Designing a diagnostic PCR kit for detection of yersiniosis (Yersinia ruckeri) in rainbow trout in Iran

Objective: In a diagnostic PCR test, the quality and quantity defect of PCR material or presence of PCR inhibitory substances in each reaction is possible and may cause for receiving false negative result. The use of internal amplification in each vial has been suggested to overcome this problem. All recent developed PCR kits for diagnosis of Y. ruckeri, lack this internal control. In this research our purpose was to minimize receiving false negative result in diagnostic PCR test. Method & Materials: Experiments steps were as followed: 1. Sampling from Y. ruckeri isolation and suspected infected fish tissue with Y. ruckeri. 2. DNA extraction from the samples of Y. ruckeri isolation and suspected infected fish tissue. 3. Designing primers for Pathogen and for rainbow troth. 4. Examination of Y. ruckeri specific PCR assay and Oncorhynchus mykiss specific PCR assay as an internal control. 5. Multiplex PCR. Two pairs of primers were designed for host and pathogen and their function were assayed in simple PCR reaction separately. Both designed primer sets were used in a Multiplex PCR. Results & Conclusion: 1- Amplification of DNA from O. mykiss, as a reaction control, and from Y. ruckeri, in other word, obtaining positive results, indicated that condition and material of PCR reaction were optimal. 2- Appearance of O. mykiss DNA amplification as an internal control showed the optimal condition of reaction. Therefore, false negatives and negative result can be differed from each other. This is one advantage of this Y. ruckeri diagnostic kit. 3- There was no band of O. mykiss DNA as the internal control and Y. ruckeri DNA. Such pathogen negative result can not be reliable and means that PCR reaction went wrong, perhaps due to various reasons such as quality and quantity defect of PCR material or presence of PCR inhibitory substances in each reaction. It seems that all different conventional Y. ruckeri diagnostic PCR assays exclude an internal amplification control, which is necessary to make a confident distinction between from truly negative and false negative result.Therefore, new PCR assay was developed by adding an internal control in each PCR reaction for recognition of the false-negative result, whereas the other Y. ruckeri diagnostic PCR assays were not able to make this distinction.