Designing Two Synthetic Constructs for Real Time PCR Detection of and Virus.

BACKGROUND

Generally, timely diagnosis of micro-organisms is very important to prevent many diseases. Many methods can detect micro-organisms like culture-based methods and molecular methods. The molecular methods are usually preferred because they provide fast and reliable results. In some cases, microbial strains are not accessible, and there is no safety to work with them; therefore, synthetic constructs which are designed according to the available sequences in databases can be used as a positive control for detection of them.

METHODS

In this study, a synthetic construct was designed for molecular detection of () and the virus by multiplex real-time PCR reaction. For this, sequences were taken from databases and then multiple alignments were done by software. Also, conventional PCR and two models of real-time PCR (SYBR green and TaqMan) were applied. Finally, multiplex real-time PCR was performed.

RESULTS

The synthetic construct was designed and used for conventional PCR and multiplex PCR. The results of common PCR showed a single band at 148 and 167 in 1.5% agarose gel stained by ethidium bromide for and virus, respectively. Also, a dual-band at 148 and 167 was observed in multiplex PCR. Results of real-time PCR showed a limit of detection about 0.1 of plasmid/.

CONCLUSION

In conclusion, the designed construct can be used as a positive control for an accurate diagnosis of these micro-organisms without any biological danger for laboratory staff. So, this method is useful for diagnosis of these agents in food, water, and blood samples.