两种HBVDNA检测试剂的比较

Comparison of two HBV DNA detection kits

目的分析两种试剂在HBVDNA定量检测中的相关性，评价其在不同病毒载量时的检测性能。方法用人AB型血清将WHO第2代HBVDNA国际标准品（编号：97／750）配制成不同浓度的10份样本，10份样本的浓度分别为1×10^6、5×10^5、1×10^5、5×10^4、1×10^4、5×10^3、1×10^3、5×10^2、1×10^2、1×10^1kIU／L。采用深圳匹基生物工程股份有限公司生产的HBVDNA荧光定量PER检测试剂（PG试剂）和美国罗氏公司生产的定量PCR试剂（罗氏试剂）检测78份HBV感染者血清、30份健康献血者血清和10份不同浓度范围的WHO标准品中HBVDNA含量。对两种试剂检测HBVDNA结果相关性进行分析；对试剂在不同病毒载量时的检测性能进行评价；并对漏检情况进行分析。两种试剂每批检测均设有阴性质控、弱阳性质控和强阳性质控。结果两种试剂对WHOHBVDNA标准物质稀释血清均能正确检出，罗氏试剂能检出最高稀释度样本浓度为2．00（klU／L，lg），PG试剂能检出最高稀释度样本浓度为3．00（kIU／L，lg）；两种试剂检测结果存在线性相关（R^2=0．9387，P〈0．01），罗氏试剂检测上限与理论值相符，大于检测上限的标本经稀释重复测定，罗氏试剂检测结果[（8．35±0．20）klU／L，lg]高于PG试剂检测结果[（7．73±0．42）klU／L，lg]，差异有统计学意义（t=3．776，P〈0．05）。两种试剂检测108份血清标本中HBVDNA含量，罗氏试剂检测值[（5．88±1．64）kIU／L，lg]高于PG试剂检测值[（5．25±1．55）klU／L．1g]，差异有统计学意义（t=12．297，P〈0．01）；检测高HBV病毒载量[（〉5．00-≤7．00）klU／L，lg和（〉7．00～≤9．00）klU／L，lg]组，两种试剂的相关性较高（R^2分别为0.7797、0．6037，P均〈0．01）；对低HBV病毒载量（〉3．00-≤5．00kIU／L，lg）组，两种试剂的相关性较低（R。=0．4173，P〈0．01）；病毒载量为〉3．00-≤4．00klU／L，lg组，PG试剂的漏检率为33．3％（5／15）；病毒载量为〉1．08-≤3．00kIU／L，lg组，PG试剂未检出。结论PG试剂与罗氏试剂检测结果虽存在一定差距，但相关性较好。两种试剂低病毒载量标本的相关性要低于高病毒载量标本，PG试剂检测HBVDNA的线性范围较窄。

Objective To evaluate clinical significance of two real-time fluorescence quantitative PCR kits for quantitative detection of HBV DNA and detection performance at different viral load levels. Methods A series of calibrators with different concentrations （ 1× 10^6 , 5 ×10^5 , 1 × 10^5 , 5 × 10^4 , 1 × 10^4 , 5× 10^3,1 ×10^3,5 × 10^2,1 × 10^2,1 × 10^1 kIU/L） were prepared with AB-type sera using the second generation WHO international standard （ NIBSC code ： 97/750 ）. HBV viral load in the sera of 78 patients, 30 healthy blood donors and IO calibrators were detected by real-time fluorescence quantitative PCR HBV DNA test kit from PIJI Bio-Technical Development Company Ltd（ PG kit） and Cobas AmpliPrep/Cobas TaqMan HBV test kit. The correlation of the two methods was evaluated, and the performance of the two kits different viral load levels was evaluated. The false negative rate was analyzed. Negative control, low positive control and high positive control were included in every batch. Results Both two kits showed the correct results for the 10 specimens from the WHO international standards. The lowest detection limit of HBV DNA for Roche Cobas AmpliPrep/Cobas TaqMan HBV test and PG kit were 2. O0 （kIU/L, lg）and 3.00 （klU/L, lg）, respectively. There was linear correlation between the results from Roche Cobas AmpliPrep/Cobas TaqMan HBV test and PG kit （R2 = 0. 938 7,P 〈 0. O1 ）, the upper limit of Roche kit had coincided with theoretical value. The samples with HBV DNA level above the upper limit of detection were diluted and retested to obtain the precise result. The result form Roche Cobas AmpliPrep/Cobas TaqMan HBV test [ （8.35 ± 0.20） kIU/L,lg; was higher than that from PG kit [ （7.73 ± 0.42） kIU/L, lg ] （ t = 3. 776, P 〈 O. 05） . The detection of 108 serum samples showed that the level of HBV DNA detected by Roche Cobas AmpliPrep/Cobas TaqMan HBV test [ （5.88 ± 1.64） kIU/L,lg; was higher than that by PG kit[ （5.25 ± 1.55 kIU/L, lg] （t = 12. 297 ,P 〈 O. 01 ）. The correlation coefficients were high in samples with high HBV viral load[ HBV DNA（ 〉 5. O0 and≤7 . 00） kIU/L,lg,R^2 = 0. 779 7, P 〈 0.01 ; HBV DNA（ 〉 7.00 and≤ 9.00） klU/L,lg,R2 = 0. 603 7, P 〈0.01 1- The correlation coefficient was low in samples with low HBV viral load[ HBV DNA（ 〉3.00 and ≤5.00） klU/L,lg,R2 =0.417 3,P〈0.01）].When HBV DNA （ 〉3.00 and ≤4.00） kIU/L,lg,the false negative rate was 33.3% （5/15）. When HBV DNA （ 〉 1.08 and ≤3.00） kIU/L,lg, none of positive samples was detected with PG kit. Conclusions PG kit is not as good as Cobas AmpliPrep/Cobas TaqMan HBV test . The linear correlation between the results from the two kits is good. The correlation between the results detected with PG kit and Cobas AmpliPrep/Cobas TaqMan HBV test is higher in the high viral load groups than in the low viral load group. It is suggested that PG kit had a narrower linear range.