[Detection of Toxoplasma gondii DNA by loop-mediated isothermal amplification].

OBJECTIVE: To detect Toxoplasma gondii DNA by loop-mediated isothermal amplification (LAMP). METHODS: DNA was extracted by phenol-chloroform extraction from T. gondii tachyzoites. Four primers which recognized 6 distinct regions on the B1 gene of T. gondii were designed and used for LAMP assay. To evaluate the specificity of the method, Plasmodium vivax, P. falciparum, Pneumocystis carinii, Schistosoma japonicum, and mouse leucocytes were used gs controls. The parasite extract (T. gondii) was 10-fold serially diluted for evaluating the sensitivity of LAMP, and was amplified by LAMP. LAMP results were read with naked eye and analyzed by electrophoresis. RESULTS: After LAMP reaction, positive amplification was observed with T. gondii, but no positive signal was noted for the negative controls in the study. The sensitivity of LAMP assay reached up to 2-3 T. gondii tachyzoites/ml per reaction. CONCLUSION: LAMP assay shows proper specificity and sensitivity for the detection of T. gondii.

[Construction of Sjcb2 DNA vaccine and its expression in HeLa cells].

A recombinant plasmid containing cathepsin B endopeptidase of Schistosoma japonicum (Sjcb2) was constructed, indentified by PCR, restrictive enzyme, digestion and DNA sequencing, and expressed into mammalian cells. Immunochemistry examination showed that the Sjcb2 gene can be expressed in the eukaryotic system, providing a basis for the development of schistosome DNA vaccine.

[Induction of protective immunity against Schistosoma japonicum by pcDNA3.1/Sj20.8 nucleic acid vaccine in BALB/c mice].

Sj20.8 gene was amplified by PCR and inserted into eukaryotic expression plasmid pcDNA3.1 to construct recombinant plasmid pcDNA3.1/Sj20.8, which was then injected into the quadriceps femoris of the BALB/c mice. Results showed that the Sj20.8 antigen was low expressed in the local tissue of the mice, and was not able to significantly reduce eggs in the liver than in the control mice.

[Acquisition and analysis of 149 ESTs and 18 novel genes of Schistosoma japonicum].

OBJECTIVE: To acquire and analyze adult stage Schistosoma japonicum (Chinese strain) expressed sequence tags and new genes from an adult S. japonicum cDNA library, and to search new vaccine candidates and drug targets. METHODS: A cDNA library was constructed from adult stage S. japonicum. Clones were selected randomly from the cDNA library and were sequenced. ESTs and new genes were acquired after analysis in GenBank databases by BLAST and other programs. All ESTs and new genes were submitted to GenBank and received accession numbers. RESULTS: 149 ESTs were acquired from a total 382 clones that were randomly selected from the adult S. japonicum cDNA library. All ESTs were successfully submitted to the dbEST at Genbank. Some of them were homologous with sequences of male, female, egg, schistosomula, cercaria and miracidia of S. japonicum. 18 new genes of adult S. japonicum were acquired. Some genes were housekeeping genes and some genes might be interesting as vaccine candidates or drugs targets. CONCLUSIONS: The EST strategy is a rapid, efficient and economical method to acquire ESTs and to discover new genes of adult stage S. japonicum from cDNA libraries.

The structural changes of the bagasse hemicelluloses during the cooking process involving active oxygen and solid alkali.

This work describes the structural changes of bagasse hemicelluloses during the cooking process involving active oxygen (O(2) and H(2)O(2)) and solid alkali (MgO). The hemicelluloses obtained from the bagasse raw material, pulp, and yellow liquor were analyzed by high-performance anion-exchange chromatography (HPAEC), gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FT-IR), and (1)H-(13)C 2D hetero-nuclear single quantum coherence spectroscopy (HSQC). The results revealed that the structure of the bagasse hemicelluloses was L-arabino-(4-O-methylglucurono)-D-xylan. Some sugar units in hemicelluloses were oxidized under the cooking conditions. Additionally, the backbones and the ester linkages of hemicelluloses were heavily cleaved during the cooking process.

[Rapid detection of norwalk GII with reverse transcription loop-mediated isothermal amplification].

To develop a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Norwalk GII. 4 primers which recognized 6 distinct regions on the RNA-dependent RNA polymerase gene of Norwalk GII were designed and used for LAMP assay. Norwalk GII RNA was amplified under isothermal conditions (65 degrees C) for 120 min, and LAMP results were then judged with naked eye, SYBR Green I staining, electrophoretic analysis and restriction digestion. To evaluate the specificity of the RT-LAMP, 48 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses were tested. To compare the sensitivity of the RT-LAMP with that of conventional RT-PCR, Norwalk GII RNA was serially diluted and amplified by RT-LAMP and RT-PCR, respectively. With 46 fecal specimens of Norwalk GII, observation with naked eyes, SYBR Green I staining and electrophoretic analysis were able to detect the PCR products in the RT-LAMP assay. The specificity of RT-LAMP products was also confirmed by digestion of the RT-LAMP products with restriction enzymes. No RNA amplification was observed in 2 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses. The specificity of the RT-LAMP assay with regard to RT-PCR were 100% for Norwalk GII. The detection limits of RT-LAMP was 15.6 pg/tube for Norwalk GII and similar to that of a RT-PCR assay. Compared to RT-PCR, the RT-LAMP assay has been proven to be a rapid, sensitive, specific and accurate method for detection of the Norwalk GII in fecal specimens, and that RT-LAMP assay is potentially useful for the rapid detection of Norwalk GII from fecal specimens in outbreaks of infectious diarrhea.