Objective:To establish fusion PCR for amplification of the full-length cDNA of dengue virus type 2. Methods:According to the published nucleotide sequence of D2-43,the primers were devised and the 5′ and 3′ half genomic cDNAs of dengue virus type 2 were amplified by long reverse transcription PCR. Using the PCR products as model,the approximate 11 kb full-length cDNA was amplified by fusion PCR. The sequence containing the 5′ noncoding region was determined by PRISMTM ABI 377 automated sequencer.Results:Using fusion PCR,the full-length cDNA of dengue virus type 2 was successfully amplified and its correctness was proved by partial nucleotide sequences analysis. To our best knowledge, this is the first report of the same kind.Conclusion:Fusion PCR is an effective method to amplify the genomic cDNA of dengue virus.

OBJECTIVETo develop an HPLC method for determining four components in Sini Tan, benzoylmesaconine, liquiritin, glycyrrhizic acid and 6-gingerol.

METHODThe Hypersil BDS column was adopted with gradient elution program at a flow rate of 1.0 mL x min(-1) and the detection wavelength of 235 nm.

RESULTBenzoylmesaconine, liquiritin, glycyrrhizic acid and 6-gingerol showed good separation, with the linear range of 0.006-0.12, 0.021-0.42, 0.012-0.24 and 0.018-0.36 g x L(-1), respectively. Their average recoveries were 99.3%, 96.9%, 100% and 100%, respectively; and RSD of the above four components were 1.5%, 0.6%, 1.3% and 2.1%, respectively.

CONCLUSIONThe method is proved to be so easy and accurate and practical that it can be used to determine the four components in Sini Tang.

Aconitine ; analogs & derivatives ; analysis ; isolation & purification ; Anti-Inflammatory Agents ; analysis ; isolation & purification ; Catechols ; analysis ; isolation & purification ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Fatty Alcohols ; analysis ; isolation & purification ; Flavanones ; analysis ; isolation & purification ; Glucosides ; analysis ; isolation & purification ; Glycyrrhizic Acid ; analysis ; isolation & purification ; Medicine, Chinese Traditional ; Reproducibility of Results

Cimicifugae Rhizoma (Sheng ma) is a Ranunculaceae herb belonging to a composite family and well known in China. has been widely used in traditional Chinese medicine. Thecontains three varieties ((Turcz.),L. andKom.) which have been used clinically as "Sheng-ma". However, the chemical constituents of three components of "Sheng-ma" have never been documented. In this study, a rapid method for the analysis of the main components of "Sheng-ma" was developed using ultra-high performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The present study reveals the major common and distinct chemical constituents of,andand also reports principal component and statistical analyses of these results. The components were identified by comparing the retention time, accurate mass, mass spectrometric fragmentation characteristic ions and matching empirical molecular formula with that of the published compounds. A total of 32 common components and 8 markers for different "Sheng-ma" components were identified. These findings provide an important basis for the further study and clinical utilities of the three "Sheng-ma" varieties.

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.
Genome, Viral ; Haplotypes ; Humans ; Mutation ; Open Reading Frames ; Phylogeny ; SARS Virus ; genetics

We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.
Base Sequence ; China ; Cluster Analysis ; Gene Components ; Genetic Variation ; Genome, Viral ; Genotype ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; Sequence Analysis, DNA ; Severe Acute Respiratory Syndrome ; genetics