Frontiers in microbiology · 2026
Development and evaluation of a multiplex PCR-based dual-platform targeted sequencing framework for precise differentiation of lumpy skin disease virus.
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Wang Yuqi, Wang Yifei, Bai Zilong, Wang Suqiu, Wang Lin, Zhang Wei, Gao Zhiqiang, Pu Jing, Du Sile, Deng Congliang, Xu Liwei, Wu Wangxue, Zhao Xiangpeng, Ren Tong, Wu Dan, Zhang Jiawei, Gu Qiang, Wang Shaolin, Shi Xiju
Аннотация
BACKGROUND: Lumpy skin disease virus (LSDV) shares over 96% genomic identity with goatpox and sheeppox viruses, presenting severe diagnostic challenges due to cross-reactivity.
METHODS: To address this bottleneck, we established a targeted sequencing framework integrating multiplex PCR with short-read and long-read platforms. By sequentially screening target pathogens, identifying low-homology genes, and designing short and gradient long-fragment primer pools, we evaluated these dual-platform panels using highly homologous poxvirus samples.
RESULTS: The short-read panel stably detected target viruses at inputs as low as 5.26 ×101 copies/μL. Under strict alignment criteria, LSDV mapping rates reached 42.91%, suppressing non-target signals to 3.05%. The Nanopore-Targeted Sequencing (NTS) long-amplicon strategy successfully eliminated homologous interference. By applying length-dependent diagnostic thresholds (≥ 100 reads for short amplicons; ≥ 50 reads for long amplicons), precise species-level identification was achieved, maintaining near-zero cross-reads (0-5) in ultra-long regions. Crucially, the field-deployable NTS workflow enabled complete detection in approximately 4 h.
CONCLUSION: This complementary strategy seamlessly meets both laboratory demands for high-sensitivity enrichment and frontline requirements for rapid typing, providing a reliable tool for LSDV surveillance, mutation tracking, and outbreak control.
METHODS: To address this bottleneck, we established a targeted sequencing framework integrating multiplex PCR with short-read and long-read platforms. By sequentially screening target pathogens, identifying low-homology genes, and designing short and gradient long-fragment primer pools, we evaluated these dual-platform panels using highly homologous poxvirus samples.
RESULTS: The short-read panel stably detected target viruses at inputs as low as 5.26 ×101 copies/μL. Under strict alignment criteria, LSDV mapping rates reached 42.91%, suppressing non-target signals to 3.05%. The Nanopore-Targeted Sequencing (NTS) long-amplicon strategy successfully eliminated homologous interference. By applying length-dependent diagnostic thresholds (≥ 100 reads for short amplicons; ≥ 50 reads for long amplicons), precise species-level identification was achieved, maintaining near-zero cross-reads (0-5) in ultra-long regions. Crucially, the field-deployable NTS workflow enabled complete detection in approximately 4 h.
CONCLUSION: This complementary strategy seamlessly meets both laboratory demands for high-sensitivity enrichment and frontline requirements for rapid typing, providing a reliable tool for LSDV surveillance, mutation tracking, and outbreak control.