JLM-Lifetech’s PCR "Twin Stars" Assist CAHS in Establishing a New "5-in-1 Multiplex" Detection Method for Sheep and Goat
In the battlefield of animal disease prevention and control, time is life, and precision is victory. Bluetongue virus (BTV), foot-and-mouth disease virus (FMDV), Schmallenberg virus (SBV), pests des petits ruminants virus (PPRV), and sheeppox/goatpox virus (SPPV-GTPV) are five major viral diseases that severely threaten the sheep and goat farming industry. Sharing similar clinical manifestations (such as high fever, mucosal lesions, and skin rashes) and being prone to mixed infections, detecting them through single-pathogen testing is both time-consuming and costly. Such traditional methods can no longer meet the urgent needs of customs quarantine and large-scale epidemiological screening.
Recently, the research team led by Shi Xiju at the China Academy of Household Animal Science (CAHS) / Chinese Academy of Customs Science and Technology published a major research breakthrough in the Chinese Journal of Veterinary Science. They successfully established a multiplex real-time fluorescence RT-PCR method capable of simultaneously detecting these five crucial small ruminant viruses. In this study, JLM-Lifetech's Digital Matrix-5000 digital PCR system and the QX600 quantitative fluorescence PCR system joined forces as a "golden duo," taking on the key responsibilities of "calibration" and "detection" respectively.
In this study, researchers first constructed recombinant plasmids containing the target genes of the five viruses. To determine the exact copy numbers contained in these "standard reference materials," the research team chose JLM-Lifetech’s Digital Matrix-5000 digital PCR system for precise quantification.
As shown in the figures below, absolute quantification was performed on the positive plasmids of BTV, FMDV, SBV, PPRV, and SPPV-GTPV. The droplet plots for each target plasmid demonstrated excellent separation between positive and negative droplets. The concentration of each standard reference material ranged from 2.13 x 103 to 1.02 x 104 copies/μL, providing a solid concentration baseline for subsequent standard curve generation and sensitivity analysis.
With the precise "ruler" provided by JLM-Lifetech’s digital PCR, the research team then utilized the JLM-Lifetech QX600 quantitative fluorescence PCR system to establish the multiplex detection assay. They successfully achieved the synchronized detection of all five viral targets within a single reaction system:
Multi-channel Simultaneous Acquisition: During the reaction, the QX600 successfully collected fluorescence signals across multiple channels, including FAM, HEX, ROX, TAMRA, CY5, and CY5.5, ensuring that the signals of different viruses were free from mutual interference and could be accurately identified.
Outstanding System Compatibility: Compared to singleplex assays, the amplification efficiency (E-value) of the multiplex system remained within the ideal range of 90%–100%, and the correlation coefficients (R2) were all greater than 0.996. This demonstrates the instrument's high stability and low error rate in multiplex detection.
The combination of JLM-Lifetech's "digital PCR calibration + QX600 detection" delivered exceptional performance for this research breakthrough:
100% Specificity: The established method generated amplification signals only for the target viruses, remaining negative for seven other common pathogens affecting sheep and goats.
Ultra-high Sensitivity: The limit of detection (LoD) for each target was below 5.31 copies/μL. Even if the viral load in a sample is extremely low, this method can sensitively capture it.
Perfect Score in Clinical Validation: In the testing of 500 clinical samples, the method successfully detected positive samples for PPRV, FMDV, and BTV, achieving 100% consistency with the results of the singleplex qPCR method.
This research achievement not only found its place in the core journal Chinese Journal of Veterinary Science but also carries profound practical significance. It condenses what would have been five separate experiments into a single run, greatly reducing the consumption of reagents and consumables, as well as labor costs. This is particularly suitable for large-scale screenings in intensive farming areas like Northwest China, and provides a powerful "smart eye" for rapid customs quarantine at ports. JLM-Lifetech, as the technical supporter behind this research, is deeply honored.
