Abstract:
Serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) is a surrogate marker for reflecting the transcriptional activity of covalently closed circular DNA. However, there is still no standardized assay for the quantitative detection of serum HBV RNA in chronic hepatitis B patients. In this study, quantitative polymerase chain reactions for detecting the preC/C-RNA (preC/C region HBV pgRNA), SF-RNA (splicing variants-free pgRNA) and XR-RNA (X region remained pgRNA) regions were set up. The dynamic changes of serum pgRNA splicing variants and 3\' terminal truncations were analysed in three retrospective cohorts: 35 treatment-naive chronic HBV-infected patients (cohort A), 52 chronic hepatitis B (CHB) patients who received nucleos(t)ide analogs (NAs) therapy for 48 weeks (cohort B) and eight CHB patients who are under long-term NAs treatment (cohort C). The accuracy and sensitivity of HBV RNA detection were assessed by the National Standard of HBV RNA. We confirmed that high proportions of pgRNA splicing variants and 3\' terminal truncations were present and significantly affect the quantitative detection of serum HBV RNA in both treatment-naive and NAs-treated CHB patients. To achieve the higher accuracy and sensitivity on the detection of HBV RNA level, the primers and probes should be designed at the 5\' terminal region of HBV genome and outside the mainly spliced sequence of pgRNA, especially for CHB patients under long-term NAs treatment. This study would help to better understand the significance of the pgRNA splicing variants and 3\' terminal truncations, and further guide the clinical detection of serum HBV RNA.
摘要:
血清乙型肝炎病毒(HBV)前基因组RNA(pgRNA)是反映共价闭合环状DNA转录活性的替代标记。然而,目前还没有标准化的定量检测慢性乙型肝炎患者血清HBVRNA。在这项研究中,定量聚合酶链反应检测preC/C-RNA(preC/C区HBVpgRNA),建立SF-RNA(无剪接变体的pgRNA)和XR-RNA(X区保持pgRNA)区。在三个回顾性队列中分析了血清pgRNA剪接变体和3'末端截短的动态变化:35例初治慢性HBV感染患者(队列A),接受核苷(t)类似物(NAs)治疗48周的52例慢性乙型肝炎(CHB)患者(队列B)和长期NAs治疗(队列C)的8例CHB患者。采用HBVRNA国家标准对HBVRNA检测的准确性和敏感性进行评估。我们证实存在高比例的pgRNA剪接变体和3'末端截短,并显着影响初治和NAs治疗的CHB患者血清HBVRNA的定量检测。为实现HBVRNA水平检测的更高准确度和灵敏度,引物和探针应设计在HBV基因组的5'末端区域和pgRNA的主要剪接序列之外,特别是长期接受NAs治疗的CHB患者。这项研究将有助于更好地理解pgRNA剪接变体和3'末端截短的意义,并进一步指导临床检测血清HBVRNA。