Journal List > Lab Med Online > v.6(1) > 1057327

Ha, Park, Shim, and Kim: Evaluation of Real-time PCR Kits for Epstein-Barr Virus DNA Assays

Abstract

Background

Epstein-Barr virus (EBV) is known to be the causative agent of infectious mononucleosis and EBV-related malignancies. In this study, we compared the results of three real-time PCR kits for EBV DNA assays.

Methods

A total of 300 whole blood samples submitted for quantitative EBV PCR between January 2013 and September 2014 at Severance Hospital were included. The samples were tested by using the Artus EBV RG PCR Kit (Qiagen, Germany), AccuPower EBV Quantitative PCR Kit (Bioneer, Korea), and Real-Q EBV Kit (BioSewoom, Korea). Samples with discordant results between the three kits were confirmed by direct sequencing.

Results

The result concordance rate and kappa coefficient (K) were 86.3% and 0.69 for Artus-AccuPower, 93.3% and 0.85 for Artus-Real-Q, and 92.3% and 0.83 for AccuPower-Real-Q, respectively. The correlations between the three kits were found to be significant, with a correlation coefficient of r=0.854 for Artus-AccuPower, -0.802 for Artus-Real-Q, and -0.977 for AccuPower-Real-Q, respectively (P<0.0001). If the real-time PCR concordant results of 258 samples and the direct sequencing results of 42 real-time PCR discordant samples were assumed to be true, the sensitivity/specificity values were 0.921/0.976 for Artus, 0.902/0.965 for AccuPower, and 0.967/1.000 for Real-Q.

Conclusions

The three real-time PCR kits showed excellent sensitivities and specificities. All these kits would be acceptable for clinical and therapeutic management of EBV. However, some discordant results between the kits indicate the need for caution in clinical diagnosis and staging. Further implementation of standardized methodology would be needed for EBV DNA assays.

Figures and Tables

Fig. 1

Correlation of EBV DNA loads or Ct values between EBV PCR kits in 178 positive samples.

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Table 1

Characteristics of the EBV PCR kits evaluated in this study

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Real-time PCR kit Artus AccuPower Real-Q
Amplification method Real-time PCR Real-time PCR Real-time PCR
Instrument Rotor-Gene Q Exicycler 96 CFX 96
Target sequence EBNA-1 Gp220 EBNA-1
Probe technology Hydrolysis Hydrolysis Hydrolysis
Type of result Quantitative Quantitative Qualitative
LOD 510 copies/mL 265.6 copies/mL 110 copies/mL
Measurement range (copies/mL) 2.5×104-2.5×107 4×102-4×1011 125-5×1011
No. of PCR cycles 45 cylcles 45 cylcles 45 cylcles
Assay time 1 hr 40 min 1 hr 40 min 2 hr

Abbreviation: LOD, limit of detection.

Table 2

Results of the EBV PCR kits

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Qualitative results N Sequencing results Quantitative results*
Artus AccuPower Real-Q Artus (log10Copies/mL) AccuPower (log10Copies/mL) Real-Q (Ct)
Positive Positive Positive 178 4.41 (3.88-5.87) 4.51 (3.91-5.48) 31.50 (28.94-33.45)
Negative Negative Negative 80 - - -
Positive Negative Positive 16 Positive: 16 3.31 (3.20-3.58) - 37.13 (36.37-37.93)
Negative Positive Positive 13 Positive: 13 - 3.84 (3.24-4.36) 34.44 (33.95-35.73)
Negative Positive Negative 6 Positive: 4, Negative: 2 - 2.79 (2.74-2.85) -
Positive Negative Negative 6 Positive: 3, Negative: 3 3.26 (3.12-3.59) - -
Negative Negative Positive 1 Positive: 1 - - 33.60
300 4.33 (3.68-5.46) 4.41 (3.79-5.38) 32.14 (29.20-34.16)

*Values are presented as median (1st to 3rd quartiles).

Notes

This article is available from http://www.labmedonline.org

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