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To study the preparation of oligonucleotide microarray for detecting vaccinia virus.Oligonucleotide probes were designed and synthesized according to the specific genes of vaccinia virus. Sample DNA of the virus and the negative control sample were obtained and labeled by restriction display technique, followed by hybridization to the oligonucleotide microarray and scanned by Agilent scanner.Strong hybridization signals were detected from the viral DNA hybridized with the microarray, but were absent in the negative sample when positive probes were not used.Distinct differences in the hybridization signals between the virus sample and negative sample and between the samples obtained in different phase of infection demonstrate high specificity and sensitivity of the microarray for vaccinia virus detection.

To amplify and clone the N gene of severe acute respiratory syndrome-associated coronavirus.Using primer Premier 5.0 software, two pairs of nested PCR primers were designed to amplify the N gene. After purification, the amplified products were cloned into pMD18-T vectors, and the positive clones with the inserted fragments were identified by sequence analysis.The amplified products was about 1 375 bp in length, and sequence analysis demonstrated that the N gene fragments had been successfully inserted into pMD18-T vectors.The successful amplification and cloning of N gene facilitates further investigation of the expression of the N protein and study of its structure and functions.

To design oligonucleotide probes for microarray detection of dengue virus.By analyzing the cDNAs of dengue viruses of 4 different serotypes with BLAST program, a group of specific sequences for the candidate probes was acquired. Oligo6.0 software was applied to analyze the candidates to select the probes with high specificity, identical length and similar melting temperature (Tm).Altogether 48 oligonucleotide probes were designed, and deposited on oligonucleotide chips as the microarray for dengue virus detection.BLAST program and Oligo6.0 software are simple and effective means for designing the oligonucleotide probes.

To study the method for rapid preparation of the gene chip probes for Bacillus anthracis protective antigen (pag).According to the sequences of pag published on PubMed, a pair of primers was designed to amplify the PA gene of Bacillus anthracis. Restriction enzyme digestion and AT clone were employed to rapidly screen out the recons of PA segments for the preparation of the gene chip probes. The DNA sequences of these segments were determined with 377 DNA Sequencer, and the resultant sequences analyzed with Bioinformatic software.With the designed primers, the pag 2,205 bp in length was amplified. After enzyme digestion and AT clone, 7 fragments of varied lengths were screened, which underwent analysis with the basic local alignment sequence tool (BLAST) and 377 DNA Sequencer. BLAST analysis showed that all the fragments cloned belonged to Bacillus anthracis.PCR in conjunction with restriction enzyme digestion and AT clone is rapid and simple to prepare the desired gene chip probes.

To explore an effective means for the detection of Severe Acute Respiratory Syndrome (SARS)- associated coronavirus.The RNAs of the virus contained in the sputum samples from established SARS patients were extracted and reversely transcripted, followed by nested PCR using the reversely transcripted cDNA as the template. The PCR products were cloned then into the pMD18-T vectors, followed by sequence analysis.Specific fragments were amplified from the sputum samples of SARS patients, which were confirmed by DNA cloning and sequencing to belong to SARS-associated coronavirus. The Result of Blast shows only the difference in one nucleic acid from the TOR2 strain of SARS-associated coronavirus.Sequence analysis has confirmed the existence of SARS-associated coronavirus in the sputum samples of SARS patients, and nested RT-PCR is a quick, easy, and convenient way for the detection of the virus.