Validation Of Multiplex PCR Assay Research Articles

Development and validation of multiplex PCR assay for differentiating tunas and billfishes.

Commercially available tunas and billfishes are generally processed as steaks, making it difficult to visually distinguish between the two. We developed and validated species-specific primers to prevent the adulteration of tunas by billfishes. Tunas and billfishes primers were designed on the cytochrome oxidase subunit I. Multiplex PCR bands obtained were 579bp, 291bp and 114bp for tunas, billfishes and internal control. Sensitivity was determined to be 5ng for tunas and billfishes. A total of 50 samples were monitored: 49 for tunas and 1 for billfish. As a result of the monitoring, the fake tunas did not show due to the agreement between product name and the raw material of the wrapping paper. Our results indicate that the species-specific primers developed in this study are suitable for differentiating tunas and billfishes. The newly developed multiplex PCR assay is a time and cost effective technique for determining the authenticity of tunas and billfishes.

Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines

This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10-6, 10-6 and 10-4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.