摘要
目的建立酸碱＂一步法＂荧光PCR快速检测乙型肝炎病毒（hepatitis B virus,HBV）核酸。方法对建立的酸碱＂一步法＂荧光PCR中的核酸释放剂组分及其浓度、释放时间,PCR缓冲液的组分、p H值及防污染体系进行优化。验证该方法的灵敏度及线性范围,与同类试剂盒进行比较,并应用于其他病原体的检测。结果最佳核酸释放剂为：0.4 mol/L KOH,0.3%十二烷基硫酸锂（lithium lauryl sulphate,LLS）,0.3%Triton X-100,0.02%Foam Ban和0.5%N-乙酰半胱氨酸（N-acetylcysteine,NAC）;最佳PCR缓冲液组分如下：0.5 mol/L Tris-醋酸（p H 7.4）,1%甘油,2.5%海藻糖,1 mol/L KCl,0.5 mol/L Mg Cl2,10 mmol/L EDTA、0.2%BSA和0.01%NaN3。建立的方法的最低检测量达1×10^2copies/ml,在1×10^2-1×10^9 copies/ml范围内参考品浓度与Ct值呈良好线性关系,回归方程为：y=-3.36 x＋39.469 9,R2=0.999 9。该方法与同类试剂盒的检测结果差异无统计学意义（P〉0.05）,不能用于检测解脲脲原体。结论酸碱＂一步法＂荧光PCR可用于HBV核酸的快速检测,该方法操作简便、快速,抗污染及干扰能力强,具有较高的临床应用价值。
Objective To develop an acid-alkali one step method-fluorescence PCR technique for rapid detection of hepatitis B virus（HBV）nucleic acid. Methods The acid-alkali one step method-fluorescence PCR technique was developed,of which the component and concentration of nucleic acid release agent, time for release, component and p H value of PCR buffer as well as contamination-preventing system were optimized. The method was verified for sensitivity and linear range,compared with the kits of the same kind, and applied to the detection of other pathogens. Results The optimal release agents of nucleic acid consisted of 0. 4 mol / L potassium chloride, 0. 3%（lithium lauryl sulphate, LLS）, 0. 3% Triton X-100, 0. 02% Foam Ban and 0. 5%（N-acetylcysteine, NAC）, while the optimal PCR buffer consisted of 0. 5 mol / L Tris-acetic acid（p H 7. 4）, 1% glycerol, 2. 5% trehalose, 1 mol / L potassium chloride, 0. 5 mol / L magnesium chloride,10 mmol / L EDTA, 0. 2% BSA and 0. 01% sodium azide. The minimum detection limit of the developed method was 1 ×10~2copies / ml. The Ct value showed good linear relationship to the reference concentration at a range of 1 × 10~2~ 1 ×10~2copies / ml, of which the regression equation was as follows： y =-3. 36 x ＋ 39. 469 9,R2 = 0. 999 9. No detection result by the method showed no significant difference with that by the kid of the same kind（P〉0. 05）. The method could not be used for detection of Ureaplasma urealyticum. Conclusion The developed acid-alkali one step method-fluorescence PCR technique may be used for the rapid detection of HBV nucleic acid. With strong abilities in preventing con-tamination or interference, the technique is simple, rapid, and of a significance in clinical application.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第12期1297-1304,共8页
Chinese Journal of Biologicals
基金
"艾滋病与病毒性肝炎等重大传染病防治"科技重大专项(2013ZX10004802)
