[show abstract][hide abstract] ABSTRACT: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA.
We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients.
The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R(2)=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (Kappa=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively).
The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.
The Korean Journal of Laboratory Medicine 08/2007; 27(4):298-304. · 0.72 Impact Factor
[show abstract][hide abstract] ABSTRACT: Based on sequence variation in the N-terminus of the UL55 gene, which encodes glycoprotein B (gB), human cytomegalovirus (CMV) can be classified into four gBn genotypes. We assessed the distribution of CMV gBn genotypes and the correlation between CMV gBn DNA (detected by real-time PCR) and CMV-positive pp65 cells (identified by immunohistochemical staining) in a cohort of hematopoietic stem cell transplant patients. The distribution of gB genotypes was as follows: gBn1, 60% of patients; gBn2, 13.3%; mixed gBn1 and gBn3 infection, 26.7%; and gBn4 and other mixed infections, 0%. CMV gBn1 was the most common genotype. The detected level of CMV gB DNA correlated well with the number of CMV-positive pp65 cells detected by immunostaining (r = 0.585).
PLoS ONE 01/2012; 7(12):e51224. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Real-time PCR has been widely considered as a powerful tool for the evaluation of Human Cytomegalovirus (CMV) DNA kinetics. Successful PCR relies on optimization, which is an extremely demanding procedure. Nevertheless, certain values could be optimal for most primers in use.
Seventeen CMV primer sets recommended in the literature were selected for optimization in terms of MgCl2 and primers concentrations as well as annealing temperature using the LightCycler instrument and SYBR Green I detection format. Optimal values were considered as those showing the lowest crossing point (Cp), the highest fluorescence intensity, the steepest sigmoid curve slope, and the absence of non-specific PCR products.
Optimal values for most studied primers were found to be 3 mM for MgCl2 concentration, 0.5 microM and 0.6 microM for primers concentration, and 55 degrees C for annealing temperature.
Adopting the resulting values for CMV-specific primers generally used in single-target real-time PCR assays with the same thermal cycler may guarantee their efficient performance minimizing cost and time needed for optimization.
The Journal of Infection in Developing Countries 02/2008; 2(5):406-10. · 1.00 Impact Factor
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