诺如病毒与磁珠偶联的组织血型抗原结合的特异性和动力学。
Specificity and kinetics of norovirus binding to magnetic bead-conjugated histo-blood group antigens.
机构信息
Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94547-1105, USA.
出版信息
J Appl Microbiol. 2010 Nov;109(5):1753-62. doi: 10.1111/j.1365-2672.2010.04812.x. Epub 2010 Aug 2.
AIMS
To characterize the specificity and effect of pH and ionic strength on the kinetics of virus binding to histo-blood group antigens (HBGA)-conjugated magnetic beads.
METHODS AND RESULTS
HBGAs from porcine gastric mucin (PGM) have been conjugated to magnetic beads (PGM-MB) for concentration of NoV. A GII.4 virus was used for the detailed binding kinetics study and a panel of genogroup I (GI) NoVs, genogroup II (GII) NoVs and recombinant NoVs (rNoVs) were used for specificity and binding efficiency assays. We determined that NoV can be captured after 15min of incubation with PGM-MB, and virus recovery efficiency is decreased after extended incubation times. rNoV binding as measured by ELISA and NoV recovery as measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), were both enhanced significantly at acidic pH conditions. rNoV binding to PGM as measured by ELISA was increased up to 66%. While real-time RT-PCR analyses suggest that NoV could be concentrated as much as 1000-fold at neutral pH, up to 3·4-fold further increase of NoV recovery was achieved by adjusting the pH of the sample to 3·0-4·2. Variation between GI and GII viral binding to the PGM-MB at basic pH was observed. All five GI rNoVs tested and 6 of 9 GII rNoVs were captured by PGM. All eight GI strains tested were concentrated by PGM-MB, ranging from 28-fold (GI.4) to 1502-fold (GI.1). Eleven of 13 GII strains were concentrated from 30-fold (GII.5) to 1014-fold (GII.4, lab strain) by PGM-MB. GI and GII rNoVs viral capsid proteins were recovered with high salt conditions, but results were inconsistent for whole virus recovery.
CONCLUSIONS
All GI and 85% of GII NoVs tested could be captured and concentrated by PGM-MB method. The binding occurred rapidly and was enhanced at low pH.
SIGNIFICANCE AND IMPACT OF THE STUDY
These results facilitated development of a prototype method for sensitive detection of NoV in samples requiring larger volumes.
目的
研究 pH 值和离子强度对病毒与结合有组织血型抗原（HBGA）的磁珠结合动力学的特异性和影响。
方法和结果
将猪胃粘蛋白（PGM）中的 HBGA 与磁珠（PGM-MB）结合，用于浓缩诺如病毒。使用 GII.4 病毒进行详细的结合动力学研究，并使用一组基因 I（GI）诺如病毒、基因 II（GII）诺如病毒和重组诺如病毒（rNoV）进行特异性和结合效率测定。我们发现，诺如病毒在与 PGM-MB 孵育 15 分钟后即可被捕获，并且在延长孵育时间后病毒回收率会降低。ELISA 法测定 rNoV 结合率和实时 RT-PCR 法测定诺如病毒回收率均在酸性 pH 条件下显著增加。ELISA 法测定 rNoV 与 PGM 的结合率增加了 66%。虽然实时 RT-PCR 分析表明，在中性 pH 值下，诺如病毒的浓度可高达 1000 倍，但通过将样品 pH 值调节至 3.0-4.2，可使诺如病毒回收率进一步增加 3.4 倍。在碱性 pH 值下，观察到 GI 和 GII 病毒与 PGM-MB 的结合存在差异。检测的 5 种 GI rNoV 和 9 种 GII rNoV 中的 6 种均可被 PGM 捕获。所有 8 种 GI 株均被 PGM-MB 浓缩，范围从 28 倍（GI.4）到 1502 倍（GI.1）。13 株 GII 株中的 11 株经 PGM-MB 浓缩 30 倍（GII.5）至 1014 倍（GII.4，实验室株）。所有 GI 和 85%的 GII NoV 均可通过 PGM-MB 方法捕获和浓缩。结合发生迅速，并在低 pH 值下增强。
结论
所有 GI 和 85%的 GII NoV 均可通过 PGM-MB 方法捕获和浓缩。结合发生迅速，并在低 pH 值下增强。
意义和影响
这些结果促进了一种用于从需要更大体积样本中灵敏检测诺如病毒的原型方法的开发。
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