3种荧光定量PCR试剂检测HBV-DNA阴性结果的比较

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

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。

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.