School of Molecular Sciences, Arizona State University, Tempe, AZ, USA.
School of Life Sciences, Arizona State University, Tempe, AZ, USA.
Virol J. 2017 Oct 13;14(1):197. doi: 10.1186/s12985-017-0863-9.
Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity to understand virus evolution and can be useful for experimental systems.
We have developed a novel molecular method for sequence-specific detection of RNA virus genetic variants called Tentacle Probes. The probes are modified molecular beacons that have dramatically improved false positive rates and specificity in routine qPCR. To validate this approach, we have designed Tentacle Probes for two different strains of Lymphocytic Choriomeningitis Virus (LCMV) that differ by only 3 nucleotide substitutions, the parental Armstrong and the more virulent Clone-13 strain. One of these mutations is a missense mutation in the receptor protein GP1 that leads to the Armstrong strain to cause an acute infection and Clone-13 to cause a chronic infection instead. The probes were designed using thermodynamic calculations for hybridization between target or non-target sequences and the probe.
Using this approach, we were able to distinguish these two strains of LCMV individually by a single nucleotide mutation. The assay showed high reproducibility among different concentrations of viral cDNA, as well as high specificity and sensitivity, especially for the Clone-13 Tentacle Probe. Furthermore, in virus mixing experiments we were able to detect less than 10% of Clone-13 cDNA diluted in Armstrong cDNA.
Thus, we have developed a fast, cost-effective approach for identifying Clone-13 strain in a mix of other LCMV strains.
病毒感染通常会导致病毒株的准种，这可能对疾病结果产生巨大影响。然而，测序病毒以确定菌株组成既耗时又往往成本过高。需要快速、经济有效的方法来准确测量病毒多样性，以了解病毒进化，并且对实验系统也很有用。
我们开发了一种新的分子方法，用于序列特异性检测 RNA 病毒遗传变异，称为 Tentacle Probes。这些探针是经过修饰的分子信标，在常规 qPCR 中大大提高了假阳性率和特异性。为了验证这种方法，我们针对两种不同的淋巴细胞性脉络丛脑膜炎病毒 (LCMV) 菌株设计了 Tentacle Probes，它们仅相差 3 个核苷酸替换，亲本 Armstrong 菌株和更具毒性的 Clone-13 菌株。这些突变中的一个是受体蛋白 GP1 中的错义突变，导致 Armstrong 株引起急性感染，而 Clone-13 株引起慢性感染。这些探针是使用目标或非目标序列与探针之间的杂交的热力学计算来设计的。
使用这种方法，我们能够通过单个核苷酸突变单独区分这两种 LCMV 菌株。该测定法显示了不同浓度病毒 cDNA 之间的高重现性，以及高特异性和灵敏度，特别是对于 Clone-13 Tentacle Probe。此外，在病毒混合实验中，我们能够检测到稀释在 Armstrong cDNA 中的不到 10%的 Clone-13 cDNA。
因此，我们开发了一种快速、经济有效的方法，用于识别混合其他 LCMV 菌株中的 Clone-13 菌株。
