Loading...

Summer Research Fellowship Programme of India's Science Academies

PCR amplification of PFO-B gene using pGEMT easy vector

Mrunal Ratnaparkhi

Department of Microbiology, Vidya Pratishthan's Arts Science and Commerce College, Baramati 413102

Daman Saluja

Professor and Director, Dr B R Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007

ABSTRACT

Trichomoniasis is among the most prevalent sexually transmitted infections, caused by a protozoan parasite Trichomonas vaginalis. It infects the urogenital tract of both women and men. Itching, burning, redness or soreness of the genitals, discomfort during urination, white, yellow or greenish vaginal discharge with unusual smell are some of the common symptoms of this infection. Once diagnosed, the infection is treatable.

There are several diagnostic techniques like microscopic methods and antibody based methods but the NAAT based diagnosis at the genetic level ensures specificity and sensitivity.

The purpose of the study was to develop a positive control for NAAT based diagnosis of the infection using a fragment of PFO-B of T.vaginalis and pGEM-T Easy vector. To serve the purpose, the gene of interest was amplified following PCR method, amplification product was extracted from the agarose gel following electrophoresis and ligated into pGEM-T Easy vector. The ligation product was further used to transform the DH5-alpha strain of E.coli cells which was allowed to grow overnight and positive clones were selected for plasmid isolation.

The yield of isolated plasmid was found out to be 48.6, 150, 16.7, ng\µL. These clones were stored at -20℃ to be used later in diagnostic research.

Cloning was performed by, amplifiction of PFO-B gene followed by Gel electrophoresis and Ligation Transformation of recombinant plasmid with competent E.coli cells.

Thus, the product rom transformation after its analysis for characterization, showed desired results.

Keywords: trichomoniasis, NAAT diagnosis, pGEMT easy vector

More
Written, reviewed, revised, proofed and published with