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一种新型的长尾引物核酸检测方法,可在即时护理点检测 HPV 16、18 和 45 DNA。

A novel tailed primer nucleic acid test for detection of HPV 16, 18 and 45 DNA at the point of care.

机构信息

Department of Bioengineering, Rice University, Houston, TX, USA.

Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD, USA.

出版信息

Sci Rep. 2023 Nov 21;13(1):20397. doi: 10.1038/s41598-023-47582-y.

Abstract

Cervical cancer is a leading cause of death for women in low-resource settings despite being preventable through human papillomavirus (HPV) vaccination, early detection, and treatment of precancerous lesions. The World Health Organization recommends high-risk HPV (hrHPV) as the preferred cervical cancer screening strategy, which is difficult to implement in low-resource settings due to high costs, reliance on centralized laboratory infrastructure, and long sample-to-answer times. To help meet the need for rapid, low-cost, and decentralized cervical cancer screening, we developed tailed primer isothermal amplification and lateral flow detection assays for HPV16, HPV18, and HPV45 DNA. We translated these assays into a self-contained cartridge to achieve multiplexed detection of three hrHPV genotypes in a disposable cartridge. The developed test achieves clinically relevant limits of detection of 50-500 copies per reaction with extracted genomic DNA from HPV-positive cells. Finally, we performed sample-to-answer testing with direct lysates of HPV-negative and HPV-positive cell lines and demonstrated consistent detection of HPV16, HPV18, and HPV45 with 5000-50,000 cells/mL in < 35 min. With additional optimization to improve cartridge reliability, incorporation of additional hrHPV types, and validation with clinical samples, the assay could serve as a point-of-care HPV DNA test that improves access to cervical cancer screening in low-resource settings.

摘要

尽管人乳头瘤病毒(HPV)疫苗接种、早期发现和癌前病变治疗可预防宫颈癌,但宫颈癌仍是资源匮乏地区女性死亡的主要原因之一。世界卫生组织建议采用高危型 HPV(hrHPV)作为宫颈癌筛查的首选策略,但由于成本高、依赖集中式实验室基础设施以及样本周转时间长,该策略在资源匮乏地区难以实施。为满足对快速、低成本和分散式宫颈癌筛查的需求,我们开发了用于 HPV16、HPV18 和 HPV45 DNA 的长尾引物等温扩增和侧向流检测分析。我们将这些分析转化为一个独立的试剂盒,以在一次性试剂盒中实现三种 hrHPV 基因型的多重检测。该检测方法在提取 HPV 阳性细胞的基因组 DNA 时,可达到 50-500 拷贝/反应的临床相关检测限。最后,我们直接对 HPV 阴性和 HPV 阳性细胞系的裂解物进行了样本至答案的测试,并证明在<35 分钟内可稳定检测到 5000-50000 个细胞/mL 的 HPV16、HPV18 和 HPV45。通过进一步优化以提高试剂盒的可靠性、纳入更多的 hrHPV 类型以及对临床样本进行验证,该检测方法可作为一种即时护理 HPV DNA 检测方法,改善资源匮乏地区宫颈癌筛查的可及性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a19b/10663460/66befd208c0e/41598_2023_47582_Fig1_HTML.jpg

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