Degenerate polymerase chain reaction strategy with DNA microarray for detection of multiple and various subtypes of virus during blood screening
Degenerate polymerase chain reaction strategy with DNA microarray for detection of multiple and various subtypes of virus during blood screening
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简并聚合酶链反应策略与 DNA 微阵列在血液筛查过程中检测多种病毒亚型
DOI：
10.1111/trf.12193
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Hamaguchi I
中科院分区：
文献类型：
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作者：
Takizawa K;Nakashima T;Mizukami T;Kuramitsu M;Endoh D;Kawauchi S;Sasaki K;Momose H;Kiba Y;Mizutani T;Furuta RA;Yamaguchi K;Hamaguchi I
背景：由于新出现和再出现的病毒，输血过程中血液传播感染的风险不断增加。需要开发一种快速筛选可能通过输血传播的新出现病毒的方法，以便在献血者筛选期间消除此类病原体。由于在器官移植和细胞治疗中越来越多地使用人体材料，供体传播病毒感染的风险也在增加。虽然核酸扩增技术（NAT）专门用于血液筛查，但在实验室和医院层面需要一种小型、方便的检测系统。研究设计与方法我们开发了一种新的病原体检测系统，该系统可以同时检测多种病毒，使用原始设计的简并聚合酶链反应引物扩增多种病毒基因型。扩增的样品使用病原体特异性探针的DNA微阵列进行鉴定。结果单片检测到人丙型肝炎病毒（HCV）、人乙型肝炎病毒（HBV）、人细小病毒B19 （PVB19）、西尼罗病毒（WNV）等多种亚型病毒的极低拷贝数。我们还检测了人类免疫缺陷病毒（HIV）的所有基因型，但敏感性低于其他病毒。结论我们开发了一种使用新型引物的微阵列检测方法，用于检测多种病原体和亚型。在特异性、敏感性和基因型包容性方面，我们的NAT系统在检测HIV、HBV、HCV、PVB19和WNV方面是准确可靠的。我们的系统可以针对新出现的病原体进行定制和扩展，适合作为未来的NAT系统。
BackgroundThe risk of transferring blood‐borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor‐transmitted viral infections is also increasing. Although nucleic acid amplification technology (NAT) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level.Study Design and MethodsWe developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen‐specific probes.ResultsWe detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus (HCV), human hepatitis B virus (HBV), human parvovirus B19 (PVB19), and West Nile virus (WNV), using a single plate. We also detected all genotypes of human immunodeficiency virus (HIV) but sensitivity was less than for the other viruses.ConclusionWe developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV, HBV, HCV, PVB19, and WNV, with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system.