New molecular diagnostic targets for Avibacterium paragallinarum and a set of single-plex and multiplex qPCR methods for the rapid differential diagnosis of Mycoplasma gallisepticum, Mycoplasma synoviae, and Avibacterium paragallinarum

Abstract

Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and Avibacterium paragallinarum (APG) are respiratory-borne bacterial pathogens that severely harm the poultry industry. The clinical symptoms caused by them share many similarities, such as respiratory disease, growth retardation, and decreased egg production. They are not suitable for rapid diagnosis through isolation and culture and often need to detect nucleic acids or antibodies for differential diagnosis. In this study, bioinformatics analyses were used, and six specific coding genes were identified as being shared among all the APG strains that were absent in other species with published genome sequences. Combined with MG- and MS-specific genes identified in previous studies, we established a set of single-plex and multiplex qPCR assays for the rapid differential diagnosis of these three pathogens. The results indicated that the correlation coefficients (R²) of the standard curve established in these methods were not less than 0.999, and the amplification efficiencies (E) were between 90 % and 110 %. In terms of specificity, with the exception of the amplification curve and C_T value generated in the positive control, other related pathogens, chicken cells, and empty plasmid did not amplify. In terms of sensitivity, the 100 % detection sensitivity of MG single-plex qPCR, MS single-plex qPCR, APG single-plex qPCR, and MG-MS duplex qPCR established in this study was 5 copies/reaction. The 100 % detection sensitivity of MG-MS-APG triplex qPCR was 5 copies/reaction in both the MG and MS detection channels and 10 copies/reaction in the APG detection channel. The detection rate of triplex qPCR in the APG detection channel at 5 copies/reaction was 80 %. The intra-group and inter-group variation coefficients of the qPCR methods established in this study were all within 2 % in the repeatability evaluation. In terms of the coincidence rate of clinical sample testing, the qPCR methods showed 100 % detection consistency for the clinical samples tested. The established qPCR methods exhibited good specificity, sensitivity, and repeatability, which provide powerful technical support for the rapid and efficient differential diagnosis of MG, MS, and APG simultaneously.

Keywords: Avibacterium paragallinarum; Mycoplasma gallisepticum; Mycoplasma synoviae; diagnostic target; molecular diagnosis.

Fig. 1

The organization of the molecular diagnostic target gene K5O18_RS13655 and its upstream and downstream genes. The K5O18_RS13655 is marked in yellow. The tRNA-Ile encoding gene is marked in green.

Fig. 2

Amplification plots and standard curves of MG, MS, and APG single-plex qPCR using the genomic DNA as the template. Panels A, B, and C show the amplification plots of MG single-plex qPCR, MS single-plex qPCR, and APG single-plex qPCR for detecting genomic DNA with 6 concentration gradients, respectively. The standard curves, equations, and R² values of MG single-plex qPCR, MS single-plex qPCR, and APG single-plex qPCR for detecting different concentrations of genomic DNA are showed in panels D, E, and F, respectively.

Fig. 3

Amplification plots and standard curves of MG and MS duplex qPCR. Panels A and B show the amplification plots of the MG and MS detection channels of duplex qPCR in detecting genomic DNA with 6 concentration gradients, respectively. The standard curves, equations, and R² values of the MG and MS detection channels of duplex qPCR for detecting different concentrations of genomic DNA are showed in panels C and D, respectively.

Fig. 4

Amplification plots and standard curves of MG, MS, and APG triplex qPCR. Panels A, B, and C show the MG, MS, and APG detection channels of triplex qPCR in detecting genomic DNA with 6 concentration gradients, respectively. The standard curves, equations, and R² values of the MG, MS, and APG detection channels of triplex qPCR for detecting different concentrations of genomic DNA are showed in panels D, E, and F, respectively.

Fig. 5

Specificity analyses of MG, MS, and APG single-plex qPCR. Panels A, B, and C show the amplification plots of MG single-plex qPCR, MS single-plex qPCR, and APG single-plex qPCR for detecting the genomic DNA or cDNA of 12 avian pathogens and chicken cells and the DNA of the plasmid pMD18-T, respectively.

Fig. 6

Specificity analyses of MG and MS duplex qPCR. Panels A, B, and C show the amplification plots of the MG and MS detection channels of duplex qPCR for detecting the genomic DNA or cDNA of 12 avian pathogens and chicken cells and the DNA of the plasmid pMD18-T, respectively.

Fig. 7

Specificity analyses of MG, MS, and APG triplex qPCR. Panels A, B, and C show the amplification plots of the MG, MS, and APG detection channels of triplex qPCR for detecting the genomic DNA or cDNA of 12 avian pathogens and chicken cells and the DNA of the plasmid pMD18-T, respectively.

Fig. 8

Sensitivity analyses of single-plex qPCR, duplex qPCR, and triplex qPCR. The sensitivities of different qPCR were evaluated using the corresponding genomic DNA at five different concentrations, with 10 replicates at each concentration (1, 5, 10, 100, and 1000 copies per reaction). Panels A, B, and C show the percentages of detection of MG single-plex qPCR, MS single-plex qPCR, and APG single-plex qPCR in detecting different concentrations of templates, respectively. Panels D and E show the percentages of detection for the MG and MS detection channels of duplex qPCR in detecting different concentrations of templates, respectively. Panels F, G, and H show the percentages of detection for the MG, MS, and APG detection channels of triplex qPCR in detecting different concentrations of templates, respectively.