的标准差对能力验证总体标准差进行评估,计算Sσ,该值小于能力验证的推荐值,该样品的均匀性可能满足能力验证的对样品要求。     

新型冠状病毒灭活疫苗抗原含量检测方法的建立

目的 建立新型冠状病毒(新冠病毒)灭活疫苗抗原含量检测的双抗体夹心ELISA方法,并对该方法检测2家企业疫苗的适用性进行验证。方法 使用羊抗S蛋白抗体作为包被抗体,兔抗S蛋白抗体作为显示抗体,建立了双抗体夹心ELISA方法,并对方法的准确度、精密度进行验证;分别使用该方法及企业自建方法对2家企业生产的各20批次疫苗产品进行检测,评价方法的一致性。结果 成功建立了新冠病毒灭活疫苗抗原含量检测方法,该方法线性良好,R²>0.99。方法验证结果显示,对于2家企业疫苗回收率均在80%～120%范围内,试验内与试验间CV均<10%。方法比对结果显示,该方法与企业方法检测结果比值对于A企业比值在0.83～1.22之间,平均为1.02;与B企业比值在0.91～1.07之间,平均为0.99;不同方法检测结果差异无统计学意义(P>0.05)。结论 成功建立了新冠病毒灭活疫苗抗原含量检测方法,该方法的准确性与精密度良好,并且对国内2家企业生产的疫苗具有较好的适用性。     

A型肉毒毒素ELISA鉴别试验方法的建立

目的建立鉴定A型肉毒毒素的ELISA鉴别试验方法以替代传统的动物试验法。方法采用现代免疫学技术,制备马源性和兔源性抗A型肉毒毒素多克隆抗体,建立了双抗体夹心ELISA,并就此初步进行方法学验证。结果所建立的ELISA具有良好的特异性、灵敏度、精密度和耐用性,具有替代动物试验方法的良好前景。结论在进一步验证和确认之后,该方法有望正式成为可用于鉴定A型肉毒毒素的试验方法以替代动物试验法。     

均匀设计超声波提取豆粕异黄酮的研究

本该采用均匀设计方法试验优化技术,进行超声波循环提取豆粕异黄酮的多因素复杂试验,通过应用统计分析软件SPSS处理试验数据,得出多因素回归方程,求出最佳试验条件,并通过试验进行验证．     

数字PCR仪校准方法研究

数字PCR仪是核酸绝对定量的重要仪器,因此确保数字PCR仪检测结果的准确性十分重要。通过对国内市场上数字PCR仪的比较分析,剖析了数字PCR仪的性能指标,对数字PCR仪的校准方法进行了探讨,设定了拷贝数浓度相对示值误差、拷贝数浓度重复性、荧光通道一致性和反应单元个数重复性作为数字PCR仪整机校准的计量技术指标,采用具有溯源性的国家有证标准物质,对方法进行了试验验证。验证结果表明了校准思路和方法的可行性,该方法操作性强,能够满足仪器技术要求以及用户需求, 提高了数字PCR仪检测结果的准确性和可靠性,对进一步拓展和深化数字PCR 技术的应用具有积极的促进作用。     

花红胶囊微生物限度检查方法学验证

目的对花红胶囊微生物限度检查方法进行方法学验证。方法按《中国药典》(2010版)一部附录ⅧC项下的常规方法检查,采用平皿计数法,分组分别使用5种试验菌验证。结果:花红胶囊中5种试验菌的回收率均达70%以上。控制菌大肠埃希菌、大肠菌群、沙门菌经方法学验证可行性强。结论采用常规检查法对花红胶囊微生物限度检查,可控制该产品的细菌、霉菌和酵母菌的总数和控制菌。     

磷酸类脂快速测定方法

磷酸类脂快速测定经修订和多年试用验证,方法简单快速,试验结果可靠。     

AFLP指纹图谱试验体系在甜菜无融合生殖系中的应用

目的：比较甜菜无融合生殖品系M14子代之间的同一性及亲缘关系,从而验证AFLP试验体系在甜菜无融合生殖系中的适用性。方法：利用已建立的AFLP试验体系,通过琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳检测以及遗传距离的比较来验证这一试验体系。结果：M14子代之间及二倍体甜菜之间的同一性较好,其亲缘关系也较近。结论：验证了所建立的AFLP试验体系的适用性,同时也确证了AFLP技术在构建DNA指纹图谱中的优越性,为无融合生殖的进一步研究奠定了基础。     

APPLICATIONS FOR BACTERIAL MEMBRANE FRACTIONS IN STERILE PHARMACEUTICAL QUALITY CONTROL

The commercially available bacterial membrane preparation Oxyrase was examined for use in quality control laboratory procedures for culturing anaerobic microorganisms, and as a media additive for parenteral product filling line validation by media fill to detect anaerobes. Comparison studies between Oxyrase products and conventional anaerobic culturing methods were performed. The results from the studies showed Oxyrase for Broth to be effective in promoting the growth of anaerobes in nonprereduced media not normally used for anaerobic cultivation.     

A Fluorescence‐Based Sensor Assay that Monitors General Protein Aggregation in Human Cells

Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ‐peptide. Exposure to proteome destabilizers lead to re‐localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide‐genetic screens to identify genes and pathways involved in protein misfolding and aggregation.     

Impaired repair activity of a truncated DNA polymerase beta protein.

DNA polymerase beta (polbeta) is an essential enzyme for gap filling synthesis in damaged DNA template involved in base excision repair pathway. A truncated polbeta protein is expressed in primary colorectal and breast adenocarcinomas. To determine a possible alteration in the functions of the enzyme, a human cell line named HeLapolbetadelta expressing the truncated form of polbeta has been established. These cells revealed a significantly reduced level of repair activity evaluated by gap filling synthesis and polbeta activity. More importantly, the HeLapolbetadelta cells are hypersensitive to MNNG, a DNA alkylating agent. It appears from the responses that the gap filling synthesis of WT cells, a HeLa cell line overexpressing wild-type polbeta protein, was inhibited by HeLapolbetadelta protein.     

Bacterial populations on brewery filling hall surfaces as revealed by next-generation sequencing

Due to the presence of moisture and nutrients, brewery filling line surfaces are susceptible to unwanted microbial attachment. Knowledge of the attaching microbes will aid in designing hygienic control of the process. In this study the bacterial diversity present on brewery filling line surfaces was revealed by next generation sequencing. The two filling lines studied maintained their characteristic bacterial community throughout three sampling times (13–163 days). On the glass bottle line, γ-proteobacteria dominated (35–82% of all OTUs), whereas on the canning line α-, β- and γ-proteobacteria and actinobacteria were most common. The most frequently detected genera were Acinetobacter, Propinobacterium and Pseudomonas. The halophilic genus Halomonas was commonly detected, which might be due to its tolerance to alkaline foam cleaners. This study has revealed a detailed overall picture of the bacterial groups present on filling line surfaces. Further effort should be given to determine the efficacy of washing procedures on different bacterial groups.     

Replication-guided nucleosome packing and nucleosome breathing expedite the formation of dense arrays

The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. How cells achieve such high coverage within a short time, e.g. after DNA replication, remains poorly understood. It is known that random sequential adsorption of impenetrable particles on a line reaches high density extremely slowly, due to a jamming phenomenon. The nucleosome-shifting action of remodeling enzymes has been proposed as a mechanism to resolve such jams. Here, we suggest two biophysical mechanisms which assist rapid filling of DNA with nucleosomes, and we quantitatively characterize these mechanisms within mathematical models. First, we show that the ‘softness’ of nucleosomes, due to nucleosome breathing and stepwise nucleosome assembly, significantly alters the filling behavior, speeding up the process relative to ‘hard’ particles with fixed, mutually exclusive DNA footprints. Second, we explore model scenarios in which the progression of the replication fork could eliminate nucleosome jamming, either by rapid filling in its wake or via memory of the parental nucleosome positions. Taken together, our results suggest that biophysical effects promote rapid nucleosome filling, making the reassembly of densely packed nucleosomes after DNA replication a simpler task for cells than was previously thought.     

神木" 全民免费医疗" 的可推广性探讨

神木模式是我国"全民免费医疗"的一次创造性探索,虽然它不是真正意义上的全民免费医疗制度,但对比目前新医改推广的"全民医保"制度而言,它在解决群众"看病难、看病贵"的问题上起到更积极的作用,对全国也起着一定的借鉴意义。     

试论中国法医学司法鉴定体制的现状及其未来改革方向

随着我国社会的发展与进步,作为我国司法鉴定工作中重要组成部分的法医学司法鉴定也在与时俱进地进行改革,以促进我国司法鉴定工作的公开、公平和公正,与此同时,这也是顺应时代发展和社会进步的需求。本文通过对当前中国法医学司法鉴定体制的种类进行总结,分析该体制在现实司法鉴定工作中的利与弊,并在此基础上对中国法医学司法鉴定体制未来的改革方向进行探讨。     

神木“全民免费医疗”的可推广性探讨

神木模式是我国＂全民免费医疗＂的一次创造性探索,虽然它不是真正意义上的全民免费医疗制度,但对比目前新医改推广的＂全民医保＂制度而言,它在解决群众＂看病难、看病贵＂的问题上起到更积极的作用,对全国也起着一定的借鉴意义。     

Two-step validation of a Monte Carlo dosimetry framework for general radiology

The Monte Carlo technique is considered gold standard when it comes to patient-specific dosimetry. Any newly developed Monte Carlo simulation framework, however, has to be carefully calibrated and validated prior to its use. For many researchers this is a tedious work. We propose a two-step validation procedure for our newly built Monte Carlo framework and provide all input data to make it feasible for future related application by the wider community. The validation was at first performed by benchmarking against simulation data available in literature. The American Association of Physicists in Medicine (AAPM) report of task group 195 (case 2) was considered most appropriate for our application. Secondly, the framework was calibrated and validated against experimental measurements for trunk X-ray imaging protocols using a water phantom. The dose results obtained from all simulations and measurements were compared. Our Monte Carlo framework proved to agree with literature data, by showing a maximal difference below 4% to the AAPM report. The mean difference with the water phantom measurements was around 7%. The statistical uncertainty for clinical applications of the dosimetry model is expected to be within 10%. This makes it reliable for clinical dose calculations in general radiology. Input data and the described procedure allow for the validation of other Monte Carlo frameworks.     

Genome-wide analysis of single nucleotide variants allows for robust and accurate assessment of clonal derivation in cell lines used to produce biologics

A clonally derived (or “monoclonal”) cell line is a cell population derived from a single progenitor cell. Clonally derived cell lines are required for many biotechnological applications. For instance, recombinant mammalian cells used to produce therapeutic proteins are expected by regulatory authorities to be clonally derived. Assurance of clonal derivation (or “clonality”) is usually obtained from the characterization of the procedure used for cell cloning, for instance by assessing the success rate of single-cell sorting but not by assessing the cell line itself. We have developed a method to assess clonal derivation directly from the genetic makeup of cells. The genomic test of clonality is based on whole-genome sequencing and statistical analysis of single nucleotide variants. This approach quantifies the clonal fractions present in nonclonal samples and it provides a measure of the probability that a cell line is derived from a single cell. Upon experimental validation of the test, we show that it is highly accurate and that it can robustly detect minor clonal fractions of as little as 1% of the cell population. Moreover, we find that it is applicable to various cell line development protocols. This approach can simplify development protocols and shorten timelines while ensuring clonal derivation with high confidence.