快速鉴定B群链球菌及检测其耐药性方法的建立和初步应用

目的建立快速鉴定B群链球菌(group B Streptococcus,GBS)的同时检测其对克林霉素、红霉素耐药基因的多重PCR方法,并初步应用。方法建立多重PCR方法,同时扩增以下4个基因位点:GBS编码CAMP因子的基因cfb、大环类脂类耐药基因erm B和mef A/E、克林霉素耐药基因lin B。对该方法的引物质量浓度、Taq酶量和退火温度进行优化,确定多重PCR最适反应体系及最低检测限;将该方法用于91株GBS临床菌株的鉴定及其耐药性的检测,并评价其与常规PCR方法结果的一致性。结果多重PCR的最适反应体系,总量25μL:多重PCR mix212.5μL、多重PCR mix1Taq酶0.23μL、lin B-F和lin B-R引物0.4μmol/L、mef A/E-F和mef A/E-R引物0.2μmol/L、erm B-F和erm B-R引物0.12μmol/L、cfb-F和cfb-R引物0.08μmol/L、DNA 10~100 ng、不足量补入无菌去离子水。扩增程序为:94℃预变性60 s;94℃变性30 s、53℃退火90 s、72℃延伸30 s,共30个循环;72℃延伸10 min。最低检测限为20 pg/μL。多重PCR方法与常规PCR方法检测91株GBS临床菌株,cfb基因结果一致率为100%,erm B、mef A/E和lin B基因的Kappa值分别为0.938、0.956和0.935。结论建立的以GBS cfb、erm B、mef A/E和lin B基因为检测位点的多重PCR方法,可在鉴定GBS的同时检测其对克林霉素、红霉素的耐药性。     

Fujenal, a diterpenoid saga of neighbouring group participation

The chemistry, the biosynthesis and the biotransformations related to fujenal and its analogues are reviewed. Despite the opportunity for free rotation about the C-9:C-10 bond, the chemistry of the ring B of this diterpenoid is dominated by neighbouring group participation between C-6, C-7 and C-19.     

肺炎球菌免疫血清库的建立及应用

目的建立24个血清型的肺炎链球菌(简称肺炎球菌)免疫血清库,为肺炎球菌疫苗中各型多糖含量准确测定提供可靠保障。方法 (1)制备肺炎球菌各型免疫血清:用肺炎球菌菌株制备免疫原,按免疫程序接种青紫蓝兔,将符合规定的免疫血清合并、除菌、分装后建档;(2)鉴定肺炎球菌各型免疫血清:全程采用速率散射比浊法进行鉴定。首先确定肺炎球菌免疫血清工作浓度,再通过特异性和线性对免疫血清进行初步筛查(将筛查出有交叉反应的免疫血清进行吸收),然后用血清交叉试验进行再次筛查,最后通过绘制反应曲线(截取多糖质量浓度1.0~5.0μg/m L的线段)对免疫血清进行线性评定;(3)肺炎球菌免疫血清库的应用:用建成的肺炎球菌免疫血清库中各型免疫血清和丹麦国家血清研究所的诊断血清,检测5个厂家的23价肺炎球菌多糖疫苗的各型多糖含量,对比结果。结果建成的肺炎球菌免疫血清库各型免疫血清效价较高;初步筛查出的有交叉反应的免疫血清,经吸收后交叉反应消失且线性良好(R20.98);通过血清交叉试验再次筛查,未见有交叉反应的免疫血清;反应曲线线段(多糖质量浓度1.0~5.0μg/m L)线性良好(R20.98);应用两套血清检测23价肺炎球菌多糖疫苗的多糖含量,差异无统计学意义(P0.05)。结论成功建立了肺炎球菌免疫血清库,且各型免疫血清稳定、无交叉反应,可用于肺炎球菌疫苗及其疫苗血清型多糖的各项免疫学检测。     

Set up and run an objective structured clinical exam.

Objective structured clinical exams are increasingly used as a way of assessing a range of clinical skills at both undergraduate and postgraduate level. To those planning to introduce such assessments, this article provides basic guidance on their development and structure and the personnel required. For those already using the assessments, our article may provide new ideas or be the impetus for an exchange of ideas. For those who are facing such formal assessment as candidates, we hope this article shows the efforts that are made to achieve the necessary structure and objectivity in this type of examination.     

Phosphoryl group participation leads to peptide formation from N-phosphorylamino acids.

N-phosphorylated amino acids without a side chain functional group can transfer themselves into peptides after prolonged standing in solvents at warm temperatures. Seven N-phosphorylpeptides and free peptides were isolated and their structures determined. The phosphoryl group participation is the key to the peptide formation. An intramolecular mixed carboxylic-phosphoric anhydride intermediate was proposed for this type of reaction which might provide a clue to the function of the phosphoryl group in the phosphorylated enzymes and in the prebiotic synthesis of protein.     

Why patient participation groups stop functioning: general practitioners' viewpoint.

Out of the 87 patient participation groups that were known to the National Association for Patient Participation as having been established by the end of 1983, 17 (25%) are not functioning. The general practitioners concerned with these non-functioning groups were interviewed to identify problems that they had had in keeping the group going and to seek possible explanations for the problems. Fourteen of the groups had stopped functioning in part owing to lack of interest by patients. Groups become non-functioning often in the first year of starting up, and this may be because of the nature of the practice population that they seek to represent.     

Neighbouring group participation in the unblocking of phosphotriesters of nucleic acids.

Two examples of neighbouring group participation during the removal of protecting groups from phosphotriesters of partially or fully protected intermediates of nucleic acids are presented. The first example shows that ammonolysis of aryl groups from phosphotriesters of partially protected - 5'- hydroxy free - nucleic acids (e.g., 4b approximately to; Ar=2C1C 6H4) gives rise to the formation of unnatural nucleic acids (e.g., 7 approximately to and 8 approximately to). The second one illustrates that fluoride ion promoted hydrolysis of 2,2,2-trichloroethyl groups from phosphotriesters of fully protected nucleic acids (e.g., 18a approximately to), having t-butyldimethylsilyl groups at the 2'-positions, leads to the formation of a considerable amount of side-products (e.g., 20 approximately to and 21 approximately to).     

Opening up the group II chaperonins

A new study in this issue of Structure (Huo et?al., 2010) reports the crystal structure of a group II chaperonin in?the open state, providing unprecedented levels of detail for the domain movements that result from ATP?hydrolysis and a clearer picture of the protein folding mechanism mediated by chaperonins.     

建立HPLC-MS检测肺炎支原体中C12-神经酰胺的方法

目的建立HPLC-MS检测肺炎支原体(Mycoplasma pneumoniae,MP)中C12-神经酰胺的方法。方法采用氯仿甲醇提取法提取MP中的C12-类神经酰胺,利用优化了质谱、色谱条件的HPLC-MS对C12-神经酰胺进行鉴定。结果质谱条件选择ESI(+)模式,离子源温度250℃,电喷雾电压5 k V;色谱条件选择0.10%甲酸水溶液(A)-甲醇(B)为流动相,洗脱程序0~7 min,95%→30%A;7~20 min,30%→0 A。C12-神经酰胺的保留时间为17.695 min,m/z为481.79。C12-神经酰胺样品的色谱峰、保留时间及二级图谱下的特征碎片离子均与C12-神经酰胺标准品吻合。MP细胞悬液、MP培养基两者均得不到二级图谱,且m/z与C12-神经酰胺标准品均不相同。结论建立了HPLC-MS检测肺炎支原体中C12-神经酰胺的方法,该方法简便快捷,结果准确可靠,同时也适用于肺炎支原体中神经酰胺类物质的检测。