干斑法保存血浆中HBV、HCV、HIV病毒核酸及其保存效期研究

The Preservation of HBV,HCV,HIV Viral Nucleic Acidsin Plasmaby Dry Spot Method and the Duration of Preservation

目的:建立一种以干斑法为基础制作的血液采集卡保存血浆中病毒核酸的方法,通过建立阿伦尼乌斯方程,预测该种保存方法保存核酸的效期,并论证该种保存方法在血站留存血液样本进行核酸复测的可行性。方法:分别将HBV、HCV、HIV核酸阳性的血浆样本制备成保存卡,以干浆斑的形式进行保存;将制备的保存卡分别放置于37、45、50和55℃条件下进行加速保存实验,不定期取出各个温度条件下的保存卡片,进行核酸提取操作,纯化出待测核酸样本,进行后续PCR测试,并记录CT值;根据阿伦尼乌斯方程建立起失效时间与储存温度之间的模型,进而推测出血液采集卡在规定保存温度条件下保存核酸的有效期。结果:HBV、HCV、HIV 3种核酸阳性的血浆样本,在使用干斑法保存时,其保存效期与温度的回归方程分别为:y=-11546x+31.74、y=-12949x+36.88、y=-12204x+34.48,线性回归的相关系数r均>0.98。预测HBV、HCV和HIV 3种病毒核酸使用干斑法保存时,在4℃的保存温度下,其保存效期分别为20792、19289和14285 d;在20℃的保存温度下,保存效期分别为2135、1502和1289 d。结论:血液样本中常见的3种病毒核酸,使用干斑法保存时,在加速破坏实验中符合一级反应模式,核酸降解速度与保存的绝对温度之间的关系符合阿伦尼乌斯方程。经过该方程的推算,使用干斑法保存血浆核酸样本,在不高于20℃的保存条件下,核酸稳定性效期最低可达3.5年,基本可满足血站血液样本留样保存年限的要求。

Objective:To establish a method for preserving viral nucleic acids in plasma using a blood collection card based on the dry spot method,to predict the duration of nucleic acid preservation by establishing the Arrhenius equation,and to demonstrate the feasibility of this preservation method for the re-testing of nucleic acids in blood samples retained by blood banks.Methods:Plasma samples positive for HBV,HCV,and HIV nucleic acids were prepared into preservation cards in the form of dry plasma spots for storage.The prepared preservation cards were placed under accelerated storage conditions at 37,45,50,and 55℃.The preservation cards were periodically retrieved from each temperature condition for nucleic acid extraction,and the nucleic acid samples were purified for subsequent PCR testing,with the recorded CT values.An Arrhenius equation model was established between the expiration time and the storage temperature,thereby predicting the validity period of nucleic acid preservation in blood collection cards under specified storage temperature conditions.Results:For the plasma samples positive for HBV,HCV,and HIV nucleic acids preserved using the dry spot method,the regression equations for the duration with temperature were as follows:y=-11546x+31.74 for HBV,y=-12949x+36.88 for HCV,and y=-12204x+34.48 for HIV,with the correlation coefficient r greater than 0.98 for all.It was predicted that at a storage temperature of 4℃,the preservation periods for HBV,HCV,and HIV viral nucleic acids using the dry spot method would be 20792 days,19289 days,and 14285 days,respectively.At a storage temperature of 20℃,the preservation periods would be 2135 days 1502 days,and 1289 days,respectively.Conclusion:The nucleic acids of the three common viral pathogens in blood samples,when preserved using the dry spot method,conform to a first-order reaction pattern in the accelerated degradation experiment.The relationship between the rate of nucleic acid degradation and the absolute temperature of storage is consistent with the Arrhenius equation.Based on the calculations using this equation,the stability and validity period of plasma nucleic acid samples preserved using the dry spot method can reach a minimum of 3.5 years under storage conditions not exceeding 20℃,which essentially meets the requirements for the preservation period of blood samples retained by blood banks.