Rajeevan Mangalathu S, Patel Sonya, Li Tengguo, Unger Elizabeth R
Centers for Disease Control and Prevention, Division of High-Consequence Pathogens & Pathology, 1600 Clifton Road, Atlanta, GA 30329, USA.
Viruses. 2021 Jan 27;13(2):188. doi: 10.3390/v13020188.
High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV vaccination. We evaluated the NanoString nCounter platform for detection and digital readout of 48 HPV types in a single reaction. NanoString (NS) used proprietary software to design CodeSets: type-specific probe pairs targeting 48 HPV types and the globin gene. We tested residual DNA extracts from epidemiologic specimens and defined samples (HPV plasmids at 10 to 10 copies/reaction) directly (No-PCR) as well as after L1 consensus PCR of 45 (PCR-45) or 15 cycles (PCR-15). Assay and interpretation followed NS recommendations. We evaluated analytic performance by comparing NanoString results for types included in prior assays: Roche Linear Array (LA) or HPV TypeSeq assay. No-PCR results on 40 samples showed good type-specific agreement with LA (k = 0.621) but sensitivity was 65% with lower limit of detection (LOD) at 10 plasmid copies. PCR-45 results showed almost perfect type-specific agreement with LA (k = 0.862), 82% sensitivity and LOD at 10 copies. PCR-15 results on 75 samples showed substantial type-specific agreement with LA (k = 0.796, 92% sensitivity) and TypeSeq (k = 0.777, 87% sensitivity), and LOD at 10 copies of plasmids. This proof-of-principle study demonstrates the efficacy of the NS platform with HPV CodeSet for type-specific detection using a low number of PCR cycles (PCR-15). Studies are in progress to evaluate assay reproducibility and analytic validation with a larger number of samples.
具有更高自动化程度、更快周转时间和特定类型数字读数的高通量HPV分型检测方法将有助于监测HPV疫苗接种影响的研究。我们评估了NanoString nCounter平台在单一反应中检测48种HPV类型并进行数字读数的能力。NanoString（NS）使用专有软件设计CodeSets：针对48种HPV类型和珠蛋白基因的特定类型探针组。我们直接（无PCR）以及在进行45个循环（PCR-45）或15个循环（PCR-15）的L1共识PCR后，测试了流行病学标本和定义样本（HPV质粒，10至10拷贝/反应）中的残留DNA提取物。检测和解读遵循NS的建议。我们通过比较NanoString结果与先前检测（罗氏线性阵列（LA）或HPV TypeSeq检测）中包含的类型来评估分析性能。对40个样本的无PCR结果显示与LA具有良好的特定类型一致性（k = 0.621），但灵敏度为65%，检测下限（LOD）为10个质粒拷贝。PCR-45结果显示与LA几乎具有完美的特定类型一致性（k = 0.862），灵敏度为82%，LOD为10个拷贝。对75个样本的PCR-15结果显示与LA（k = 0.796，灵敏度92%）和TypeSeq（k = 0.777，灵敏度87%）具有实质性的特定类型一致性，质粒拷贝数为10时的LOD。这项原理验证研究证明了NS平台与HPV CodeSet在使用少量PCR循环（PCR-15）进行特定类型检测方面的有效性。正在进行研究以评估大量样本的检测重现性和分析验证。
