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Analytical specificity of the quadruplex qPCR assay. (A) Analytical specificity for Mhp detected through the FAM channel (red). (B) Analytical specificity for Mhr detected through the ROX channel (black). (C) Analytical specificity for APP detected through the VIC channel (blue). (D) Analytical specificity for Gps detected through the Cy5 channel (green). In (A–D): 1 = p-Mhp recombinant plasmid; 2 = p-Mhr recombinant plasmid; 3 = p-APP recombinant plasmid; 4 = p-Gps recombinant plasmid; 5 = Mhp (strain J); 6 = Mhr (positive clinical sample); 7 = APP (trivalent vaccine containing serotype 1 strain JL9901, serotype 2 strain XT9904, and serotype 7 strain GZ9903); 8 = Gps (quadrivalent vaccine containing serotype 4 strain SD02, serotype 5 strain HN02, serotype 12 strain GZ01, and serotype 13 strain JX03); 9–12 = Positive clinical samples of Mhp, Mhr, APP, and Gps, respectively; 13 = CSFV; 14 = PCV2; 15 = PRRSV; 16 = PRV; 17 = SIV; 18 = E. coli ; 19 = Group C Streptococcus and S. suis serotype 2; 20 = S. choleraesuis ; 21 = E. rhusiopathiae ; 22 = P. multocida ; 23 = Nuclease-free distilled water. qPCR = Quantitative polymerase chain reaction; Mhp = Mycoplasma hyopneumoniae ; Mhr = Mycoplasma hyorhinis ; APP = Actinobacillus pleuropneumoniae ; Gps = Glaesserella parasuis ; FAM = 6-Carboxyfluorescein; ROX = Carboxy-X-rhodamine; VIC = VIC fluorescent dye; Cy5 = Cyanine 5; CSFV = Classical swine fever virus; PCV2 = Porcine circovirus type 2; PRRSV = Porcine reproductive and respiratory syndrome virus; PRV = Pseudorabies virus; SIV = Swine influenza virus.

Journal: Veterinary World

Article Title: Development and validation of a quadruplex real-time quantitative polymerase chain reaction assay for the simultaneous detection of Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Actinobacillus pleuropneumoniae, and Glaesserella parasuis

doi: 10.14202/vetworld.2026.3944-3960

Figure Lengend Snippet: Analytical specificity of the quadruplex qPCR assay. (A) Analytical specificity for Mhp detected through the FAM channel (red). (B) Analytical specificity for Mhr detected through the ROX channel (black). (C) Analytical specificity for APP detected through the VIC channel (blue). (D) Analytical specificity for Gps detected through the Cy5 channel (green). In (A–D): 1 = p-Mhp recombinant plasmid; 2 = p-Mhr recombinant plasmid; 3 = p-APP recombinant plasmid; 4 = p-Gps recombinant plasmid; 5 = Mhp (strain J); 6 = Mhr (positive clinical sample); 7 = APP (trivalent vaccine containing serotype 1 strain JL9901, serotype 2 strain XT9904, and serotype 7 strain GZ9903); 8 = Gps (quadrivalent vaccine containing serotype 4 strain SD02, serotype 5 strain HN02, serotype 12 strain GZ01, and serotype 13 strain JX03); 9–12 = Positive clinical samples of Mhp, Mhr, APP, and Gps, respectively; 13 = CSFV; 14 = PCV2; 15 = PRRSV; 16 = PRV; 17 = SIV; 18 = E. coli ; 19 = Group C Streptococcus and S. suis serotype 2; 20 = S. choleraesuis ; 21 = E. rhusiopathiae ; 22 = P. multocida ; 23 = Nuclease-free distilled water. qPCR = Quantitative polymerase chain reaction; Mhp = Mycoplasma hyopneumoniae ; Mhr = Mycoplasma hyorhinis ; APP = Actinobacillus pleuropneumoniae ; Gps = Glaesserella parasuis ; FAM = 6-Carboxyfluorescein; ROX = Carboxy-X-rhodamine; VIC = VIC fluorescent dye; Cy5 = Cyanine 5; CSFV = Classical swine fever virus; PCV2 = Porcine circovirus type 2; PRRSV = Porcine reproductive and respiratory syndrome virus; PRV = Pseudorabies virus; SIV = Swine influenza virus.

Article Snippet: Each 20 μL reaction contained 10 μL of 2× Premix Ex Taq (Probe qPCR) (Code No. RR390A; TaKaRa, Dalian, China), 0.1-0.6 μL each of primers and probes (20 μM), 2.0 μL of the mixed standard plasmids (1 × 108 copies/μL), and nuclease-free water (Code No. 9012; TaKaRa, Dalian, China) to a final volume of 20 μL.

Techniques: Recombinant, Plasmid Preparation, Real-time Polymerase Chain Reaction, Virus