Background
Methods
Groups with EBV-associated diseases or immune disorders
Population sample
Serology testing
EBV-specific antibody detection in the population sample
Molecular testing
EBV gene targets, beta-globin and PCR controls
Target | Primer Name | Oligonucleotide Sequence 5'-3' | Amplicon Length | GenBank Accession (position) | Reference | Optimised PCR Thermal Cycling Conditions |
---|---|---|---|---|---|---|
EBNA-1
| QP1 | GCC GGT GTG TTC GTA TAT GG | 213 bp | AJ507799 (97174-97386) | Stevens et al, 1999 | 95°C initial denaturation for 10 mins; 58°C annealing |
QP2 | CAA AAC CTC AGC AAA TAT ATG AG | |||||
BHRF-1
| EA-1F | GGA GAT ACT GTT AGC CCT G | 208 bp | AJ507799 (42105-42312) | Custom | 98°C initial denaturation for 13 mins; 60°C annealing |
EA-2R | GTG TGT TAT AAA TCT GTT CCA AG | |||||
Plasmid construct (randomised primers in bold)
| EA-F | CTA TAT GTC TGC TTA CTC CGG CG /G GAG ATA CTG TTA GCC CTG | 554 bp | N/A | Custom | 95°C initial denaturation for 10 mins; 55°C annealing |
EB-R | CGC CGG AGT AAG CAG ACA TAT AG /CAA AAC CTC AGC AAA TAT ATG AG | 95°C initial denaturation for 10 mins; 55°C annealing | ||||
Beta-Globin TAL57
| BG-1F | TAG CAA CCT CAA ACA GAC ACC A | 247 bp | EU760960 (171-417) | Custom | 95°C initial denaturation for 10 mins; 61°C annealing |
BG-1R | CAG CCT AAG GGT GGG AAA AT |
DNA extraction and molecular assay design
Cloning of EBNA-1 and BHRF-1 DNA targets into plasmid vector pGEM and standard curve construction
Product identification, reproducibility, sensitivity, limit of detection and specificity
Viral load calculation and result interpretation
Statistical calculations
Results
Performance of EBV QPCR assays: reproducibility, sensitivity, detection limit and specificity
DNA Target (copies/5 ul) | Mean CT | Mean R-G 6000™ Results (copies/5ul) | Standard Deviation of R-G 6000™ Results (copies/5ul) | Mean % Variation | COV (%) | Mean R2 |
---|---|---|---|---|---|---|
EBNA-1 Intra-Assay Variation (same day)
| ||||||
100,000 | 18.03 | 87,329 | 6,670 | 12.68% | 7.64 | 0.991 |
10,000 | 21.28 | 11,735 | 3,092 | 26.30% | 26.34 | |
1,000 | 25.21 | 1,057 | 100 | 7.00% | 9.50 | |
100 | 29.10 | 103 | 38 | 32.12% | 37.25 | |
10 | 32.91 | 11 | 6 | 46.16% | 57.50 | |
EBNA-1 Intra-Assay Variation (different days)
| ||||||
100,000 | 16.94 | 89,643 | 8,164 | 11.00% | 9.11 | 0.998 |
10,000 | 20.31 | 10,678 | 1,207 | 10.00% | 11.31 | |
1,000 | 23.85 | 1,133 | 129 | 16.00% | 11.41 | |
100 | 27.65 | 102 | 4 | 3.00% | 3.90 | |
10 | 31.42 | 10 | 2 | 17.88% | 18.95 | |
BHRF-1 Intra-Assay Variation (same day)
| ||||||
100,000 | 17.23 | 97,884 | 9,144 | 8.08% | 9.34 | 0.994 |
10,000 | 20.91 | 9,852 | 542 | 4.45% | 5.50 | |
1,000 | 24.38 | 1,146 | 202 | 16.12% | 17.64 | |
100 | 28.43 | 94 | 19 | 17.70% | 20.51 | |
10 | 31.95 | 11 | 5 | 35.38% | 41.91 | |
BHRF-1 Intra-Assay Variation (different days)
| ||||||
100,000 | 18.05 | 105,387 | 4,621 | 6.02% | 4.38 | 0.997 |
10,000 | 21.75 | 9,779 | 818 | 6.23% | 8.37 | |
1,000 | 25.23 | 1,042 | 141 | 11.63% | 13.55 | |
100 | 29.06 | 89 | 18 | 13.76% | 19.88 | |
10 | 32.66 | 12 | 2 | 25.30 | 16.53 | |
EBNA-1 Inter-Assay Variation
| ||||||
100,000 | 19.87 | 101,644 | 14,058 | 10.99% | 28.35 | 0.990 |
10,000 | 23.75 | 10,660 | 1,471 | 13.65% | 13.80 | |
1,000 | 27.68 | 1,084 | 191 | 18.67% | 17.60 | |
100 | 31.71 | 111 | 49 | 33.58% | 43.75 | |
10 | 35.86 | 12 | 9 | 65.03% | 75.85 | |
BHRF-1 Inter-Assay Variation
| ||||||
100,000 | 17.30 | 109,065 | 14,266 | 10.01% | 13.08 | 0.990 |
10,000 | 21.49 | 9,209 | 2,154 | 16.84% | 23.39 | |
1,000 | 25.49 | 860 | 251 | 22.19% | 29.15 | |
100 | 29.01 | 108 | 49 | 35.53% | 45.33 | |
10 | 32.29 | 15 | 8 | 69.41% | 57.23 |
EBV detection and load in EBV-associated disease states and immunocompromised individuals
Group | Patient ID | Sex/Age | Condition | Specimen (Positive/n Tested) | Target | Detectable EBV DNA Load (copies/ml) | Clinical Notes |
---|---|---|---|---|---|---|---|
1. | A. | M/46y | PTLD | Plasma (5/6) | EBNA-1 | Day +32 - 8.0 × 102 | MUD HSCT for AML; EBV VCA IgG positive pre-Tx; Plasma collected on Days +32, +39, +46, +60, +75 and +81 for EBV QPCR; Plasma EBV (qualitative) PCR positive on Days +75, +78 and +81; Treatment with Foscarnet and Rituximab after Day +75; Died of pneumonia on Day +88 |
Day +46 - 1.0 × 103 | |||||||
Day +60 - 8.8 × 103 | |||||||
Day +75 - 1.1 × 106 | |||||||
Day +81 - 2.3 × 105 | |||||||
CSF (2/2) | EBNA-1 | Day +75 - 1.3 × 106 | CSF collected on Days +75 and +78 | ||||
Day +78 - 2.7 × 106 | |||||||
B. | M/42y | PTLD | Whole Blood (1/5) | EBNA-1 | Day +95 - 2.0 × 107 | MUD HSCT for AML; Plasma EBV (qualitative) PCR positive Day +96; Plasma collected on Day +95 for EBV QPCR; Died on Day +99 due to multi-organ failure | |
C. | F/59y | PTLD | Plasma (3/6) | EBNA-1 | Day +45 - 2.2 × 105 | MUD HSCT for AML; CMV reactivation on Day +44, Treatment with Foscarnet and ganciclovir on Day +52; Plasma collected Days +38, +40, +45, +52 and +59; Died on Day +66; EBV VCA IgG positive, HHV6 IgG positive and CMV IgG positive pre-Tx | |
Day +52 - 9.6 × 103 | |||||||
Day +59 - 3.0 × 105 | |||||||
Whole Blood (1/8) | EBNA-1 | Day +46 - 6.6 × 104 | EDTA collected Days +3, +5, +10, +17, +26, +31, +33, +46 | ||||
D. | M/48y | PTLD | Plasma (4/6) | EBNA-1 | Day +40 - 3.4 × 103 | MUD HSCT for AML; EBV VCA IgG positive pre-Tx; Plasma collected Days +28, +33, +40, +47, +54, +61; Plasma EBV (qualitative) PCR negative on Day +62; Died Day +72 of multi-organ failure | |
Day +47 - 3.6 × 104 | |||||||
Day +54 - 3.4 × 106 | |||||||
Day +61 - 6.3 × 106 | |||||||
Whole Blood (2/2) | EBNA-1 | Day +62 - 1.3 × 108 | EDTA collected Days +62 and +63. | ||||
Day +63 - 1.8 × 107 | |||||||
E. | F/57y | PTLD | Whole Blood (1/5) | EBNA-1 | 9.5 × 104 | No serology results available however clinical notes indicate EBV reactivation; Plasma EBV (qualitative) PCR positive 9-16 days after VL testing done; negative at 1-7 months thereafter. | |
2. | F. | Unknown | IM | Plasma (1/1) | EBNA-1 | 3.7 × 104 | EBV VCA IgM positive |
BHRF-1 | 1.6 × 104 | ||||||
G. | Unknown | IM | Plasma (0/1) | EBNA-1 | 0 | EBV VCA IgM positive | |
BHRF-1 | 0 | ||||||
H. | Unknown | IM | Plasma (1/1) | EBNA-1 | 7.6 × 103 | EBV VCA IgM positive | |
BHRF-1 | 1.5 × 103 | ||||||
I. | Unknown | IM | Plasma (1/1) | EBNA-1 | 2.3 × 103 | EBV VCA IgM positive | |
BHRF-1 | 8.7 × 104 | ||||||
J. | M/17y | IM | Whole Blood (1/1) | EBNA-1 | 1.0 × 105 | EBV VCA IgM positive | |
BHRF-1 | 1.8 × 103 | ||||||
K. | F/19y | IM | Whole Blood (1/1) | EBNA-1 | 2.2 103 | EBV VCA IgM positive | |
BHRF-1 | 5.6 × 104 | ||||||
L. | F/53y | IM | Whole Blood (1/1) | EBNA-1 | 2.0 × 105 | EBV VCA IgM positive; acute glandular fever | |
BHRF-1 | 1.8 × 104 | ||||||
3. | M. | M/36y | EBVAHS | Whole Blood (1/1) | EBNA-1 | 7.5 × 104 | EBV (qualitative) PCR positive; died of EBVAHS |
BHRF-1 | 1.1 × 105 | ||||||
4. | N. | Unknown | HIV | Plasma (1/1) | EBNA-1 | 0 | HIV plasma VL 324, 000 RNA copies/ml |
BHRF-1 | 1.0 × 103 | ||||||
O. | Unknown | HIV | Plasma (0/1) | EBNA-1, | 0 | HIV plasma VL 13, 000 RNA copies/ml | |
BHRF-1 | 0 | ||||||
P. | Unknown | HIV | Plasma (0/1) | EBNA-1, | 0 | HIV plasma VL 26, 800 RNA copies/ml | |
BHRF-1 | 0 | ||||||
Q. | Unknown | HIV | Plasma (0/1) | EBNA-1, | 0 | HIV plasma VL 21, 300 RNA copies/ml | |
BHRF-1 | 0 | ||||||
R. | Unknown | HIV | Plasma (0/1) | EBNA-1, | 0 | HIV plasma VL 12, 700 RNA copies/ml | |
BHRF-1 | 0 | ||||||
S. | Unknown | HIV | Plasma (0/1) | EBNA-1, | 0 | HIV plasma VL 1, 040, 000 RNA copies/ml | |
BHRF-1 | 0 | ||||||
T. | Unknown | HIV | Plasma (0/1) | EBNA-1, | 0 | HIV plasma VL 17, 700 RNA copies/ml | |
BHRF-1 | 0 | ||||||
U. | Unknown | HIV | Plasma (0/1) | EBNA-1, | 0 | HIV plasma VL 47, 500 RNA copies/ml | |
BHRF-1 | 0 | ||||||
V. | Unknown | HIV | Plasma (1/1) | EBNA-1 | 5.6 × 103 | HIV plasma VL 16, 400 RNA copies/ml | |
BHRF-1 | 3.0 × 103 | ||||||
W. | Unknown | HIV | PBMC (1/1) | EBNA-1 | < 2.0 × 102 | HIV PBMC VL 12, 800 RNA copies/ml | |
BHRF-1 | < 2.0 × 102 | ||||||
X. | Unknown | HIV | PBMC (1/1) | EBNA-1 | < 2.0 × 102 | HIV PBMC VL 12, 700 RNA copies/ml | |
BHRF-1 | 0 | ||||||
Y. | Unknown | HIV | PBMC (0/1) | EBNA-1, | 0 | HIV PBMC VL 118, 000 RNA copies/ml | |
BHRF-1 |
EBV detection and load in the population sample
Target | Positive n (%) | Detectable Range | Spearman correlation (p) | |||
---|---|---|---|---|---|---|
EBNA-1 DNA load
|
BHRF-1 DNA load
|
Combined EBV Targets DNA load
|
VCA IgG
| |||
EBV EBNA-1 DNA load (copies/ml) | 24 (11.0%) | 2.0 × 102 - 9.1 × 104 | 1.00 | |||
EBV BHRF-1 DNA load (copies/ml) | 47 (21.6%) | 2.0 × 102 - 3.3 × 104 | 0.63 p < 0.001 | 1.00 | ||
Combined EBV targets DNA load (mean of BHRF & EBNA loads where both positive) (copies/ml) | 49 (22.5%) | 2.0 × 102 - 6.2 × 104 | 0.73 p < 0.001 | 0.97 p < 0.001 | 1.00 | |
Viral capsid antigen IgG (titres) | 209 (95.9%) | 1:10 - 1:5120 | 0.11 p = 0.11 | 0.13 p = 0.05 | 0.14 p = 0.04 | 1.00 |