痰液保存条件对恒温扩增荧光法核酸检测结果的影响  

Effect of sputum preservation conditions on the results of nucleic acid detection through isothermal amplification method using fluorescence

作　　者：佘军[1] 高迎春 来汉江[1] 王贤 饶焕新 贺君丽 孙刚 周艳琼 SHE Jun;GAO Yingchun;LAI Hanjiang;WANG Xian;RAO Huanxin;HE Junli;SUN Gang;ZHOU Yanqiong(Department of Clinical Microbiology,the First People's Hospital of Xiaoshan District of Hangzhou City,Zhejiang,Hangzhou 311200,China;Ustar Biotechnologies(Hangzhou)Ltd.,Zhejiang,Hangzhou 310053,China)

机构地区：[1]杭州市萧山区第一人民医院临床微生物科,浙江杭州311200 [2]杭州优思达生物技术有限公司,浙江杭州310053

出　　处：《中国医药科学》2020年第21期176-179,229,共5页China Medicine And Pharmacy

基　　金：浙江省杭州市科技发展计划后补助项目(20162013A02)。

摘　　要：目的评估痰液保存条件对恒温扩增荧光法的结核分枝杆菌核酸检测结果的影响,为痰液保存方式和有效期的确定提供参考依据。方法选取2018年11月~2019年1月杭州市萧山区第一人民医院诊断治疗肺结核的患者104例剩余痰液样本为研究对象。将痰液样本分成4组,分别给予室温(14~22℃)、冷藏(4~8℃)、冷冻(-18^-23℃)以及反复冻融的条件进行保存。在不同的时间点,用恒温扩增荧光法检测痰液样本中的结核分枝菌复合群的核酸。以0d检测结果为对照,根据检测结果以及扩增曲线的Tt值变化,分析痰液样本在不同条件下的保存效期。结果痰样本室温保存5d,冷藏保存5d,未发现核酸降解,继续延长保存时间,核酸降解的风险显著升高。冷冻条件下反复冻融4次,未发现核酸降解。结论痰液样本应尽可能当天检测,获得最准确的核酸检测结果。当天不进行检测,痰液样本应保存于冷冻条件,时间不超过1个月且避免反复冻融,或保存于冷藏条件,时间不超过5d。Objective To evaluate the influence of sputum preservation conditions on the results of mycobacterium tuberculosis nucleic acid detection through isothermal amplification method using fluorescence,so as to provide reference for the determination of sputum preservation mode and the period of validity.Methods The residual sputum samples of 104 patients diagnosed with pulmonary tuberculosis and treated in the First People's Hospital of Xiaoshan District of Hangzhou City from November 2018 to January 2019 were selected as the subjects of research.The sputum samples were divided into four groups,which were respectively stored at room temperature(14-22℃),cold storage(4-8℃),freezing(-18--23℃)and conditions of repeated freezing and thawing.At different time points,the nucleic acid of mycobacterium tuberculosis complex in sputum samples was detected through isothermal amplification method using fluorescence.With the test results of Day 0 being taken as control,the period of validity for preservation of sputum samples under different conditions was analyzed according to the test results and the changes of the Tt value of amplification curve.Results When the sputum samples were stored at room temperature for 5d and cold storage conditions for 5d,no nucleic acid degradation was found,but the risk of nucleic acid degradation was significantly increased with the prolongation of the storage time.Under freezing conditions,repeated freezing and thawing for 4 times incurred no nucleic acid degradation.Conclusion For the purpose of obtaining the most accurate results of nucleic acid detection,sputum samples had better be detected on the same day.If no test is carried out on the same day,sputum samples should be stored in freezing conditions for no more than 1 month and avoid repeated freezing and thawing,or stored in cold storage conditions for no more than 5d.

关 键 词：结核分枝杆菌 痰液 保存条件 核酸检测 

分 类 号：R446.11[医药卫生—诊断学]