乙型肝炎患者血清及外泌体内HBV-DNA提取及检测的方法学研究

Methodoiogical study on extraction and detection of HBV-DNA in serum and exosomes of patients with hepatitis B

目的:比较煮沸法与苯酚-氯仿法提取血清及外泌体内HBV-DNA,Taqman法与SYBR法qPCR检测HBV-DNA表达的优缺点,并寻找更为准确敏感的诊疗参考指标。方法:收集高病毒载量组与低病毒载量组的血清,提取血清外泌体并鉴定,分别提取血清与外泌体内HBV-DNA,比较分析煮沸法与苯酚-氯仿法提取及qPCR Taqman法与SYBR法检测之间的表达差异。结果:高病毒载量组(HBV-DNA>5E+02 U/ml)中煮沸法提取血清HBV-DNA,Taqman法检测值高于SYBR法(P<0.05);Taqman测定血清HBV-DNA、SYBR测定外泌体HBV-DNA,煮沸法提取效率均高于苯酚-氯仿法(P<0.05);血清HBV-DNA表达高于外泌体(P<0.05)。低病毒载量组(HBV-DNA<5E+02 U/ml)中煮沸法与苯酚-氯仿法提取、Taqman法与SYBR法qPCR检测血清及外泌体HBV-DNA差异均无统计学意义。外泌体HBV-DNA表达高于血清(P<0.05)。结论:两种提取及检测方法均具有一致的灵敏度与特异度。外泌体包裹的HBV-DNA水平可能是临床诊断和治疗慢性乙肝中不可或缺的关键因素。

Objective:To compare the advantages and disadvantages of boiling method and phenol-chloroform method for extraction of HBV-DNA in serum and exosomes,Taqman method and SYBR qPCR for detection of HBV-DNA expression,and search for more accurate and sensitive diagnosis and treatment reference index.Methods:Collected serum samples from high viral load and low viral load groups,extracted serum exosomes and identified them.Then,extracted HBV-DNA from both serum and exosomes,and compared expression differences between boiling method and phenol-chloroform method for extraction,as well as qPCR Taqman method and SYBR method for detection.Results:In high viral load group(HBV-DNA>5E+02 U/ml),serum HBV-DNA was extracted by boiling method,Taqman method was higher than SYBR method(P<0.05);Taqman was used to detect HBV-DNA in serum,SYBR was used to detect HBV-DNA in exosomes,the extraction efficiency of boiling method was both higher than that of phenol-chloroform method(P<0.05);HBV-DNA expression in serum was higher than that in exosomes(P<0.05).In low viral load group(HBV-DNA<5E+02 U/ml),there was no statistical difference between boiling method and phenol-chloroform method,and between Taqman method and SYBR method for serum and exosome HBV-DNA qPCR detection.HBV-DNA expression in exosomes was higher than that in serum(P<0.05).Conclusion:Both extraction and detection methods have the same sensitivity and specificity.Level of HBV-DNA encapsulated by exosomes may be an indispensable key factor in clinical diagnosis and treatment of chronic hepatitis B.