环介导等温扩增(LAMP)试剂盒检测石蜡包埋肝组织中乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)

 目的  建立一种检测石蜡包埋的肝组织中乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)的新方法,并探讨其应用价值和临床意义。方法  提取30例慢性HBV感染患者的石蜡包埋肝组织中的DNA,采用质粒安全ATP依赖性DNA酶(plasmid-safe ATP-dependent DNase,PSAD)消化,去除非cccDNA后,加入引物及Bst DNA酶进行环介导等温扩增(loop-mediated isothermal amplification,LAMP),将检测结果与临床实验室资料结合并进行分析,以判断其临床应用价值。结果  30例HBV感染患者中,有17例HBV cccDNA阳性,阳性检测率56.67%。其中肝癌5例、肝硬化4例、重型乙型肝炎5例、慢性乙型肝炎(轻-中度)3例。提示肝组织HBV cccDNA阳性可能与疾病进展密切相关,与血清HBV cccDNA阳性有一定的相关性,与乙型肝炎病毒血清免疫标志物以及肝功能状况无明显相关。结论  LAMP法能检测到石蜡包埋肝组织中的HBV cccDNA,结合临床实验室资料,对判断抗病毒药物疗效及研究乙型肝炎致病机制具有一定的意义。

A detection method of the loop-medidated isothermal amplification (LAMP) kit for hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in paraffin embedded liver tissues established

Objective  To establish a novel method for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) of paraffin embedded liver tissues, and to discuss its application value and clinical significance. Methods  The extraction DNA of 30 patients with chronic HBV infection in paraffin embedded liver tissues was digested by plasmid-safe ATP-dependent DNase (PSAD). Non cccDNA was removed, and then the necessary primers and Bst DNA polymerase were added together for loop mediated isothermal amplification (LAMP). The detection results combined with clinical laboratory data were analyzed to estimated its clinical application valus. Results  Among the 30 HBV infected patients,17 cases were detected as HBV cccDNA positive with the positive detection rate of 56.67%, of which 5 cases were liver cancer, 4 cases were cirrhosis, 5 cases were severe chronic hepatitis B, and 3 cases were mild to moderate charonic hepatitis B. It revealed that positive HBV cccDNA might have a close link with the progress of the disease, also had a certain correlation with the serum HBV DNA loading, but had no obvious correlation with HBV serum markers and liver function. Conclusions  LAMP detection method can indicate HBV cccDNA in the paraffin embedding liver tissue, which combined with clinical laboratory data has a certain significance in judging the antiviral efficacy and researching pathogenic mechanism of HBV.