Elimination of false negativity of internal amplification control in PCR assay
Abstract number: 1734_113
Sayan M., Yumuk Z., Çelebi S., Willke A.
Objectives: Polymerase chain reaction (PCR) is a nucleic acid amplification technique commonly used in routine laboratories as a rapid, sensitive and specific diagnostic tool. However PCR inhibitory components may effect the PCR assay and false negative results may be obtained. To detect false negativity, internal amplification control (IAC) is used in diagnostic laboratories, but various PCR inhibitors (such as heparin, heme, leukocyte DNA etc.) may adversely effect amplification procedure. In this study we evaluated the effectiveness of various pre-PCR processing procedures on IAC in paralel with the serum samples of hepatitis B and hepatitis C patients.
Methods: From January 2005 through October 2006, hepatitis C virus (HCV) RNA (fluorion HCV QNP v2.1 kit-Iontek Ltd. Sti, Istanbul-Turkey) and hepatitis B virus (HBV) DNA (fluorion HBV QNP v2.0 kit-Iontek Ltd. Sti, Istanbul-Turkey) tests were performed by quantitative real-time PCR (iCycler IQ, v 3.0a BioRad Laboratories, Hercules, CA-USA) to 1440 and 2754 patient samples respectively. We used silica-gel based spin column (SC) and silica-coated magnetic particle (MP) as extraction methods. After then, in the sera which was seen no amplification of IAC, we re-performed PCR tests after using; (1) freeze-thawing, (2) sample dilution, (3) a new sample and (4) a new lot of IAC consecutively.
Results: Totally 211/1440 (14.6%) of HCV RNA PCR assay (SC: 122/905, MP: 89/535) it was seen no amplification in the IAC. Considering HBV DNA PCR results, false negativity was found to be 15/2754 (0.5%) (SC: 6/1724, MP: 9/1030). Comparison of two extraction methods namely SC and MP for both HBV DNA and HCV RNA PCR tests there was no statistically significance related with false negativity. False negativity ratio in IAC has decreased from 14.6% to 6.0% in HCV RNA group and from 0.5% to 0.0% HBV DNA group after applying all four inhibitor elimination methods.
Conclusion: In routine laboratories in PCR assay, inhibitory substances may lead to several problems such as re-test the same sample, higher cost and delayed results. Therefore in this case, the test should be re-performed after freeze-thawing and diluted procedure. If no result obtained, it will be beneficial to re-performed the test with a new sample or new lot of internal control.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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