Molecular cloning of virB12 gene of Brucella melitensis 16M strain in pET28a vector

Objective:To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.Methods: Brucella melitensis(B.melitensis) 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep~ Genomic DNA Extraction Kit.Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII restriction site at 5’ end of the forward and reverse primers,respectively.DNA amplification was performed using PrimSTAR~ HS DNA polymerase and the PCK product was purified by DNA AccuPrepGel Purification Kit.Purified DNA was cloned into pJET1.2 cloning vector.VirB12 gene fragment was excised from pJET1.2 asing BamHI/HindIII and subsequendy subcloned into pET28a(+).Results:Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a(+) plasmids.PCR and restriction enzyme digestion confirms the procedure.Conclusion:We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.

A comparison of culture and PCR methods for identification of Aggregatibacter actinomycetemcomitans isolated from acute necrotizing ulcerative gingivitis

Objective: To determine Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) isolated from periodontal patients and healthy subjects using culture and PCR methods. Methods: Duplicate paper point needles were taken from 100 samples (50 healthy subjects and 50 patients), who referred to the specialized dental clinic from Oct. 2015 to Mar. 2016. In laboratory after incubation period and observing the star-shaped colony A. actinomycetemcomitans, the confirmation tests, including gram staining and catalase test were carried out. For PCR, samples were analyzed with genus specific primers. These primers set, amplified a 500 bp fragment. Results: Of the 100 samples, A. actinomycetemcomitans was isolated from 31 patients (31%), (24 isolate of patients, and 7 isolate of healthy subjects) by using a selective Aggregatibacter isolation medium. Using PCR, a total of 49 (49%) samples were found to be positive for A. actinomycetemcomitans (35 isolate of patients, and 14 isolate of healthy subjects). Conclusion: PCR was found to be highly sensitive when genus specific primers were used for diagnosis of A. actinomycetemcomitans in comparison with culture method.