乙型肝炎病毒聚合酶链反应液相杂交酶免疫检测的定性判断值研究

Study on the qualitative decision value in PCR-ELISA with liquid hybridization for detecting HBV

目的探讨乙型肝炎病毒 (HBV)聚合酶链反应 (PCR)液相杂交酶免疫检测的定性判断值设立和灰区范围的计算方法。方法以 HBV为目的靶 DNA,引物 P1 5’端标记 Bio,PCR扩增 35个循环。扩增产物与 5’端标记Dig的探针混合进行液相杂交 ,杂交后产物被 SA酶标板固定 ,经酶标记抗 Dig抗体结合、显色。结果实验结果显示 ,本试剂的检测敏感度在 3× 10 4拷贝 /m l。 3种参比品 A450 值的 95 %、99%分布范围之间不存在交叉区 ,其中临界阳性参比品 95 %分布范围下限值 (A450 )为 0 .182。 5 85例献血员血清标本的 A450 均值为 0 .0 37,P99上限值为 0 .12 3,灰区范围在 0 .12 3～ 0 .182。本试剂对“HBs Ag+/HBe Ag+/抗 - HBc+”标本的阳性检出率为 98.8% ,“HBs Ag+/抗 - HBe+/抗 - HBc+”标本的检出率为 5 4.8% ,阴性标本的检测有较高的特异性。结论本试剂设立的定性判断值(灰区计算方法 )具有很高的正确性。

Objective To search for setting a qualitative decision value and a calculating method of ambiguous (borderline) region for PCR ELISA with liquid hybridization technique for detecting HBV. Methods HBV DNA was used as a target DNA and its primer was labeled with biotin,and the PCR was amplified by 35 cycles. After the amplified products were mixed with a 5' Digoxingenin labeled probe, its liquid hybridization was carried out in a thermocycler for 2 minutes at 90℃ and for l minutes at 55℃. Then the hybridized products were captured on microplate wells coated with streptavidin and monitored with an peroxidase labeled anti digoxigenin antibody. Results Results showed that the detection sensitivity is 3×10 4 copies/ml of HBV. There are no cross regions in 95% and 99% confidence interval among three control sera, and the lower limit of 99% confidence interval of critical positive control serum is 0 182(A 450 ). The average(A 450 )is 0 037, the P 99 is 0 123 in 585 normal serum samples. The ambiguous region is 0 123～0 182. The clinical application results showed that the positive ratio for HBsAg(+),HBeAg(+), anti HBc antibody(+) serum samples was 99 8%; HBsAg(+), anti HBe antibody(+),anti HBc antibody(+) samples, 54 8%, and the specificity for negative smaples was 100%. After having been redetected 13 samples of ambiguity region only 2 showed positive results. Conclusion The qualitative decision value was developed that showed very good rightness, operation and clinical practicable application.