Evaluation of a new PCR assay with competitive internal control sequence for blood donor screening
Evaluation of a new PCR assay with competitive internal control sequence for blood donor screening
复制标题
DOI：
10.1046/j.1537-2995.2000.40060718.x
复制
发表时间：
2000-06-01
期刊：
影响因子：
2.9
通讯作者：
Roth, WK
中科院分区：
文献类型：
--
作者：
Drosten, C;Weber, M;Roth, WK
背景：采用PCR技术进行输血相关病毒的高通量核酸检测需要具有高灵敏度和高效度的防污染方法。一种新的PGR试剂盒（TaqMan, PE BioSystems）通过消除pcr后处理降低了携带污染的风险。研究设计和方法：寡核苷酸设计是用专门设计试验（TaqMan）引物和探针的软件完成的。通过位点定向诱变设计的模板衍生竞争性内控序列用于揭示扩增失败。测定单供体和单患者检测的灵敏度，并通过样品小池进行测定。测试了三个血清转换面板。结果：灵敏度高，直接检测单个患者材料每公吨可达300个HBV基因组。在96个合并样本中，每毫升供体血浆的检测限达到1000个HBV基因组当量。在三个血清转化供者中，HBV感染的窗口期分别比HBsAg筛查减少了17天、10天和63天。结论：该方法具有足够的灵敏度，在输血医学中优于HBsAg筛查，且易于操作，可用于临床实验室。
BACKGROUND: High-throughput nucleic acid testing for transfusion-relevant viruses by PCR requires contamination-proof methods with high sensitivity and validity. A new PGR reagent kit (TaqMan, PE BioSystems) reduces the risk of carry-over contamination by eliminating post-PCR processing.STUDY DESIGN AND METHODS: 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.RESULTS: Sensitivity is high, reaching 300 HBV genomes per mt of single-patient material on direct testing. A detection limit of 1000 HBV genome equivalents per mi 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.CONCLUSION: 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.