A dry-reagent assay to rapidly detect Mycobacterium tuberculosis using loop-mediated isothermal amplification / 中华预防医学杂志

A molecular diagnostic assay which could be stored at room temperature was developed to rapidly detect Mycobacterium tuberculosis (MTB) based on loop-mediated isothermal amplification (LAMP) technology and dry-reagent process. LAMP uses 4 or 6 primers and Bst DNA polymerase to amplify DNA at a constant temperature. The results showed that the LAMP assay could detect the amplification of IS6110 target gene within 20 min using real-time fluorescence signal detection. The sensitive of LAMP assay was similar to the PCR technology while the precision of PCR was better than LAMP (coefficient of variation, LAMP 18.9%, PCR 3.4%), meaning LAMP was more suitable for qualitative detection. The LAMP assay did not amplify DNA of other 10 types of pathogens, including Neisseria meningitidis, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Rubivirus, mumps virus, adenovirus (type 3), adenovirus (type 7), respiratory syncytial virus B and parainfluenza virus type 2, indicating a good specificity. Furthermore, a dry-reagent assay was developed using air-drying and freeze-drying process. The performance of dried reagents did not change after 10 days storage at 50 ℃, meaning the dried reagents could be stored at room temperature (25 ℃) for more than six months. The dry-reagent LAMP assay also successfully amplified MTB DNA from several clinical samples within 20 min. In conclusion, the developed LAMP assay together with isothermal amplifier could rapidly detection MTB.

A dry-reagent assay to rapidly detect Mycobacterium tuberculosis using loop-mediated isothermal amplification / 中华预防医学杂志

A molecular diagnostic assay which could be stored at room temperature was developed to rapidly detect Mycobacterium tuberculosis (MTB) based on loop-mediated isothermal amplification (LAMP) technology and dry-reagent process. LAMP uses 4 or 6 primers and Bst DNA polymerase to amplify DNA at a constant temperature. The results showed that the LAMP assay could detect the amplification of IS6110 target gene within 20 min using real-time fluorescence signal detection. The sensitive of LAMP assay was similar to the PCR technology while the precision of PCR was better than LAMP (coefficient of variation, LAMP 18.9%, PCR 3.4%), meaning LAMP was more suitable for qualitative detection. The LAMP assay did not amplify DNA of other 10 types of pathogens, including Neisseria meningitidis, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Rubivirus, mumps virus, adenovirus (type 3), adenovirus (type 7), respiratory syncytial virus B and parainfluenza virus type 2, indicating a good specificity. Furthermore, a dry-reagent assay was developed using air-drying and freeze-drying process. The performance of dried reagents did not change after 10 days storage at 50 ℃, meaning the dried reagents could be stored at room temperature (25 ℃) for more than six months. The dry-reagent LAMP assay also successfully amplified MTB DNA from several clinical samples within 20 min. In conclusion, the developed LAMP assay together with isothermal amplifier could rapidly detection MTB.

Comparison of TTR and CMV promoters in vivo and in vitro via a secreted luciferase reporter system / 病毒学报

GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.

Transgenic 4-1-BB ligand therapy induces tumor specific immune response in oral squamous cell carcinoma / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To examine the activation and cytotoxicity of human peripheral blood T lymphocyte induced in vitro by human 4-1-BB ligand (4-1-BBL) gene transfected into tumor Tca8113 cells.</p><p><b>METHODS</b>The eukaryotic expression vector pEGFP-h4-1-BBL was transfected into human oral carcinoma cell line Tca8113 by Lipofectamine 2000. The transfected cells were then selected in medium containing G-418, cloned by limited dilution and named as Tca8113-4-1-BBL. Human 4-1-BBL mRNA and protein expression of transfected cells was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting respectively. The tumor cell vaccines (TCV) were obtained by treatment with mitomycin (MMC). Human peripheral blood mononuclear cells (PBMC) were prepared from lymphoprep, and then stimulated with anti-CD-3 mAb and incubated with non-transfected or transfected TCV-Tca8113 cells, respectively. The proliferation of T cells was evaluated by trypan blue exclusion; the CCK-8 was used to detect the cytotoxic effect of T lymphocytes. Meanwhile, the secretion of interferon-gamma (IFN-gamma) and interleukin (IL)-2 in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The Tca8113 cells transfected by pEGFP-h4-1-BBL could express human 4-1-BBL efficiently. As compared with wild type Tca8113 cells, the transfected Tca8113 cells could markedly promote proliferation, IL-2 and IFN-gamma production and cytotoxic activity of lymphocytes.</p><p><b>CONCLUSIONS</b>The transfection of human 4-1-BBL gene in Tca8113 cells is effective in enhancing its immunogenicity and inducing antitumor immune response in vitro.</p>