Validation and optimization of a novel internal control for the TaqMan qPCR in Hepatitis B viral load diagnostic

Development of rapid amplification assays for the detection and identification of biological threat agents has become a focus of diagnostic efforts in recent years. The use of Real-Time PCR as diagnostic tools depends upon two critical processes. Differentiation must be made between results achieved due to the lack of target nucleic acid and those produced due to the inability to amplify target DNA so confidence in negative reactions is possible. False negatives can occur when inhibitors are present in the sample being tested, especially if clinical samples such as blood are analyzed. To address the problem of detecting inhibition in purified nucleic acids, an exogenous internal control (IC) based on TaqMan chemistry was developed. The 71bp gene construct was designed as a loop. The construct has a universal forward primer a probe for the sense region and did not match the specificity of any genomic microorganisms. At the beginning of the 3’ construct, a part of the gene region of the HBV has been designed, whose primer is in reverse mode with a specific HBV primer. If there is a HBV genome in the patient s sample, gene construct competed with primers of the targeted region of the HBV virus. If the sample is negative, the JOE signal will be released at the optimized concentration of the gene construct. This contact has been detecting one copy of target as high sensitive limit of detection by validation approach. Using an internal control in the molecular detection of HBV as an IC, it can detect errors and standardize this technique.

Keywords:

HBV , Internal Control , TaqMan , Probe , Real Time PCR , validation