Rapid genotyping of varicella-zoster virus vaccine and wild-type strains with fluorophore-labeled hybridization probes
Rapid genotyping of varicella-zoster virus vaccine and wild-type strains with fluorophore-labeled hybridization probes
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DOI：
10.1128/jcm.38.12.4315-4319.2000
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发表时间：
2000-12-01
影响因子：
9.4
通讯作者：
Schmid, DS
中科院分区：
文献类型：
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作者：
Loparev, VN;McCaustland, K;Schmid, DS
我们开发了一种单管快速检测和区分水痘-带状疱疹病毒（VZV）疫苗和野生型毒株的方法，该方法将快速循环PCR与野生型特异性荧光探针熔解图谱相结合，用于产品基因分型。在两个荧光标记的杂交探针存在下，扩增包括VZV开放阅读框（ORF）62中的多态性位点的区域。在热循环的退火步骤期间，两种探针都与扩增子中的互补序列结合，导致共振能量转移，从而提供PCR的实时荧光监测。在PCR完成时的解链曲线分析过程中连续采集荧光数据揭示，当与野生型VZV ORF 62区完全互补的检测探针从模板上解链时，以菌株特异性方式发生荧光损失。使用这种方法可以在PCR完成后几分钟内对样品进行基因分型，从而消除了对PCR后样品操作的需要。除了减少产生结果所需的时间外，该方法还大大降低了最终产品污染的风险以及样品跟踪错误的风险。79 VZV阳性样品的基因型确定的荧光共振能量转移（FRET）方法是相同的基因型通过常规PCR和限制性片段长度多态性分析。通过FRET方法对VZV毒株进行基因分型是一种快速可靠的方法，适用于fbr分型，也适用于大量标本的处理。
We developed a single-tube rapid method for the detection and differentiation of varicella-zoster virus (VZV) vaccine and wild-type strains that combines rapid-cycle PCR with wild-type-specific fluorescent probe melting profiles for product genotyping. A region including the polymorphic site in VZV open reading frame (ORF) 62 was amplified in the presence of two fluorescence-labeled hybridization probes. During the annealing step of the thermal cycling, both probes bound to their complementary sequences in the amplicon, resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous acquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in a strain-specific manner as the detection probe, which was fully complementary to the wild-type VZV ORF 62 region, melted off the template. Use of this method allowed genotyping of samples within minutes after the completion of PCR, eliminating the need for post-PCR sample manipulation. In addition to reducing the time required to produce a result, this method substantially reduces the risk of contamination of the final product as well as the risk of sample tracking errors. The genotypes of 79 VZV-positive samples determined by this fluorescent resonance energy transfer (FRET) method were identical to the genotypes obtained by conventional PCR and restriction fragment length polymorphism analysis. The genotyping of VZV strains by the FRET method is a rapid and reliable method that is suitable fbr typing and that is also practical for use for the processing of large numbers of specimens.