聚合酶链反应检测细菌16S rRNA基因

根据细菌１６ＳｒＲＮＡ基因的高度保守性,设计合成所有细菌、革兰氏阳性细菌及革兰氏阴性细菌的共同引物,采用聚合酶链反应检测已知细菌１３株,三对引物分别扩增的阳性率为１００％,倍比稀释法能检出细菌的最低浓度为４ＣＦＵ·ｍｌ－１,同时检测临床样本４０份,阳性率为６７５％（２７／４０）,同期细菌培养阳性率为４５％（１８／４０）,二者比较差异有显著性（Ｐ＜０．０５）。结果提示聚合酶链反应检测细菌１６ＳｒＲＮＡ基因具有高度的特异性和敏感性。     

Prospective study of microchimerism in transplant recipients

BACKGROUND: We evaluated peripheral blood microchimerism in 48 consecutive organ transplant recipients (35 kidneys, ten livers, one kidney-liver, one kidney-pancreatic islet, one kidney pancreas) up to 12 months post-transplantation. Patients were categorized according to the presence or absence of rejection episodes, and the patterns of microchimerism in the two groups were then compared. METHODS: DNA was extracted from donor, pre-transplant, and post-transplant peripheral blood samples. Several polymerase chain reaction (PCR)-based assays were developed for the detection of microchimerism. Assay sensitivities ranged from 0.0001 to 3%. RESULTS: Microchimerism was detected only in sex-mismatched cases (male donors and female recipients) using nested PCR for a Y-chromosome marker. There were ten such cases (six kidneys, two livers, and two combined organ transplants). In patients without rejection (n = 7), there was a peak of donor-DNA at 1-3 wk post-transplantation followed by a second peak between 3 wk and 4 months. In patients with biopsy-proven rejection (n = 3), the peaks were absent and the levels of microchimerism were extremely low (< 0.001%). Microchimerism levels declined in all 10 patients and were barely detectable 1 yr post-transplantation. Microchimerism was not detected in the remaining 38 patients despite using a battery of sensitive PCR-based assays. CONCLUSIONS: In our study, microchimerism was detected using the Y-chromosome PCR assay only and the level of donor-DNA in a given patient varied over time. This study highlights the difficulties in establishing a correlation between microchimerism and transplant tolerance.     

Experience with the PCR-based HLA-DQα DNA typing system in routine forensic casework

Summary The results of HLA-DQ typing from 42 routine forensic cases using the polymerase chain reaction (PCR) were analyzed regarding the reliability, discrimination efficiency and informative value of this system in a given case. The cases included stain typing from a variety of different substrates, i.e. blood and semen stains, mixed body fluids, single hairs, cigarette butts, material from fingernail scratches, as well as identification and paternity cases on postmortem and fixed tissue. A total of 125 individual stain and tissue samples were included. PCR amplification was achieved in 70% of these samples. In cases with mixed body fluids, e.g. sperm and vaginal cells from rape cases, DQ typing was always carried out successfully. However, only approx. 42% of all samples that could be typed were relevant regarding the inclusion or exclusion of a suspect. This was mostly due to the limited number of alleles that can be typed at the HLA-DQ locus or to the fact that the stain or hair samples did not originate from the perpetrator, but from the victim or from other persons not related to the crime.     

DNA测序建立甘肃当归、大黄种子rRNA基因图谱的研究

目的：对甘肃地道性中药材当归、大黄种子中rRNA基因内转录间隔区进行碱基序列测定，以期建立从分子水平对中草药种子进行鉴定的一种新方法。方法：提取当归、大黄种子中核DNA，利用特异性引物对其rRNA基因内转录间隔区进行套式PCR扩增，并将扩增产物进行碱基序列测定。结果：经琼脂糖凝胶电泳证实：以上述方法可获得当归、大黄种子DNA中rRNA基因内转录间隔区PCR扩增产物；并经测序后得到了当归、大黄种子rRNA基因内转录间隔区的碱基序列，且具有明显的差异和可对比性。结论：不同植物性中药材种子rRNA基因内转录间隔区碱基序列可做为从分子水平进行鉴定的标记。     

小鼠胚泡着床相关基因的差减筛选

目的:筛选新的小鼠胚泡着床相关基因。方法:对孕4.5 d小鼠着床和非着床位点的子宫内膜组织进行PCR差减。获得了2个新的在小鼠胚泡着床位点高表达的EST(EST8和EST81)。结果:EST8在孕4.5 d小鼠着床位点、肝脏中表达较高,在非着床位点和卵巢中亦有微量表达。EST81主要在孕4.5 d小鼠子宫着床组织和卵巢中表达,其它各种组织中也有微量表达。用PCR方法获得了相应的全长。cDNA,长度分别为1665bp和1364bp。结论:用差减筛选获得的两种cDNA是孕小鼠着床相关基因,其功能有待进一步研究。     

高效液相-荧光检测法测定兔血浆羟基喜树碱方法的研究

目的 :为了测定羟基喜树碱在人体内的血药浓度 ,建立高效液相 -荧光检测法以测定兔血浆中羟基喜树碱的含量。方法 :色谱柱为DiscoveryC18(15cm× 4 6cm ,5 μm) ,流动相为柠檬酸缓冲液 -乙腈 - 75nmol·L-1磷酸二氢钾 (70∶2 3∶7) ,75nmol·L-1磷酸二氢钾中含 1%三乙胺。流速为 1 0mL·min-1,柱温为 4 0℃ ,荧光检测波长为λex36 3nm和λem5 30nm。结果 :该方法的线性范围为 13 76 5 6～ 195 7 4 4 6 8ng·mL-1(r =0 9993) ;提取回收率和方法回收率分别为 80 90 %～ 10 3 5 9% ,10 2 72 %～ 10 8 16 % ;日内RSD≤ 7 4 9% ,日间RSD≤ 9 4 0 %。最低检测限为 5 2ng·mL-1。结论 :主效渡相—荧光法专属性强 ,重现性好 ,操作简便 ,可用于羟基喜树碱的药代动力学研究和血药浓度监测。     

Quantitative Detection of Interleukin 8 Gene Expression in Lung Cancer by Real-time Polymerase Chain Reaction

Objective To develop a method for absolute quantification of interleukin 8 (IL—8) mRNA by using real-time polymerase chain reaction (PCR). Methods The IL —8 mRNA and protein expression in 2 human lung cancer cell lines, H460 and A549, were evaluated by real-time PCR and ELISA. The IL-8 mRNA expression in 9 cases of normal lung tissue and 44 cases of non-small cell lung cancer (NSCLC) were examined. Results The IL—8 mRNA copy number in a given sample can be measured by real-time PCR. The gene expression of IL—8 is correlated with its protein secretion. The normalized value of IL—8 expression was 4.87±1.69 (copies/ 10⁴ GAPDH copies) in normal lung tissue and 17.04 ±23.96 in NSCLC, respectively. The difference between these two groups is statistically significant (P=0.002). Using 9.74 and 19.48 as cut-off points for positive expression and overexpression of IL—8, 52.3%(23/44cases) of NSCLC were found to express an increased level of IL-8, among which 29.5% (13/ 44cases) were defined as positive expression and 22.7%(10/44cases) as overexpression. Statistical analysis indicated that IL—8 overexpression was significantly increased in female cancers, squamous carcinoma, and in late stages of disease (P<0.05). Conclusion The IL—8 gene expression can be determined by a real-time PCR technique. IL -8 overexpression is correlated with gender, histopathology and stages of the disease.     

Evaluating sex chromosome content of sorted human sperm samples with use of dual-color fluorescence in situ hybridization

OBJECTIVE: Although most methods for selecting the sex of offspring by sorting spermatozoa are ineffective at shifting the ratio of Y- to X-containing cells, some commercial sources continue to offer such services. Our objective was to evaluate commercially “sorted” samples with use of dual-color fluorescence in situ hybridization and to identify variations in assessment by comparing motile and total sperm populations, donors, observers, and fluorescence in situ hybridization probes.STUDY DESIGN: Cryopreserved sperm from seven anonymous donors were processed as for insemination. Sperm cells from each total sample or motile subfraction were prepared for fluorescence in situ hybridization by incubation with disulfide-reducing agents to expand sperm nuclei. Two sets of X and Y chromosome–specific, fluorophore-labeled deoxyribonucleic acid probes were used. At least 400 nuclei from each preparation were classified independently by three blinded observers. Hybridization efficiency, aneuploidy, and sex chromosome content were evaluated in subsets of five unsorted, five female-oriented, and five male-oriented samples. Total and motile subfractions were compared with eight samples. Fluorescence in situ hybridization probes were compared in five paired unsorted samples.RESULTS: No differences were detected between washed samples and paired motile subfractions. No differences in hybridization and aneuploidy were detected between groups of sorted samples. The Y/X ratio was significantly different between the sorted groups. However, male-oriented samples had a lower Y/X ratio than female-oriented samples did. Observer and probe choice accounted for small but significant variations that did not alter conclusions about the Y/X ratio for sorted samples.CONCLUSION: In a series of 10 sorted samples from one commercial source, dual-color fluorescence in situ hybridization demonstrated a small but significant shift in the sex chromosome ratios among samples. However, this shift was opposite to that expected by the orientation of the sorted samples. (Am J Obstet Gynecol 1997;176:1172-80.)