In genome sequencing project, we encounter the DNA regions that often contain stable secondary structure with high GC content. These regions are difficult to not only amplify by PCR for template preparations, but also deter mine the DNA sequences using standard Cycle sequencing (CS) method. Transcriptional sequencing (TS) is a unique DNA sequencing method using RNA polymerase, and is based on the principles of the chain-termination method, which is a powerful method to analyze GC-rich sequences. In this study, we examined the multiple displacement amplification (MDA) to overcome low efficiency of PCR amplification in GC-rich regions and subjected to TS reaction. Combination of MDA and TS (MDA-TS) was extremely successful with GC content ranging from 65 % to 85%, which are difficult to analyze with PCR and CS. We also report plasmid vector, pTS1, which has the stronger T7 and T3 promoters than those of conventional vectors, and the sequence that decreases transcriptional efficiency was removed from its multiple cloning sites. pTS1 resulted in the improved sequencing accuracy and reduced reaction time up to 5 min. These results showed that MDA-TS is a rapid and accurate method for the analysis of GC-rich templates.

To develop ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the carboxyl termini of the 34 kDa proteins of M. paratuberculosis and Mycobacterium avium subsp. avium (M. avium) were expressed in Escherichia coli expression system. Antibodies specific to M. paratuberculosis were detected with the truncated 34 kDa protein of M. paratuberculosis in ELISA after pre-absorption of serum samples with the truncated 34 kDa protein of M. avium. All the serum samples from cattle confirmed to be infected with M. paratuberculosis were positive and those from healthy cattle were negative in the present ELISA system. These results indicate that the established ELISA detects antibodies specific to M. paratuberculosis with high specificity and sensitivity and is an useful tool for the screening of Johne's disease.

Bovine nasal and oral discharges were used as samples for bovine viral diarrhea virus (BVDV) gene detection. Viral genes in serum (S), nasal discharge (N) and oral discharge (O) were quantified with real-time polymerase chain reaction using SYBR Green by the relative quantification method, and findings were compared among samples. Although the quantity of the BVDV gene in S was greater than those in N and O, all samples were available to identify persistently infected (PI) cattle with BVDV by reverse transcription polymerase chain reaction (RT-PCR). The swab samples were able to be stored for a few days at 4degC with a little decrease of amplification signal in RT-PCR. Oral swab sampling was easier than nasal swab sampling, and was also less uncomfortable for the cattle than other sampling methods without pain or unnecessary retention. This sampling method can be performed without any special technique and equipment. Therefore, the oral swab sampling method is useful for screening to detect BVDV PI cattle by RT-PCR.