[Elimination of PCR inhibitors in routine diagnostic real-time PCR assay and results of internal amplification control].
Polymerase chain reaction (PCR) inhibitors which can be found in the clinical specimens lead to increased cost of these tests and also cause delay in the results particularly in the routine laboratories. Lysis/dilution methods are effective on the removal of PCR inhibitors and the performance of internal amplification control (IAC). This study was aimed to evaluate the effects of some methods, such as lysis (freezing and thawing) and dilution (1/10) of serum samples, used for the removal of PCR inhibitors, on IAC results. This evaluation was done by investigating the results of 1440 HCV-RNA and 2754 HBV-DNA (Fluorion HCV QNP and HBV QNP; Iontek, Turkey) tests that were performed in our laboratory during January 2005-October 2006 period. The nucleic acid isolation was done by "spin colon" (Qiagen, QIAamp DNA Mini Kit, Germany) and "magnetic particle" (Qiagen, BioRobot EZ1, Germany) technologies and PCR was performed by real-time PCR (iCycler IQ - BioRad Lab., USA). False negative IAC was detected in 211 samples during HCV-RNA tests and correct results were obtained in 66.4% of these when inhibitors were removed by lysis in 121 and by serum dilution in 19 of the samples. For HBV-DNA tests false negative IAC was detected in 15 samples and application of lysis method yielded correct results in 73.3% (11/15) of these. By the application of inhibitor removal methods the rate of false negative IAC decreased from 14.6% to 4.9% (71/1440) in HCV PCR and from 0.5% to 0.1% (4/2754) in HBV PCR. These data indicated that lysis/dilution methods were simple, economical and effective methods that could be used in routine PCR laboratories for the removal of PCR inhibitors and to achieve effective IAC.
Sayan
Murat
M
Meriç
Meliha
M
Celebi
Suna
S
Willke
Ayşe
A