2.4. Quantitative PCR of Epstein‐Barr virus DNA load

The EBV DNA loads in NP brushing samples from all participants were quantified by quantitative PCR (Q‐PCR). Our real‐time Q‐PCR was based on a previously mature fluorogenic PCR reaction system.⁷ In this system, amplification primers targeting the BamHI‐W region of the EBV DNA genome and a dual‐labeled hybridization probe were included. Two primers and a probe targeting the β‐globin gene were used as a reference for quality control. All the sequences of primers and probes used are listed in Table S1. The standard sample ladders (10³, 10⁴, 10⁵, 10⁶ and 10⁷ copies/μL) were used to obtain the standard curve. Each PCR reaction was set up in a volume of 8 μL, including 4 μL PCR master mix, 1 μL primer, 0.2 μL probe, 0.8 μL water and 2 μL DNA template. Thermal cycling was initiated with a denaturation step of 5 minutes at 95°C, then 45 cycles of 95°C for 15 seconds, 60°C for 30 seconds and 72°C for 15 seconds, and then 72°C for 5 minutes. The EBV DNA and β‐globin levels in brushing samples were expressed as copy/ng DNA.

A comparative EBV DNA Q‐PCR analysis was also performed, as described in previous studies,²⁷ to distinguish the intact DNA or the fragmented DNA. In brief, dye‐based Q‐PCR, respectively, targeting a 99 and 213 bp region of Epstein‐Barr virus nuclear antigen‐1 (EBNA1) was simultaneously performed in one sample. The sequences of the two paired primers targeting EBNA1 are listed in Table S1. During PCR amplification, EBV DNA from EBV positive cell lines C666 was used as the positive control. The standard sample ladders (10¹, 10², 10³ and 10⁴ copies/μL) were used to obtain the standard curve. Each PCR reaction was set up in a volume of 10 μL including 5 μL PCR master mix, 1 μL primer, 3 μL water and 1 μL DNA template. Thermal cycling was initiated with a denaturation step of 5 minutes at 95°C, then 40 cycles of 95°C for 10 seconds, 55°C for 15 seconds and 72°C for 30 seconds, and then 72°C for 7 minutes. The EBV DNA in brushing samples was expressed as copy/μL. When the ratio (99 bp EBNA1 expression divided by 213 bp EBNA1 expression) was over 1.5, the sample was thought to have fragmented EBV DNA.