HLA-B27基因检定技术

目的：用聚合酶链反应（ＰＣＲ） 和血清学方法检测ＨＬＡＢ２７ ，并进行比较。方法：采用ＰＣＲ 技术检测７８ 例可疑为强直性脊椎炎患者样本的ＨＬＡＢ２７ 基因，并与血清学结果比较。结果：３６ 例具有Ｂ２７ 基因者中２４ 例同时作血清学试验，５ 例血清学难以判定结果，并与ＰＣＲ 结果不一致。４２ 例无Ｂ２７ 基因者中２１ 例同时作血清学试验，３ 例血清学结果为可疑。结论：Ｂ２７ 基因检定技术与血清学方法比较，有快速、简便、准确性高等优点。

PCR生物学领域的革命性突破

从痕迹斑或精斑、单根毛发中识别案犯个体或进行亲子鉴定;对β地中海贫血的基因检定以实施婚配指导及产前诊断:从极少许粪便中分离轮状病毒;从丙型肝炎感染早期(未产生抗体时)血清中检出HCV RNA等等一系列令人惊呀的痕迹分析技术,这就是PCR。这种技术较目前广泛使用的酶联免疫(EIA)及放射免疫分析(RIA)

A Method for Detecting Adhesive Related-Factors of Streptococcus suis Serotype 2 by Real-time PCR

[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []Vlethod] The gene fragments en- coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan- dard curves and melting curves of the adhesive related-factors and aroA were ob- tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0×102 copies in 1 μl of initial templates. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.

Expression Detection of DMRTs and Two sox9 Genes in Takifugu rubripes (Tetraodontidae, Vertebrata)

Sex determination and sex differentiation are important phenomena in fish, but the mechanisms of sex determination in Takifugu rubripes are poorly understood. In our study, the expression patterns of genes for DMRTs （DMRT1, DMRT2 and DMRT3）,sox9a and sox9b in T. rubripes tissues were verified with the Reverse Transcription （RT）-PCR detection. It is showed that DMRT1 expressions in testis and ovaries were much lower, and no expressions were fotmd in muscle, blood and tailfin. However, expressions for DMRT2 and DMRT3 were not found in the tissues stated above. Transcripts of sox9a were detected in muscle, fin, ovary and testis, but not in blood, whereas sox9b expression was only detected in ovary. The expression patterns of DMRTs, sox9a and sox9b in T. rubripes gonads suggest that these genes may not be sex-specific.

KRAS mutation testing in metastatic colorectal cancer

The KRAS oncogene is mutated in approximately 35%-45% of colorectal cancers, and KRAS mutational status testing has been highlighted in recent years. The most frequent mutations in this gene, point substitutions in codons 12 and 13, were validated as negative predictors of response to anti-epidermal growth factor receptor antibodies. Therefore, determining the KRAS mutational status of tumor samples has become an essential tool for managing patients with colorectal cancers. Currently, a variety of detection methods have been established to analyze the mutation status in the key regions of the KRAS gene; however, several challenges remain related to standardized and uniform testing, including the selection of tumor samples, tumor sample processing and optimal testing methods. Moreover, new testing strategies, in combination with the mutation analysis of BRAF , PIK3CA and loss of PTEN proposed by many researchers and pathologists, should be promoted. In addition, we recommend that microsatellite instability, a prognostic factor, be added to the abovementioned concomitant analysis. This review provides an overview of KRAS biology and the recent advances in KRAS mutation testing. This review also addresses other aspects of status testing for determining the appropriate treatment and offers insight into the potential drawbacks of mutational testing.

Detection of PRL-2 gene expression in hepatocellular carcinoma by real-time fluorescence quantitative PCR

Objective:To quantitatively detect the expression level of PRL-2 in primary hepatocellular carcinoma using real-time fluorescence quantitative PCR.Methods:Total RNA isolated from human HCC and liver tissue adjacent to the tumor was reversely transcribed into cDNA.Real-time fluorescence quantitative PCR(Q-PCR) method was used to analyze the expres-sion level of PRL-2 gene.Results:The Q-PCR method was performed successfully to precisely detect RNA level.PRL-2 was expressed in all portal vein tumor thrombosis(PVTT) and HCC,but only in some paratumor tissue.The highest expression level of PRL-2 gene was recorded in PVTT;meanwhile expression level of PRL-2 was higher than that in paratumor liver tis-sues and in HCC(P < 0.01),and it was higher in HCC than that in paratumor liver tissues.Conclusion:The Q-PCR may be the most precise method to quantitatively detect RNA level and can be used in gene expression changes.The PRL-2 gene has higher expression in PVTT than that in HCC and in paratumor liver tissue cells,indicating that it plays an important role in the development and metastasis of the HCC.

Quantitative Real-Time Reverse Transcription-PCR Assay for the Expression of Tob mRNA in Human Colorectal Cancer

OBJECTIVE Tob is a member of Tob/BTG antiproliferative family. To date, Tob expression in human carcinoma using clinical specimens has not been studied in depth except for lung carcinoma and thyroid carcinoma. This study is the first to investigate the expression levels of Tob gene in human colorectal cancer tissues, and their corresponding para-cancerous tissues. The correlation of expression of the Tob gene with clinicopathological characteristics of colorectal cancer was also analyzed. METHODS Quantitative real time RT-PCR was used to detect the expression of Tob mRNA in 31 colorectal cancers. RESULTS Compared with normal tissues, up-regulation of Tob mRNA was observed in 31 colorectal cancer tissues （P = 0.020）. The expression level of Tob at Dukes C ＋ D phase was higher than Dukes A ＋ B phase, and the difference was significant （P 〈 0.05）. However, in this study, it was found that the expression of Tob mRNA was not related with age, gender, and pathological type of colorectal cancer. CONCLUSION The up-regulation of Tob may be closely associated with tumorigenesis of colorectal carcinoma.

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.

Effect of Lichong Decoction on expression of IGF-I and proliferating cell nuclear antigen mRNA in rat model of uterine leiomyoma

OBJECTIVE:To study the effect of Lichong Decoction(Lichong Decoction for strengthening anti-pathogenic Qi and eliminating blood stasis) on the expression of insulin-like growth factor-I(IGF-I) and proliferating cell nuclear antigen(PCNA) mRNA in a rat model of uterine leiomyoma.METHODS:Fifty female Wistar rats were randomized into a normal control group,model group,Lichong Decoction group,Guizhifuling Capsule(Capsule containing Cassia Twig and Poria) group,and Mifepristone group.The uterine leiomyoma model was established by peritoneal injections of exogenous estrogen and progesterone hormone.The ultrastructural changes in cells of rat uterine tissues were observed with transmission electron microscopy,and the expression of IGF-I and PCNA mRNA was detected by real-time fluorescent quantitative PCR.RESULTS:Following treatment,cells in the Lichong Decoction group appeared to be arranged normally,the cellular morphology were almost in a normal state,hyperplasia and hypertrophy of the chondriosome was reduced,collagen fibers were arranged in a regular manner,without obvious hyperplasia,and the expression of IGF-I and PCNA mRNA was significantly decreased compared with the model group(P<0.01).CONCLUSIONS:The effect of Lichong Decoction on uterine leiomyoma is related to its function in reducing the expression of IGF-I and PCNA mRNA.

Electrochemical single nucleotide polymorphisms genotyping on surface immobilized three-dimensional branched DNA nanostructure

An electrochemical assay for single nucleotide polymorphisms (SNPs) genotyping is reported. Although electrochemical method is sensitive for DNA detection on surfaces, the ability of surface assay to precisely recognize DNA hybridization event is sacrificed to some extent due to the crowded confined surfaces environments that disfavor DNA hybridization. In the present study, we employed branched tetrahedron structure probes (TSPs) to replace regular linear single stranded DNA capture probes that were immobilized on solid surfaces. This three-dimensional DNA nanostructure lowers the density of immobilized DNA probes on confined surfaces, providing a hybridization environment that is similar to homogenous solution. This TSP-based electrochemical assay reveals excellent performance for SNPs genotyping with concentration as low as 1 nM.

Clones identification of Sequoia sempervirens (D. Don) Endl. in Chile by using PCR-RAPDs technique

A protocol of polymerase chain reaction-random amplified polymorphic DNAs (PCR-RAPDs) was established to analyse the gene diversity and genotype identification for clones of Sequoia sempervirens (D. Don) Endl. in Chile. Ten (out of 34) clones from introduction trial located in Voipir-Villarrica, Chile, were studied. The PCR-RAPDs technique and a modified hexadecyltrimethylammonium bromide (CTAB) protocol were used for genomic DNA extraction. The PCR tests were carried out employing 10-mer random primers. The amplification products were detected by electrophoresis in agarose gels. Forty nine polymorphic bands were obtained with the selected primers (BG04, BF07, BF12, BF13, and BF14) and were ordered according to their molecular size. The genetic similarity between samples was calculated by the Jaccard index and a dendrogram was con- structed using a cluster analysis of unweighted pair group method using arithmetic averages (UPGMA). Of the primers tested, 5 (out of 60) RAPD primers were selected for their reproducibility and high polymorphism. A total of 49 polymorphic RAPD bands were detected out of 252 bands. The genetic similarity analysis demonstrates an extensive genetic variability between the tested clones and the dendrogram depicts the genetic relationships among the clones, suggesting a geographic relationship. The results indicate that the RAPD markers permitted the identification of the assayed clones, although they are derived from the same geo- graphic origin.

On-chip detection of a single nucleotide polymorphism without polymerase amplification

A nanoparticle-assembled photonic crystal （PC） array was used to detect single nucleotide polymorphism （SNP）. The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair （bp） difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease （PKD） with a sensitivity of 0.9 fg/mL （50 aM）-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA （gDNA） extracted from the urine and blood of two PKD-wild type and three PKD positive cats. The standard curves for PKD positive （PKD＋） and negative （PKD-） DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection （LOD） was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction （PCR）. The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analvsis is desirable.