Designing and optimization of internal control for herpes simplex virus PCR test

Herpes Simplex Virus (HSV) is a member of the herpes virus family, Herpesviridae, which infect humans. There are different methods for detection of this virus like cultural ways which is time consuming, but molecular methods such as PCR are more appropriate for detection of HSV. Although nowadays molecular assays are using in laboratories but different results are achieved because of lack of standard methods which are counted as disadvantageous of molecular methods. For solving this issue in this study, designing of competitive Internal control(IC) through PCR-cloning has been attempted. primers for PCR test were optimized. Besides, the composite primers for IC-HSV were designed then the PCR was optimized. The IC-HSV which amplified, was ligated in pTZ57R plasmid then transformed in E.coli JM107 and was cloned. Specificity and sensitivity of test were determined. PCR amplicon for HSV and IC-HSV with special primers were 454bp and 662bp respectively, so there was a significant different between their size. The appropriate concentration of IC used in each reaction was 50 fg and the sensivity of HSV DNA with IC was between 1 million to 1000 virus particles. Despite of high speed and accuracy of PCR, false positive and negative results which are caused by PCR inhibitors, are the most important problems of this technique that can reduce its efficiency. Using another DNA as an internal control can detect these inhibitors. Indeed, amplification of this DNA shows correct amplification and detection steps.