A dual-target real-time PCR for proactive detection of Mpox variants

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods Pub Date : 2025-09-05 DOI:10.1016/j.jviromet.2025.115260

Tracy D. Lee , Alan O’Dwyer , Michael Chan , Branco Cheung , Frankie Tsang , John R. Tyson , Kathleen L. Kolehmainen , Natalie A. Prystajecky , Agatha N. Jassem

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引用次数: 0

Abstract

In 2022, cases of Monkeypox virus (MPXV) in California contained a mutation in the TNF receptor gene (GR2G) that rendered the virus undetectable using a widely adopted public health diagnostic qPCR assay. This underscored the need for a dual-target PCR approach and prompted validation of a second target by the BCCDC Public Health Laboratory. In addition to the GR2G target validated in the original qPCR assay (and duplexed with the endogenous target human β-globin (HBG)), GP113 (OPG128) was identified and validated using both clinical samples and MPXV DNA controls. Mutations in GR2G and GP113 (found in the emerging clade Ib) were also addressed along with the updated target. The new triplex assay (GR2G/GP113/HBG) had 100 % inclusivity and 100 % accuracy with all clinical samples tested and did not cross-react with herpes simplex virus-1 or −2, varicella zoster virus, or enterovirus. It showed < 5 % coefficient of variance between replicates and had a limit-of-detection of 10 copies/μL for GR2G and GP113. The use of two targets presents redundancy against further mutations in MPXV and is recommended for use with all viral qPCR assays moving forward.

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主动检测m痘变异的双目标实时PCR。

2022年，加利福尼亚州的猴痘病毒（MPXV）病例包含TNF受体基因（GR2G）突变，这使得使用广泛采用的公共卫生诊断qPCR检测无法检测到该病毒。这强调了双靶点PCR方法的必要性，并促使BCCDC公共卫生实验室对第二个靶点进行验证。除了在最初的qPCR实验中验证的GR2G靶点（并与内源性靶点人β-珠蛋白（HBG）双活）外，GP113 （OPG128）在临床样本和MPXV DNA对照中被鉴定和验证。GR2G和GP113的突变（在新兴分支Ib中发现）也与更新的靶标一起得到了解决。新的三重检测（GR2G/GP113/HBG）对所有临床样本检测具有100%的包容性和100%的准确性，并且不会与单纯疱疹病毒-1或-2、水痘带状疱疹病毒或肠病毒发生交叉反应。它显示

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来源期刊

Journal of virological methods 医学-病毒学

CiteScore

5.80

自引率

0.00%

发文量

209

审稿时长

41 days

期刊介绍： The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. The methods may include, but not limited to, the study of: Viral components and morphology- Virus isolation, propagation and development of viral vectors- Viral pathogenesis, oncogenesis, vaccines and antivirals- Virus replication, host-pathogen interactions and responses- Virus transmission, prevention, control and treatment- Viral metagenomics and virome- Virus ecology, adaption and evolution- Applied virology such as nanotechnology- Viral diagnosis with novelty and comprehensive evaluation. We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.