An ultra-robust fingerprinting method for quality assessment of traditional Chinese medicine using multiple reaction monitoring mass spectrometry

Chromatographic fingerprinting has been perceived as an essential tool for assessing quality and chemical equivalence of traditional Chinese medicine. However, this pattern-oriented approach still has some weak points in terms of chemical coverage and robustness. In this work, we proposed a multiple reaction monitoring (MRM)-based fingerprinting method in which approximately 100 constituents were simultaneously detected for quality assessment. The derivative MRM approach was employed to rapidly design MRM transitions independent of chemical standards, based on which the large-scale fingerprinting method was efficiently established. This approach was exemplified on QiShenYiQi Pill (QSYQ), a traditional Chinese medicine-derived drug product, and its robustness was systematically evaluated by four indices: clustering analysis by principal component analysis, similarity analysis by the congruence coefficient, the number of separated peaks, and the peak area proportion of separated peaks. Compared with conventional ultraviolet-based fingerprints, the MRM fingerprints provided not only better discriminatory capacity for the tested normal/abnormal QSYQ samples, but also higher robustness under different chromatographic conditions (i.e., flow rate, apparent pH, column temperature, and column). The result also showed for such large-scale fingerprints including a large number of peaks, the angle cosine measure after min-max normalization was more suitable for setting a decision criterion than the unnormalized algorithm. This proof-of-concept application gives evidence that combining MRM technique with proper similarity analysis metrices can provide a highly sensitive, robust and comprehensive analytical approach for quality assessment of traditional Chinese medicine.     

黄苓苷对HaCaT细胞影响的蛋白组学研究

目的 本研究旨在观察黄苓苷对永生化人表皮角质形成细胞HaCAT株差异表达蛋白的影响.方法 培养HaCaT细胞,并加入黄苓苷进行干预.培养24h后收集细胞总蛋白,利用双向电泳方法分离细胞蛋白质,并用ImageMaster图像分析软件比较各组细胞的蛋白质图谱,筛选出的差异蛋白质点应用基质辅助激光解吸离子化-飞行时间质谱(MALDI-TOF-MS)测定其胶内酶解后的肽质量指纹图谱,然后利用Mascot查询软件搜寻Swiss Prot数据库鉴定蛋白质.结果 黄苓苷使HaCaT细胞蛋白质组发生改变,对其中18个差异蛋白质点进行质谱分析,获得15个点的肽质量指纹图,初步鉴定为热休克蛋白beta-1,肌动蛋白、二氢嘧啶酶调节蛋白2等.结论 黄苓苷可通过影响细胞中多种蛋白的表达,增强细胞的稳态和抵抗环境刺激损伤的能力,发挥抗肿瘤活性.     

Detection of genomic alterations in carcinogen-induced mouse liver tumors by DNA fingerprint analysis

DNA fingerprint analysis was used to study structural abnormalities in the genome of mouse liver tumor cells. Liver tumors were induced in three strains of mice, namely C57BL/6J, C3H/He and B6C3F1, by a single injection of 20 micrograms/g body wt. diethylnitrosamine on day 15 after birth. DNA from liver tumors was digested with Hinfl restriction enzyme and hybridized on Southern blots with wild-type bacteriophage M13 DNA as probe. The resulting fingerprints of tumor DNA were compared with those of DNA from normal liver tissue. Genomic aberrations were detected in two out of 68 tumors analyzed, one stemming from a C57BL/6J and the other from a C3H/He mouse.     

DNA指纹图与生化标记分析在近交系大鼠遗传检测中的比较研究

目的 :建立DNA指纹图技术实现对近交系大鼠遗传物质的精确检测。方法 :采用DNA指纹图法对国内已知的 6个品系 8个近交系大鼠群体进行分析 ,并与常规生化标记分析法进行比较。结果 :①生化标记分析中除 2个群体SHR(哈 )和WKY(哈 )在Es_3位点出现可疑带外 ,其他位点均与国标标准相一致。②DNA指纹图在 4.0～ 2 3.1kb间的平均图带数为 14～ 19条。③同一品系不同个体间DNA指纹图带的相似系数 (F)及共有带率 (X) ,除SHR(哈 )和WKY(哈 )在 0 .7以下外 ,其他均在 0 .9以上。④不同品系个体之间DNA指纹图带的相似系数和共有带率在0 .0 7以下 ,相同DNA指纹图概率小于 10 _2 2 。⑤相同DNA不同次制作的DNA指纹图谱基本一致。结论 :经比较 ,DNA指纹图在动物遗传检测中比生化标记分析显示出较大的优势 ,它不但能区分品系与品系、品系与亚系间 ,而且还能反映出动物个体间、群体间及品系间的变异程度、亲缘关系及遗传距离 ,具有较高的灵敏度和稳定性     

乌拉尔甘草优良品系选育研究(Ⅰ)——4个来源甘草遗传基础的AFLP分析

目的研究4个来源甘草属植物的遗传基础,初步探讨扩增片段长度多态性(AFLP)分子标记技术在甘草品系选育中的应用。方法采用AFLP标记技术对乌拉尔甘草栽培新品系"民勤1号"、"喀什1号"、"阿克苏1号"和常规栽培品系内蒙古乌拉尔甘草进行基因组DNA多态性、指纹图谱和UPGMA聚类分析。结果从64对引物组合中筛选出了8对引物组合进行多态性分析,以多态位点百分率最高的E-AAC/M-CAG组合构建4个来源甘草的指纹图谱。UPGMA聚类分析将4个来源甘草分为4组,表现出不同的亲缘关系。结论"民勤1号"、"喀什1号"、"阿克苏1号"形成了独特的基因构成,可以作为乌拉尔甘草优良栽培新品系选育目标进行深入研究。     

IS6110-限制性片段长度多态性DNA分型在结核分子流行病学研究中的应用

目的 建立宁夏、北京和上海等地结核分支杆菌发离株IS6110－RFLP DNA指纹图谱，观察其流行病学特征。方法 提取结核分支杆菌基因组DNA，经限制性内切酶PvuⅡ切割、琼脂糖凝胶电泳和Southern转印后，用荧光标记的IS6110DNA序列中245bp探针杂交，以核酸化学发光试剂盒探测荧光信号，比较各菌株指 IS6110拷贝数和带型，分析不同地理区域流行菌株的特点。结果 103例结核患的临床分离株，经245bp探针杂交后的指纹图谱显示大部分菌株含8－21个IS6110拷贝，在宁夏和北京地区流行的结核分支杆菌带型具有共同特点，并有成簇分布的现象，在上海分离株中发现1株零拷贝株和1株单拷贝株。结论 IS6110－RFLP DNA分型方法可用于我国流行的结核分支杆菌分子流行病学研究；宁夏分离株与北京分离株在基因上亲缘关系较相近。     

Molecular Typing in Public Health Laboratories: From an Academic Indulgence to an Infection Control Imperative

Using three Austrian case studies, the variegated applications of molecular typing in today''s public health laboratories are discussed to help illustrate preventive management strategies relying on DNA subtyping. DNA macrorestriction analysis by pulsed field gel electrophoresis has become the gold standard for subtyping of food borne pathogens like listeria, salmonella, campylobacter and Bacillus cereus. Using a Salmonella Mbandaka outbreak from the year 2010 as example, it is shown how the comparison of patterns from human isolates, food isolates, animal isolates and feed isolates can allow to identify and confirm a source of disease. An epidemiological connection between the simultaneous occurrence of tuberculosis in cattle and deer with cases of human tuberculosis due to Mycobacterium caprae in 2010 was excluded using mycobacterial interspersed repetitive units variable-number tandem repeats subtyping. Also in 2010, multilocus sequence typing with nonselective housekeeping genes, the so-called sequence based typing protocol, was used to elucidate connections between an environmental source (a hospital drinking water system) and a case of legionellosis. During the last decades, molecular typing has evolved to become a routine tool in the daily work of public health laboratories. The challenge is now no longer to simply type microorganisms, but to type them in a way that allows for data exchange between public health laboratories all over the world.     

Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus

Identifying Lactobacillus species using only phenotypic and genotypic (16S rDNA sequence analysis) techniques yields inaccurate results. The objective of this study was to develop species-specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting to distinguish species within the closely related Lactobacillus plantarum group. One of these primers, OPD-3, produced a species-specific band that was found only in the tested Lactobacillus pentosus. This specific fragment was isolated from agarose gel and ligated into a vector for DNA sequencing. A pair of primers, SpOPD3Lpen-F1/R1, that were highly specific sequence-characterized-amplified-regions (SCARs) were designed according to the nucleotide sequences of the specific RAPD marker. These primers were used for PCR analysis of the template DNA of the Lactobacillus strains, and a single 542 bp species-specific band was found only in L. pentosus. Using PCR, a novel species-specific primer pair is shown to rapidly, accurately and effectively distinguish L. pentosus from other species in the L. plantarum group of probiotic bacteria.     

光疗前后新生儿的DNA指纹分析

目的：了解光疗对新生儿的ＤＮＡ是否有损伤。方法：采用ＬＥ１１．８小卫星探针，经Ｓｏｕｔｈｅｒｎ印迹法检测接受光疗前后新生儿外周血的ＤＮＡ指纹谱带。结果：２２例接受光疗前后的新生儿ＤＮＡ指纹相比，均未见到任何谱带的改变。结论：光疗未引起新生儿的ＤＮＡ损伤，应用国产蓝光治疗器对高胆红素血症新生儿在４８ｈ内治疗是安全的。