Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology

AIM: To develop a real-time PCR for detecting hepatitis B virus-(HBV) DNA based on TaqMan technology using a new MGB probe.METHODS: Plasmid containing the sequence of X gene (1414-1744 nt) was constructed as HBV-DNA standard for quantitative analysis. A TaqMan-MGB probe between primers for amplification was designed to detect PCR products. The interested sequence contained in the plasmid and in clinical specimens was quantitatively measured.RESULTS: The detection limit of the assay for HBV DNA was 1 genome equivalent per reaction. A linear standard curve was obtained between 100 and 109 DNA copies/reaction (r＞0.990). None of the negative control samples showed false-positive reactions in duplicate. HBV DNA was detected in 100% (50/50) of HBV patients with HbeAg, and in 72.0% (36/50) with HBsAg, HBeAb and HBcAb. The coefficient of variation for both intra- and inter-experimental variability demonstrated high reproducibility and accuracy.CONCLUSION: Real-time PCR based on TaqMan-MGB probe technology is an excellent method for detection of HBV DNA.

Development of a Real-Time PCR Method (Taqman) for Rapid Identification and Quantification of Prorocentrum donghaiense

Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.

CXCR4/SDF-1 axis is involved in lymph node metastasis of gastric carcinoma

AIM:To investigate the role of CXC chemokine receptor-4 (CXCR4) and stromal cell-derived factor-1 (SDF-1) in lymph node metastasis of gastric carcinoma.METHODS:In 40 cases of gastric cancer,expression of CXCR4 mRNA in cancer and normal mucous membrane and SDF-1 mRNA in lymph nodes around the stomach was detected using quantitative polymerase chain reaction (PCR) (TaqMan) and immunohistochemistric assay.SGC-7901 and MGC80-3 cancer cells were used to investigate the effect of SDF-1 on cell proliferation and migration.RESULTS:Quantitative reverse transcription PCR and immunohistochemistry revealed that the expression level of CXCR4 in gastric cancer was significantly higher than that in normal mucous membrane (1.6244 ± 1.3801 vs 1.0715 ± 0.5243,P < 0.05).The expression level of CXCR4 mRNA in gastric cancer with lymph node metastasis was also significantly higher than that without lymph node metastasis (0.823 ± 0.551 vs 0.392 ± 0.338,P < 0.05).CXCR4 expression was significantly related to poorly differentiated,high tumor stage and lymph node metastasis.Significant differences in the expression level of SDF-1 mRNA were found between lymph nodes in metastatic gastric cancer and normal nodes (0.5432 ± 0.4907 vs 0.2640 ± 0.2601,P < 0.05).The positive expression of SDF-1 mRNA in lymph nodes of metastatic gastric cancer was consistent with the positive expression of CXCR4 mRNA in gastric cancer (r=0.776,P < 0.01).Additionally,human gastric cancer cell lines expressed CXCR4 and showed vigorous proliferation and migratory responses to SDF-1.AMD3100 (a specific CXCR4 antagonist) was also found to effectively reduce the migration of gastric cancer cells.CONCLUSION:The CXCR4/SDF-1 axis is involved in the lymph node metastasis of gastric cancer.CXCR4 is considered as a potential therapeutic target in the treatment of gastric cancer.

Identification of deregulated miRNAs and their targets in hepatitis B virus-associated hepatocellular carcinoma

AIM： TO identify the differentially expressed miRNAs and their targets in hepatitis B virus （HBV）-associated hepatocellular carcinoma （HCC）. METHODS： Six hundred and sixty seven human miRNAs were quantitatively analyzed by Taqman lowdensity miRNA array （TLDA） in HBV-HCC tissues. Gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analyses were used to analyze the significant function and pathway of the differentially expressed miRNAs in HBV-HCC. TargetScan software was used to predict the targets of deregulated miRNAs. Western blotting and luciferase assay were performed to verify the targets of these miRNAs.RESULTS： Ten up-regulated miRNAs （miR-217, miR- 518b, miR-517c, miR-520g, miR-519a, miR-522, miR- 518e, miR-525-3p, miR-512-3p, and miR-518a-3p） and 11 down-regulated miRNAs （miR-138, miR-214, miR-214#, miR-199a-5p, miR-433, miR-511, miR-592, miR-483-3p, miR-483-5p, miR-708 and miR-1275） were identified by Taqman miRNAs array and confirmed quantitatively by reverse transcription polymerase chain reaction in HCC and adjacent non-tumor tissues. GO and KEGG pathway analysis revealed that ＂regulation of actin cytoskeleton＂ and ＂pathway in cancer＂ are most likely to play critical roles in HCC tumorigenesis. MiR- 519a and ribosomal protein S6 kinase polypeptide 3 （RPS6KA3） were predicted as the most significant can-didates by miRNA-mRNA network. In addition, cyclin D3 （CCND3） and clathrin heavy chain （CHC）, usually up-regulated in HCC tissues, were validated as the di- rect target of miR-138 and miR-199a-5p, respectively.

A real-time PCR targeted to the upstream regions of HlyB for specific detection of Edwardsiella tarda

Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry. Therefore, a rapid, reproducible, and sensitive method for detection and quantification of this pathogen is needed urgently. To achieve this purpose, we developed a TaqMan-based real-time PCR assay for detection and quantification orE. tarda. The assay targets the hemolysin activator HlyB domain protein of E. tarda. Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction. A standard curve was generated from the threshold cycle values （y） against log10 （E. tarda genomic DNA concentration） as x. The intra- and inter-assay coefficient of variation （CV） values were less than 2.06% and 1.05% respectively, indicating that the assay had good reproducibility. This method is highly specific to E. tarda strains, as it shows no cross-reactivity to Edwardsiella ictaluri, a member of the same genus, or to nine other fish-pathogenic bacteria species belonging to three other genera. This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E. tarda in clinical samples.

Cloning, characterization, and expression of Cytochrome b (Cytb)——a key mitochondrial gene from Prorocentrum donghaiense

Mitochondrial cytochrome b （Cytb）, one of the few proteins encoded by the mitochondrial DNA, plays an important role in transferring electrons. As a mitochondrial gene, it has been widely used for phylogenetic analysis. Previously, a 949-bp fragment of the coding gene and mRNA editing were characterized from Prorocentrum donghaiense, which might prove useful for resolving P. donghaiense from closely related species. However, the full-length coding region has not been characterized. Ih this study, we used rapid amplification of cDNA ends （RACE） to obtain full-length, 1 124 bp cDNA. Cytb transcript contained a standard initiation codon ATG, but did not have a recognizable stop codon. Homology comparison showed that the P. donghaiense Cytb had a high sequence identity to Cytb sequences from other dinoflagellate species. Phylogenetic analysis placed Cytb from P. donghaiense in the clade of dinoflagellates and it clustered together strongly with that from P. minimum. Based on the full-length sequence, we inferred 32 editing events at different positions, accounting for 2.93% of the Cytb gene. 34.4% （11） of the changes were A to G, 25% （8） were T to C, and 25% （8） were C to U, with smaller proportions of G to C and G to A edits （9.4% （3） and 6.2% （2）, respectively）. The expression level of the Cytb transcript was quantified by real-time PCR with a TaqMan probe at different times during the whole growth phase. The average Cytb transcript was present at 39.277.46 copies of cDNA per cell during the whole growth cycle, and the expression of Cytb was relatively stable over the different phases. These results deepen our understanding of the structure and characteristics of Cytb in P. donghaiense, and confirmed that Cytb in P. donghaiense is a candidate reference gene for studying the expression of other genes.

CYP1A2*1F基因多态性的检测方法建立及其对阿戈美拉汀药代动力学的影响

建立实时荧光定量PCR方法检测CYP1A2*1F基因多态性,并探讨CYP1A2*1F基因多态性对阿戈美拉汀药代动力学的影响。该方法采用扩增阻滞突变系统(ARMS)结合TaqMan探针技术(ARMS-TaqMan法)同时检测野生和突变两个反应体系,根据野生引物循环阈值(Ct_w)与突变引物循环阈值(Ct_m)的差值绝对值(△Ct)来判断CYP1A2*1F的基因型。同时进行口服阿戈美拉汀片25mg的药代动力学试验,采用液相色谱-串联质谱法测定血浆中阿戈美拉汀浓度,采用WinNonlin 6.3软件处理血浆中药物浓度-时间数据,非房室模型方法计算药代动力学参数T_(max)、C_(max)、AUC_(0→1)、AUC_(0→∞)和T_(1/2)。结果表明,在55名健康受试者中,CYP1A2*1F野生型共12例(21.8%),其△Ct值为9.89±0.56;杂合型22例(40.0%),其△Ct值为0.41±0.14;纯合突变型21例(38.2%),其△Ct值为6.85±0.32。统计分析显示,CYP1A2*1F3种基因型组间各药代动力学参数比较皆无显著性差异(P>0.05),但所建立的用于CYP1A2*1F基因多态性检测的ARMS-TaqMan法具有准确、操作简单、高通量等优势,便于进一步筛查基因多态性对阿戈美拉汀药代动力学的影响,适合临床应用。本方法检测原理可用于各种单碱基突变率的检测,具有广泛的应用前景。