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Seminar 763  
Date:2008-03-12
Update:2014-01-09

Seminar:  763  

1

Speaker(s)

CH Pan

Topic

A Visual DNA Chip for Simultaneous Detection, Genotyping and Differentiation of Wild-type and Vaccine-type Classical Swine Fever Virus.

Abstract

Routinely, RT-PCR followed by DNA sequencing has been the method used to detect classical swine fever virus (CSFV) and exclude the interference of vaccine viruses for the field cases. Here, a DNA Chip assay was developed to enable simultaneous detection, genotyping and differentiation between wild and vaccine-type CSFV. Specific oligonucleotide primers and probes were designed in the 3' non-translated region of the CSFV genome. One-step RT-PCR amplification was performed with biotin-labeled primers, followed by hybridization to the DNA probe immobilized on the plastic chip. The DNA chip can not only accurately differentiate between the three major genotypes of CSFV, but can also discriminate between wild-type and vaccine-type CSFV. The limit of detection for wild-type virus was 10 TCID 50 /mL for RT-PCR and 1 TCID 50 /mL for the DNA Chip. The sensitivity of the visual DNA Chip was 10 times higher than that of the RT-PCR confirmed by agarose gel. We conclude that RT-PCR coupled with DNA probe hybridization provides a highly sensitive diagnostic tool for genotyping CSFV and discriminating between wild-type and vaccine-type CSFV in clinical samples. The whole procedure, including the RT-PCR and the 2 hours hybridization, takes just 6 hours, which is significantly faster than the VI or RT-PCR followed by DNA sequencing.

2

Speaker(s)

F Lee

Topic

Use of analytical sensitivity for evaluating the detection of antibodies to foot-and-mouth disease virus and bluetongue virus

Abstract

The commonly refered terms "sensitivity" and "specificity" are diagnostic sensitivity and diagnostic specificity used to calculate probabilities of currectly differentiating diseased animals from non-diseased ones by a given assay. In contrast to the diagnostic sensitivity, analytical sensitivity represents the smallest amount of substance in a sample that can accurately be measured by an assay. Analtyical sensitivity is different from but can influence diagnostic sensitivity. In today's presentation, a few examples of use of analytical sensitivity to evaluate ELISAs for antibody detection will be adduced. It is believed that the analytical sensitivity, as the same as the diagnostic sensitivity, is an important indication for evaluating serological assay.

3

Speaker(s)

Ching-hui Kuo

Topic

Development of reverse transcription real-time PCR for animal influenza viruses

Abstract

An epidemiological analysis was performed to determine the genotypes of 43 Salmonella enterica serovar Choleraesuis (6,7: c:1,5) isolates identified by biochemical characteristics and serological examinations. Genotyping was done by digestion of restriction enzyme ( Xba I, Spe I and Avr II) of whole genome and p ulsed-field gel electrophoresis (PFGE). Seven, eight and thirty five different genotypes were obtained individually, using the above three enzymes digestions and the PFGE analysis . One of seven patterns digested with Xba I shows the major group containing thirty five strains ( 81.3 ); 

One of eight patterns digested with Spe I shows the major group containing twenty seven strains ( 62.8 ) ; one of twenty five patterns digested with Avr II shows the major group containing only five strains ( 11.6 ). Twenty five different restricted patterns were found in combination with the results of the above three enzyme-digested patterns and the major genotype only contained five strains. T he discriminate index was 0.95. This study shows that PFGE is a highly stable and reproducible technique . In the future, we will use standardized protocols for PFGE to analyze strains and establish database for epidemiological study.