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Updated: Sep 10, 2026
Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
Published on: March 13, 2018
Development and evaluation of a multiplex PCR-based dual-platform targeted sequencing framework for precise
Yuqi Wang1, Yifei Wang1, Zilong Bai2
1State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, China.
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.
