Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes
Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes
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DOI：
10.1007/s00216-006-0872-9
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发表时间：
2006-12-01
影响因子：
4.3
通讯作者：
Millard, Paul J.
中科院分区：
文献类型：
--
作者：
McCarthy, Erik L.;Egeler, Teressa J.;Millard, Paul J.
采用表面结合分子锁DNA探针（MPPs）和滚环扩增（RCA）技术，对传染性造血细胞坏死病毒（IHNV）和传染性鲑鱼贫血病毒（ISAV）RNA的特异性碱基序列进行了检测和鉴定。具有与从IHNV或ISAV获得的RNA相同的碱基序列的DNA寡核苷酸被MPP识别。然后通过固定的DNA寡核苷酸引物将环化的MPP捕获在玻璃微毛细管的内表面上。通过等温RCA延伸固定的引物产生DNA多联体，其用荧光SYBR绿色II核酸染料标记，并通过显微荧光法测量。分子挂锁探针，结合这种方法的表面相关的等温RCA，表现出高选择性，而不需要热循环。该方法适用于设计低功率场传感器，其能够在微毛细管的局部区域内多重检测病毒、细菌和原生动物病原体。
Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers, which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial, and protozoan pathogens within localized regions of microcapillary tubes.