背景:来自快速诊断测试(RDT)的SARS-CoV-2测序可以支持病毒基因组监测工作;然而,从RDT中最大化和标准化病原体基因组恢复的方法仍然不发达。我们旨在系统地优化RDT成分中遗传物质的洗脱,并评估RDT测序对暴发调查的有效性。
方法:在这项实验室和基于队列的研究中,我们用灭活的SARS-CoV-2接种RDT,以优化RDT侧向流条上基因组材料的洗脱。我们测量了缓冲区类型变化的影响,缓冲区中的时间,和旋转PCR循环阈值(Ct)值。我们招募了居住在大波士顿地区的18岁以上的人,MA,美国,从2022年7月18日至11月5日,通过电子邮件广告向哈佛大学的学生和教职员工,MA,美国,通过广泛的社交媒体广告。招募的所有个体均在SARS-CoV-2阳性诊断测试的5天内;未应用其他相关排除标准。每个个体完成两个RDT和一个PCR拭子。在2022年12月29日,我们还从对SARS-CoV-2呈阳性并与MA高级住房设施爆发有关的个人的便利样本中收集了RDT。美国。我们提取了所有返回的PCR拭子和RDT组件(即,拭子,strip,或缓冲液);Ct小于40的样品进行扩增子测序。我们比较了RDT品牌和组件的洗脱和测序的功效,并使用RDT衍生的序列来推断高级住房设施爆发期间的传播联系。我们对居住在高级住房设施中的有症状个体的阴性RDT进行了宏基因组测序。
结果:大于10分钟的洗脱持续时间和洗脱期间的旋转均不影响病毒滴度。在AVL缓冲液(Ct=31·4)和Tris-EDTA缓冲液(Ct=30·8)中的洗脱是等效的(p=0·34);AVL在裂解缓冲液和50%裂解缓冲液(Ct=40·0,两者p=0·0029)以及通用病毒转运培养基(Ct=36·7,p=0·079)中的洗脱优于AVL。RDT条的性能比匹配的PCR拭子的性能差(平均Ct差异10·2[SD4·3],p<0·0001);然而,RDT拭子与PCR拭子相似(平均Ct差异4·1[5·2],p=0·055)。没有RDT品牌的表现明显优于其他品牌。跨样本类型,病毒载量预测病毒基因组组装长度。我们从17个RDT衍生的拭子中的12个组装了超过80%的完整基因组,18条中的3条,和11个剩余缓冲液中的4个。我们使用扩增子和宏基因组测序产生了爆发相关的SARS-CoV-2基因组,并鉴定了导致下游传播的病毒的多个引入。
结论:RDT来源的拭子是用于病毒基因组监测和暴发调查的PCR拭子的合理替代方法。RDT衍生的侧向流条产量准确,但是少得多,病毒读取比匹配的PCR拭子。阴性RDT的宏基因组测序可以识别可能是患者症状基础的病毒。
背景:美国国家科学基金会,赫兹基金会,国家普通医学科学研究所,哈佛医学院,霍华德·休斯医学院,美国疾病控制和预防中心,广泛研究所和国家过敏和传染病研究所。
BACKGROUND: Sequencing of SARS-CoV-2 from rapid diagnostic tests (RDTs) can bolster viral genomic surveillance efforts; however, approaches to maximise and standardise pathogen genome recovery from RDTs remain underdeveloped. We aimed to systematically optimise the elution of genetic material from RDT components and to evaluate the efficacy of RDT sequencing for outbreak investigation.
METHODS: In this laboratory and cohort-based
study we seeded RDTs with inactivated SARS-CoV-2 to optimise the elution of genomic material from RDT lateral flow strips. We measured the effect of changes in buffer type, time in buffer, and rotation on PCR cycle threshold (Ct) value. We recruited individuals older than 18 years residing in the greater Boston area, MA, USA, from July 18 to Nov 5, 2022, via email advertising to students and staff at Harvard University, MA, USA, and via broad social media advertising. All individuals recruited were within 5 days of a positive diagnostic test for SARS-CoV-2; no other relevant exclusion criteria were applied. Each individual completed two RDTs and one PCR swab. On Dec 29, 2022, we also collected RDTs from a convenience sample of individuals who were positive for SARS-CoV-2 and associated with an outbreak at a senior housing facility in MA, USA. We extracted all returned PCR swabs and RDT components (ie, swab, strip, or buffer); samples with a Ct of less than 40 were subject to amplicon sequencing. We compared the efficacy of elution and sequencing across RDT brands and components and used RDT-derived sequences to infer transmission links within the outbreak at the senior housing facility. We conducted metagenomic sequencing of negative RDTs from symptomatic individuals living in the senior housing facility.
RESULTS: Neither elution duration of greater than 10 min nor rotation during elution impacted viral titres. Elution in Buffer AVL (Ct=31·4) and Tris-EDTA Buffer (Ct=30·8) were equivalent (p=0·34); AVL outperformed elution in lysis buffer and 50% lysis buffer (Ct=40·0, p=0·0029 for both) as well as Universal Viral Transport Medium (Ct=36·7, p=0·079). Performance of RDT strips was poorer than that of matched PCR swabs (mean Ct difference 10·2 [SD 4·3], p<0·0001); however, RDT swabs performed similarly to PCR swabs (mean Ct difference 4·1 [5·2], p=0·055). No RDT brand significantly outperformed another. Across sample types, viral load predicted the viral genome assembly length. We assembled greater than 80% complete genomes from 12 of 17 RDT-derived swabs, three of 18 strips, and four of 11 residual buffers. We generated outbreak-associated SARS-CoV-2 genomes using both amplicon and metagenomic sequencing and identified multiple introductions of the virus that resulted in downstream transmission.
CONCLUSIONS: RDT-derived swabs are a reasonable alternative to PCR swabs for viral genomic surveillance and outbreak investigation. RDT-derived lateral flow strips yield accurate, but significantly fewer, viral reads than matched PCR swabs. Metagenomic sequencing of negative RDTs can identify viruses that might underlie patient symptoms.
BACKGROUND: The National Science Foundation, the Hertz Foundation, the National Institute of General Medical Sciences, Harvard Medical School, the Howard Hughes Medical Institute, the US Centers for Disease Control and Prevention, the Broad Institute and the National Institute of Allergy and Infectious Diseases.