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. 2017 Mar 14;109(4):djx007. doi: 10.1093/jnci/djx007

Table 1.

Laboratory-developed tests for quantification of EBV DNA

Assay parameters Sites/laboratories
Stanford University Medical Center University of Washington/Harborview Medical Center Viracor-IBT Laboratories Primex Clinical Laboratories Mayo Clinic Brigham and Women’s Hospital
Specimen type EDTA plasma Blood, plasma, serum, CSF, DBS, tissue Plasma, serum, whole blood, bone marrow, CSF, lower respiratory sample, tissue, fecal Nasopharyngeal swab Whole blood (for quantitative PCR); whole blood, body fluid (eg, CSF, amniotic, ocular), respiratory fluid (eg, bronchial washing, BAL fluid, NP aspirate, sputum), bone marrow, fresh tissue (for qualitative PCR) Whole blood
Specimen volume 1 mL 3 mL blood, 1 mL CSF, 1 full circle DBS Varies 500 µL 200 µL required 50 µL
EBV DNA extraction method

  • Automated magnetic bead-based method

  • QIAsymphony DSP Virus/Pathogen Midi kit

  • Cellfree 1000 protocol

 Automated magnetic bead-based method, with pre-extraction lysis step if DBS or tissue is the source material, depends on specimen type Easy mag or QiaCube, optimized by specimen type Automated Roche Roche MagNA Pure LC 2.0 Automated total nucleic acid isolation
Extraction instrument (if automated) Qiagen QIASymphony SP/AS Roche MP96 or Magnapure, Promega Maxwell 16 Easy mag or QiaCube- optimized by specimen type Roche Magnapure Roche MagNA Pure LC 2.0 Roche MagNA Pure 2.0
Elution volume 90 µL 100 µL varies by specimen type 100 µL 100 µL 100 µL
PCR reaction volume 25 µL 50 µL; 32.2 µL (for EBV IR1 [large internal repeat]) _* 25 µL 20 µL 20 µL
Eluate input volume 10 µL 20 µL; 15 µL (for EBV IR1) _ 10 µL, samples standardized for DNA content prior to PCR 5 µL 5 µL
PCR enzyme Qiagen Artus EBV (for routine); Roche FastStart Taqman Probemaster (for BamHI W assay for NPC) Applied Biosystems AmpliTaq DNA Polymerase; Bio-Rad Sso7d fusion polymerase (for EBV IR1) _ Taq polymerase Roche FastStart Taq polymerase
PCR mastermix Qiagen Artus EBV (for routine); Roche FastStart Taqman Probemaster (for BamHI W) In-house PCR mix; Bio-Rad SsoAdvanced Probes Supermix with ROX (2x) (for EBV IR1) _

  • Thermo Taqman Universal Mastermix

  • includes Uracil N-glycosylase (UNG)

 Roche analyte-specific reagents Qiagen artus EBV LC PCR kit (product has been discontinued by manufacturer)
EBV gene target(s) for PCR amplification EBNA-1 (for routine); BamHI W (multicopy) EBER (single copy); EBV IR1 gene (multicopy) EBNA-1 and EBER - dual target EBNA-1; EBER1/EBER2 (single copy) Latent membrane protein (single copy) 97 bp region of EBNA-1
PCR chemistry TaqMan TaqMan TaqMan _ FRET Hydrolysis
PCR instrument Qiagen Rotor-Gene Q ABI 7500; ABI StepOne (for EBV IR1) ABI QuantStudio 12K LightCycler 2.0 LightCycler 2.0
Cycling conditions

  • 95C (10 min),

  • 45 cycles (95C [15 sec], 65C [30 sec]) (touchdown 10 cycles 1C) 72C [20 sec]) (for routine); 95C (10 min), 45 cycles (95C [15 sec], 56C [30 sec])

  • (for BamHI W)

 50C (2 min), 95C (2 min), 45 cycles (95C [20 sec], 60C [1 min]); (for EBV IR1) 50C (2 min), 95C (20 sec), 45 cycles (95C (3 sec), 60C [30 sec]) _ 50C (2 min), 95C (10 min), 40 cycles (95C (10 sec), 60C (1 min) _ 45 cycles (3 steps each [95C, 55C, 72C])
Calibrator composition Primary standard WHO, secondary standard = plasmid, Namalwa Acrometrix EBV Panel – used to calibrate plasmid standard curve, Raji Primary standard WHO, secondary standard = plasmid

  • EBV virus particles in plasma

  • (Acrometrix EBV plasma panel)

  • 100 to 1 000 000 copies

 Roche calibrators (200 000 000 copies/mL to 2000 copies/mL), Acrometrix EBV DNA plasmid
Calibrator extracted/not extracted Extracted (WHO standard), not extracted (plasmid) Extracted Extracted (WHO standard), but not with every run Extracted Not extracted Not extracted
Harmonization to IU/mL units (yes/no) Yes (calibrated to WHO standard but awaiting international sites to adopt IU/mL units before implementing) Yes Yes No Not currently (data acquired to convert to IU/mL using either WHO or Acrometrix standards) No
Unit Copies/mL and IU/mL IU/mL IU/mL Copies/mL, Ct value Copies/mL _
Assay contains internal control (yes/no) Yes (for routine), no (for BamHI W) Yes Yes Yes Yes Yes (provided in Qiagen PCR kit)
Quantitative/qualitative Quantitative for routine tests; reported as qualitative for NRG-HN001 trial Quantitative for plasma, CSF, DBS; qualitative for tissue Quantitative for multiple specimen types; qualitative for fecal and tissue specimens Quantitative Quantitative for whole blood; qualitative for multiple specimen types _
*

No response provided in survey. CSF = cerebrospinal fluid; DBS = dried blood spots; EBNA-1 = Epstein-Barr nuclear antigen 1; EBV = Epstein-Barr virus; IU = international unit; NPC = nasopharyngeal carcinoma; PCR = polymerase chain reaction; WHO = World Health Organization.