Journal: Transboundary and Emerging Diseases

Article Title: A RNase H2‐Linked TaqMan‐MGB Quantitative Real‐Time PCR Assay for Differential Detection of Mycoplasma hyopneumoniae Live‐Attenuated Vaccine Strains

doi: 10.1155/tbed/5823134

Figure Lengend Snippet: The optimization results of the RNase H2‐linked qPCR assay. (A) Probe concentration gradient (0.4/0.45/0.5/0.55/0.6 µM) for the 168‐L strain. (B) Probe concentration gradient (0.4/0.45/0.5/0.55/0.6 µM) for the RM48 strain. (C) Evaluation of nonspecific amplification of the wild‐type strain 168 under different probe concentrations (0.4/0.45/0.5/0.55/0.6 µM). (D) Annealing temperature gradient (58/59/60/61/63°C) for the 168‐L strain. (E) Annealing temperature gradient (58/59/60/61/63°C) for the RM48 strain. (F) Evaluation of nonspecific amplification of the wild‐type strain 168 under different annealing temperatures (58/59/60/61/63°C).

Article Snippet: Then, specific primers (Supporting Information Table ) for the selected candidate targets were designed to amplify the live‐attenuated vaccine strain RM48, and the amplification products were sent to Novogene Co., Ltd. for sequencing.

Techniques: Concentration Assay, Amplification

Journal: Transboundary and Emerging Diseases

Article Title: A RNase H2‐Linked TaqMan‐MGB Quantitative Real‐Time PCR Assay for Differential Detection of Mycoplasma hyopneumoniae Live‐Attenuated Vaccine Strains

doi: 10.1155/tbed/5823134

Figure Lengend Snippet: Sensitivity validation of the RNase H2‐linked differential qPCR assay. (A) Amplification curves of the standard plasmid pUC57‐vaccine at serial concentrations: 1 × 10 8 copies/µL, 1 × 10 7 copies/µL, 1 × 10 6 copies/µL, 1 × 10 5 copies/µL, 1 × 10 4 copies/µL, 1 × 10 3 copies/µL, 1 × 10 2 copies/µL, 10 copies/µL and 1 copy/µL. (B) Standard curve of the pUC57‐vaccine plasmid, plotted as log 10 copies/µL versus cycle threshold (Ct) value. (C) Amplification curves of the live‐attenuated vaccine strain 168‐L genomic DNA at serial dilutions: 10 0.5 CCU 50 /mL, 10 1.5 CCU 50 /mL, 10 2.5 CCU 50 /mL, 10 3.5 CCU 50 /mL, 10 4.5 CCU 50 /mL, 10 5.5 CCU 50 /mL, 10 6.5 CCU 50 /mL, and 10 7.5 CCU 50 /mL. (D) Standard curve of strain 168‐L, plotted as log 10 CCU 50 versus Ct value. (E) Amplification curves of the live‐attenuated vaccine strain RM48 genomic DNA at serial dilutions: 10 0.5 CCU 50 /mL, 10 1.5 CCU 50 /mL, 10 2.5 CCU 50 /mL, 10 3.5 CCU 50 /mL, 10 4.5 CCU 50 /mL, 10 5.5 CCU 50 /mL, 10 6.5 CCU 50 /mL, and 10 7.5 CCU 50 /mL. (F) Standard curve of strain RM48, plotted as log 10 CCU 50 versus Ct value.

Article Snippet: Then, specific primers (Supporting Information Table ) for the selected candidate targets were designed to amplify the live‐attenuated vaccine strain RM48, and the amplification products were sent to Novogene Co., Ltd. for sequencing.

Techniques: Biomarker Discovery, Amplification, Plasmid Preparation

Journal: Transboundary and Emerging Diseases

Article Title: A RNase H2‐Linked TaqMan‐MGB Quantitative Real‐Time PCR Assay for Differential Detection of Mycoplasma hyopneumoniae Live‐Attenuated Vaccine Strains

doi: 10.1155/tbed/5823134

Figure Lengend Snippet: Repeatability validation of the RNase H2‐linked qPCR assay: Ct value distribution and coefficient of variation (CV) for the standard plasmid pUC57‐vaccine, 168‐L, and RM48 (10 technical replicates per template).

Article Snippet: Then, specific primers (Supporting Information Table ) for the selected candidate targets were designed to amplify the live‐attenuated vaccine strain RM48, and the amplification products were sent to Novogene Co., Ltd. for sequencing.

Techniques: Biomarker Discovery, Plasmid Preparation