Cell Engineering Division -CELL BANK-

Epstein-Barr virus (EBV) is a pathogenic virus belonging to the Herpesviridae family. It spreads through saliva and primarily infects B cells. Over 95% of healthy adults are infected with EBV, and it is a virus that is commonly carried throughout life in a symbiotic manner. While EBV-related disease onset is rare, it has been found to be associated with the development of certain malignant lymphomas, such as Burkitt’s lymphoma, as well as nasopharyngeal cancer.

Our bank uses qPCR to detect EBV genomes in human cell lines derived from blood (excluding EBV-transformed B-cell lines) and in certain human cancer cell lines; oral cancer, esophageal cancer and gastric cancer that have been reported to be associated with EBV infection as a cause of cancer development. Our bank focuses on the latent infection-associated gene EBNA1^*1 and the lytic infection-associated gene BALF5^*2 as indicators for detecting the EBV genome in cell lines.
^*1 EBNA1: Epstein-Barr Nuclear Antigen 1
^*2 BALF5: the BamHI-A fragment containing the fifth leftward open reading frame

1. Preparation
(1) Sample: Genomic DNA extracted from cells
(2) Equipment: Applied Biosystems QuantStudio3 Real-Time PCR System
(3) Software: QuantStudio Design & Analysis Software, ThermoFisher SCIENTIFIC
(4) PCR Reagents: Applied Biosystems PowerUp SYBR Green Master Mix (Cat No. A25777)
(5) PCR Primers
　　• EBNA1 primers
　　　　EBNA1-F : 5’-ATCAGGGCCAAGACATAGAGATG-3’
　　　　EBNA1-R : 5’-GCCAATGCAACTTGGACGTT-3’
　　• BALF5 primers
　　　　BALF5-F : 5’-CGGAAGCCCTCTGGACTTC–3’
　　　　BALF5-R : 5’-CCCTGTTTATCCGATGGAATG–3’
(6) Standards: Synthetic DNA for each target gene

2. Method
(1) Preparation of Standards: Prepare the following copy numbers for each target gene:
　　• EBNA1；1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹ (copies)
　　• BALF5；2.5×10⁶、2.5×10⁵、2.5×10⁴、2.5×10³、2.5×10²、2.5×10¹ (copies)
(2) Preparation of Samples: Prepare genomic DNA solution at 2.5 ng/uL
(3) Preparation of PCR Master Mix

DDW	4 uL
Primer FW (10 pmol/uL)	1 uL
Primer RV (10 pmol/uL)	1 uL
PowerUp SYBR Green Master Mix (2x)	10 uL
Total	16 uL /Sample

(4) Dispense 16 uL of master mix into each well of a PCR 96-well plate.
(5) Add 4 uL of the prepared sample to each well.
(6) PCR Program:
• UDG Activation: 50°C for 2 minutes (hold)
• Dual-Lock DNA Polymerase: 95°C for 2 minutes (hold)
• Reaction Cycles (Denaturation → Annealing/Extension): 95°C for 15 seconds → 60°C for 1 minute * (40 cycles)
• Melt Curve Analysis: 95°C for 15 seconds → 60°C for 1 minute → 95°C for 15 seconds ^* (1 cycle)
^* Fluorescent data collection points
(7) Result Analysis

3. Interpretation of the result
After confirming the validity of the test (Efficiency, R², and Melt Curve TM values of the standards must be within valid conditions), the results are assessed.

4. References
• References on PCR
　　　　QuantStudio 3/QuantStudio 5 リアルタイム PCR システム 簡易操作ガイド(Japanese only)
• References on determining detection limits
　　　　血液製剤のウイルスに対する安全性確保を目的とした核酸増幅検査(NAT)の実施に関するガイドライン (厚生労働省)(Japanese only)
• References on primers and standards
　　　　Generation of two human induced pluripotent stem cell lines derived from two juvenile nephronophthisis patients with NPHP1 deletion. Stem Cell Res. 2020 May; 45:101815. PMID: 32361464.