Naked-eye detection of plant viral disease using polymerase chain reaction amplification and DNAzyme.

Plant viral diseases can seriously affect the yield and quality of crops. In this work, a convenient and highly sensitive biosensor for the visual detection of plant viral disease is proposed by the PCR-induced generation of DNAzyme. In the absence of nucleic acid for a target plant virus, the primers prohibited the production of G-quadruplex by forming a hairpin structure. However, PCR amplification occurred and generated a number of specific PCR products with free G-quadruplex sequences at both ends in the presence of the target cDNA. A catalytically active G-quadruplex DNAzyme was formed with the help of K+ and hemin, resulting in the formation of colored products visible to the naked eye and a strong absorbance by the addition of ABTS2- and H2O2. The absorbance and the logarithm of target cDNA concentrations showed a good linear relationship in the range of 10 fM-1.0 nM, with a linear regression equation of A = 0.1402 lg c + 0.3761 (c: fM) and a detection limit of 0.19 fM. This method was successfully applied to the analysis of emerging tobacco mosaic virus (TMV) infections in tobacco leaf samples collected in the field due to its flexibility and convenience, indicating a potential application for the early detection of plant viral disease.

[Expression and significance of hTERT, c-myc and Ki-67 in hepatocellular carcinoma].

AIM: To study the correlation of expression of hTERT, c-myc and Ki-67 with the clinical pathological feature of hepatocellular carcinoma and explore the relationship among the three factors. METHODS: The expressions of hTERT, c-myc and Ki-67 in 37 cancer tissues and 5 normal tissues were assessed by immunohistochemical method. RESULTS: The positive rates of hTERT, c-myc and Ki-67 in the cancer tissue were higher than those in normal tissues (P<0.05). The expressions of hTERT, c-myc and Ki-67 had nothing to do with age, sex, metastasis and the volume of tumor(P>0.05). With histology stage reducing, the expression of hTERT, c-myc and Ki-67 increased obviously. There is no correlation between hTERT and c-myc(P>0.05), but it's positive correlation between hTERT and Ki-67(P<0.05). With the expression of Ki-67 increasing, hTERT and c-myc increased. CONCLUSION: The over-expression of hTERT, c-myc and Ki-67 may play a vital role in the progress of hepatocellular carcinoma developing. The expressions of three factors in hepatocellular carcinoma are closely associated.

[Expression of GFP gene in mouse early embryo produced by sperm-mediated gene transfer].

The efficiency of the exogenous DNA transfecting mouse sperm was studied by the DIG end labeled and immunohistochemistry technology. The results suggested that: the efficiency of transfecting positive rate of individual mouse sperm was distinct difference (P < 0.01), and the average rate was 13%. The acrosomal reaction was evaluated using the technology of Coomassie brilliant blue stained, and the appropriate in vitro fertilization (IVF) medium TYH was elected. Mouse sperms were transferred with GFP gene in vitro, and the mature oocytes were fertilized using IVF, and then the zygotes were cultured in vitro. The embryos were observed using the fluorescence microscopy, and the transgenic rate was 4.7%. The results suggested that sperm mediated gene transfer (SMGT) was an effective and feasible method.

[Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.