3种荧光定量PCR试剂检测HBV-DNA阴性结果的比较

目的：为了解实时荧光定量聚合酶链反应（FQ-PCR）不同试剂间的假阴性差异,分析其原因,寻找消除办法。方法：用深圳匹基基因诊断技术有限公司的FQ-PCR试剂（A方法）检测2 333份血清病毒学标志为HBsAg阳性、HBeAg阳性、抗HBc阳性病人血清中的HBV-DNA,对HBV-DNA阴性标本,用2种试剂盒进行复核。结果：2 333份HBeAg阳性标本A方法检出48份HBV-DNA阴性标本,阴性率为2.06%,经广州中山大学达安基因股份有限公司试剂（B方法）复核,有28份转为HBV-DNA阳性;上海申友基因技术诊断公司试剂（C方法）复核,有31份转为HBV-DNA阳性。结论：FQ-PCR不同试剂检测HBV-DNA存在假阴性,且假阴性率较高,其原因可能与试剂引物设计保守序列与HBV某些变异造成不相匹配有关,可以采用多种试剂进行复核的方法弥补。当FQ-PCR检测HBV-DNA低于检测值时,应用其他试剂进行复核,以减少假阴性错误的发生,为临床诊断提供可靠的依据。

Comparison of results analyzed by 3 kinds of fluorescent quantitative PCR reagents

Objective:To find out the cause of false negative results of various fluorescent quantitative PCR(FQ PCR) reagents and the method to eliminate.Methods:Hepatitis B virus(HBV) DNA in 2 322 serum samples with HBsAg,HBeAg and anti-HBc positive were tested with FQ-PCR reagent(method A,Shenzhen PG biotech Co.,Ltd.) and re-checked with two other methods.Results:Forty-eight samples in 2 322 serum samples with HBeAg positive were proved HBV-DNA negative(2.06%) when detected with method A.Twenty-eight samples in these 48 negative results became HBV-DNA positive when re-tested with reagent from DaAn Gene Co.,Ltd.of Sun Yat-sen University,while 31 samples turned HBV-DNA positive re-tested with reagent from Shanghai shenyou biotech Co.,Ltd..Conclusion:There are false negative results tested with different reagents,which might be caused by the discrepancy between primers in reagents and mutations in HBV.Re-testing with various reagents could lessen this shortcoming.More than 2 kinds of methods should be used to re-check the negative results to decrease the false negative results and provide reliable experimental data.