Drosten C, Weber M, Seifried E, Roth W K
Institute of Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service of Hesse, Frankfurt am Main, Germany.
Transfusion. 2000 Jun;40(6):718-24. doi: 10.1046/j.1537-2995.2000.40060718.x.
High-throughput nucleic acid testing for transfusion-relevant viruses by PCR requires contamination-proof methods with high sensitivity and validity. A new PCR reagent kit (TaqMan, PE BioSystems) reduces the risk of carry-over contamination by eliminating post-PCR processing.
Oligonucleotide design was done with software specialized for designing the assays' (TaqMan) primers and probes. A template-derived competitive internal control sequence designed through site-directed mutagenesis was used to reveal failures in amplification. Assay sensitivity was determined for single-donor and single-patient testing and by spiking sample mini-pools. Three seroconversion panels were tested.
Sensitivity is high, reaching 300 HBV genomes per mL of single-patient material on direct testing. A detection limit of 1000 HBV genome equivalents per mL of donor plasma is achieved for 96 pooled samples. The window period for HBV infection was reduced by 17, 10, and 63 days from that for HBsAg screening in three seroconverting donors.
The assay provides sufficient sensitivity to be superior to HBsAg screening in transfusion medicine and will be useful in clinical laboratories because of its ease of handling.
通过聚合酶链反应（PCR）对与输血相关病毒进行高通量核酸检测需要具备高灵敏度和有效性的防污染方法。一种新的PCR试剂试剂盒（TaqMan，PE生物系统公司）通过消除PCR后处理降低了污染残留风险。
使用专门设计检测（TaqMan）引物和探针的软件进行寡核苷酸设计。通过定点诱变设计的模板衍生竞争性内部对照序列用于揭示扩增失败情况。通过对单供体和单患者检测以及对样本小池加样来确定检测灵敏度。对三个血清转化样本组进行了检测。
灵敏度很高，直接检测时每毫升单患者样本材料中可检测到300个乙肝病毒基因组。对于96个混合样本，每毫升供体血浆的检测限达到1000个乙肝病毒基因组当量。在三个血清转化供体中，乙肝病毒感染的窗口期比乙肝表面抗原筛查的窗口期分别缩短了17天、10天和63天。
该检测方法具有足够的灵敏度，在输血医学中优于乙肝表面抗原筛查，且因其操作简便在临床实验室中将很有用。
