Assessing oligonucleotide designs from early lab developed PCR diagnostic tests for SARS-CoV-2 using the PCR_strainer pipeline
Assessing oligonucleotide designs from early lab developed PCR diagnostic tests for SARS-CoV-2 using the PCR_strainer pipeline
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DOI：
10.1016/j.jcv.2020.104581
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发表时间：
2020-10-01
影响因子：
8.8
通讯作者：
Prystajecky, Natalie A.
中科院分区：
文献类型：
--
作者：
Kuchinski, Kevin S.;Jassem, Agatha N.;Prystajecky, Natalie A.
简介：在SARS-CoV-2爆发的第一个月，快速开发基于PCR的诊断测试成为全球优先事项，以便及时诊断，隔离和接触者追踪可以最大限度地减少大流行的激增。设计这些广泛的，长期的检测测试是复杂的有限的信息，新病毒的基因组序列，以及它如何可能在全球传播和适应human.Methods突变：我们评估了8个广泛采用的实验室开发的PCR检测SARS-CoV-2对15，001 SARS-CoV-2基因组序列。使用自定义的生物信息学管道称为PCR_strainer，我们确定了所有的错配和序列变异的基因组位置所针对的15套引物/探针寡核苷酸从这些assays.Results：对于12的15个引物/探针组，超过98%的SARS-CoV-2基因组没有错配。两个引物/探针组在反向引物中含有单一错配，其存在于超过99%的基因组中。一组引物/探针针对具有广泛多态性的位置，在正向引物位置观察到23个序列变体。其中一种变体包含三个核苷酸错配，作为病毒Glade出现的一部分于2月份出现，并存在于我们分析的18.8%的基因组中。讨论：大多数SARS-CoV-2的早期PCR诊断测试仍然包括循环病毒的多样性，但三种具有广泛错配的检测方法突出了新型病原体的检测方法设计挑战，并为PCR检测方法设计提供了宝贵的经验教训。未来的爆发。我们的生物信息学管道也被认为是一个有用的通用工具，用于评估针对循环菌株的PCR诊断检测。
Introduction: During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing these tests for broad, long-term detection was complicated by limited information about the novel virus' genome sequence and how it might mutate during global spread and adaptation to humans.Methods: We assessed eight widely adopted lab developed PCR tests for SARS-CoV-2 against 15,001 SARS-CoV-2 genome sequences. Using a custom bioinformatic pipeline called PCR_strainer, we identified all mismatches and sequence variants in genome locations targeted by 15 sets of primer/probe oligonucleotides from these assays.Results: For 12 out of 15 primer/probe sets, over 98 % of SARS-CoV-2 genomes had no mismatches. Two primer/probe sets contained a single mismatch in the reverse primer that was present in over 99 % of genomes. One primer/probe set targeted a location with extensive polymorphisms with 23 sequence observed variants at the forward primer location. One of these variants, which contains three nucleotide mismatches, arose in February as part of the emergence of a viral Glade and was present in 18.8 % of the genomes we analyzed.Discussion: Most early PCR diagnostic tests for SARS-CoV-2 remain inclusive of circulating viral diversity, but three assays with extensive mismatches highlight assay design challenges for novel pathogens and provide valuable lessons for PCR assay design during future outbreaks. Our bioinformatics pipeline is also presented as a useful general-purpose tool for assessing PCR diagnostics assays against circulating strains.