The specificity of the multiplex real-time qPCR assay was determined by utilizing the positive DNA or cDNA of the above-mentioned swine pathogens PCV1, PCV3, PCV4, PRRSV, CSFV, PPV, Chlamydia suis, and Toxoplasma gondii as detection templates, the plasmid standards as positive controls, and ddH2O as a negative control. All templates and ddH2O were repeated three times under the optimal reaction conditions for specificity of the multiplex PCR.
The detection limit determines the occurence of false negatives during detection. To analyze the sensitivity of the established multiplex real-time qPCR, the linearization standard plasmids, pEasy-ASFV, pEasy-PCV2, and pEasy-PRV, prepared above were diluted 10-fold serially to a final concentration between 1 × 107 copies/μL and 1 × 101 copies/μL in nuclease-free water. The diluted standard plasmids were used as templates for multiplex qPCR amplification in the optimal reaction conditions.
To analyze the repeatability and reproducibility of the established multiplex real-time qPCR, the linearization standard plasmids, pEasy-ASFV, pEasy-PCV2, and pEasy-PRV, were diluted by a factor of 10 from 107 copies/μL to 104 copies/μL using nuclease-free water. The diluted standard plasmids were used as templates for multiplex real-time qPCR amplification. Using the optimized amplification conditions, each diluted standard plasmid was amplified three times repeatedly and tested every other week. The different concentration groups were compared, and their intra-group and inter-group coefficients of variation were determined to confirm their reproducibility.
The detection limit determines the occurence of false negatives during detection. To analyze the sensitivity of the established multiplex real-time qPCR, the linearization standard plasmids, pEasy-ASFV, pEasy-PCV2, and pEasy-PRV, prepared above were diluted 10-fold serially to a final concentration between 1 × 107 copies/μL and 1 × 101 copies/μL in nuclease-free water. The diluted standard plasmids were used as templates for multiplex qPCR amplification in the optimal reaction conditions.
To analyze the repeatability and reproducibility of the established multiplex real-time qPCR, the linearization standard plasmids, pEasy-ASFV, pEasy-PCV2, and pEasy-PRV, were diluted by a factor of 10 from 107 copies/μL to 104 copies/μL using nuclease-free water. The diluted standard plasmids were used as templates for multiplex real-time qPCR amplification. Using the optimized amplification conditions, each diluted standard plasmid was amplified three times repeatedly and tested every other week. The different concentration groups were compared, and their intra-group and inter-group coefficients of variation were determined to confirm their reproducibility.
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