HBV DNA免提取核酸荧光定量PCR检测法应用评价

目的评价乙型肝炎病毒核酸免提取荧光定量PCR方法(一步法)对不同病毒载量时检测性能。方法评价试剂为圣湘公司HBV DNA荧光定量PCR检测免提取试剂盒,对照试剂为匹基公司、科发公司、达安公司3家煮沸核酸提取法试剂盒。将4种试剂盒按各自要求对配套的不同浓度标准品、阴性、临界值的反应曲线考核其线性和灵敏度。另检测1份已知浓度质控品、5份未知浓度样本血清考核其准确性、可比较性。检测仪器为瑞士罗氏公司Light Cycler荧光定量仪。结果一步法、匹基公司、科发公司、达安公司4家标准品浓度相关性R2分别为0.992、0.998、0.999及0.963,线性均满意。4个不同浓度标准品扩增曲线图形:一步法曲线比其他3家更为光滑,呈间距均匀的S形抛物线,2次结果重复曲线更趋重迭。对已知定值为(1.5E+06)IU/mL的质控品检测:一步法、匹基公司、科发公司、达安公司相应结果为(1.16E+06)、(5.27E+05)、(4.32E+05)及(4.90E+06)IU/mL,一步法最接近靶值。5份血清结果显示:4种试剂盒除P公司对2号样本结果为低于检出下限外,其他对1号、2号、3号、5号检出结果均与其他3家接近,可为临床接受。4号样本为一个低值临界范围,出现低于报告限、低值及临界追踪值3种结果现象。结论 4家HBV DNA荧光定量PCR检测检测试剂线性结果均满意,对中、高浓度样本检测结果较接近,但在低浓度临界范围检测报告可能会对临床诊断带来误导,需追踪复查。HBV PCR一步法检测技术免除了核酸高温裂解提取步骤,操作简便,人为误差减少,性能稳定,有利提高检测灵敏度、结果准确性。

Evaluation of the method on HBV DNA with nonextracting nucleic acid real-time quantitative PCR

【Objective】 To evaluate the performance of HBV DNA non-extracting nucleinic acid real-time fluorescence quantitative PCR on different viral load.【Methods】 The evaluated reagent kit with non-extracting nucleic acid was from Shengxiang company Ltd.The control reagent were boiling extracting reagent from PiJi company,KeFa company and DaAn company.According to the four kits’ instructions,four different known-concentration standard,negative,position and critical positive of HBV DNA samples were detected to assess the amplification curves’ linear and sensitivity.At the same time,the quality control material with known concerntration,five serum samples were detected for accuracy and comparability by Light Cycler Quantitative Instrument(American).【Results】 Each kit standard concentration correlation R2 from Shengxiang company,PiJi company,KeFa company and DaAn company was 0.992,0.998,0.999,0.963 respectively with a good linner.Comparison of standard amplification curve graphic: one step curve was more smoothly,space evenly and with S-type,and the repeatability of the curve being detected secondly was better.The results of detecting control quality materials for(1.5E+06) IU/mL were(1.16E+06) IU/mL,(5.27E+05) IU/mL,(4.32E+05) IU/mL and(4.90E+06) IU/mL respectively.One step was closest to the target value.Five serum samples’ results showed that the four kits’ detecting results were similar except the No 2 sample’s result from Piji company lower than the lowest limit,which was clinically acceptable.No 4 sample as a low value critical range appear the following results: below the reporting limit,low value and critical tracking value.【Conclusion】 Four HBV DNA quantitative PCR linear results were satisfactory.For medium and high concentrations of the test results was approaching,but detection of low concentrations was different.The Results may bring misunderstanding for clinic diagnose and need Follow-up review.One step for HBV PCR had avoided the regular tedious methods containing heating cracking nucleinic acid procedure,made the steps simple,reduced personal errors and had stable performance,that were advantageous to the sensitivity and accuracy.