A TaqMan-MGB real-time PCR for discriminating between MS-H-live vaccine and field Mycoplasma synoviae strains

Abstract

Mycoplasma synoviae (MS) is an important pathogen in the poultry industry and has caused significant economic losses. Worldwide, the use of live attenuated vaccine for the MS-H strain has increased to prevent MS infection. However, there is no test available to discriminate the MS-H vaccine strains from the MS strains that are causing field infection. In this study, a TaqMan-MGB real-time PCR method (qPCR) was established, validated, and evaluated to discriminate between MS-H-live vaccine and field strains based on nucleotide differences in the hlyC gene. The validation was performed for sensitivity and reproducibility by constructing recombinant plasmids. The limits of detection were 1.07 × 10¹ copies/µL for the MS-H and 1.95 × 10¹ copies/µL for field strains, respectively. The intra- and inter-assay results were less than 2.5% based on the reproducibility test. No cross-amplification signals from other common chicken pathogens were detected. Thus, our data indicated that this qPCR is sensitive, specific, and reproducible. In addition, 709 chicken clinical samples were used to evaluate this qPCR test. The results showed that positive signals could be detected from the chicken choanal cleft swabs and are 100% in concordance with the PCR sequencing method. To the best of our knowledge, we found for the first time that both L- and C-type field MS were present in flocks immunized against the MS-H vaccine strain during the validation process. In addition, this is the first report of a field strain of C-type in China.IMPORTANCEMycoplasma synoviae (MS) is an important pathogen in the poultry industry and has caused significant economic losses. Worldwide, an increasing number of farms are using the live attenuated vaccine MS-H strain to prevent MS infections. In order to monitor vaccinated and naturally infected flocks and to continue the MS control and eradication program, a differentiation of infected from vaccinated animals (DIVA) test for MS is urgently needed. We developed a TaqMan-MGB real-time qPCR (qPCR) method with a pair of primers and two competitive TaqMan-MGB probes. We performed an evaluation that can discriminate between the MS-H-live vaccine and field MS strains based on nucleotide differences in the hlyC gene. It has great sensitivity and reproducibility, and greater specificity than other methods which were established by SNP sites of the obg gene and oppF gene.

Keywords: DIVA; MS-H vaccine; Mycoplasma synoviae; hlyC gene; real-time PCR.

Fig 1

Working principle diagram of this qPCR.

Fig 2

Preparation of the plasmid standards. (A and B) Amplification curves (X-axis: cycle, Y-axis: △Rn, fluorescence intensity) of MS-H and field MS for each standard plasmid with concentrations from 1 × 10⁹ copies/µL to 1 × 10¹ copies/µL. (C and D) Standard curves of the standard plasmids of pMD19-T/MS-H and pMD19-T/XL3.

Fig 3

The result of both pMD19-T/MS-H and pMD19-T/XL3 is positive by qPCR. Blue line: FAM; red line: ROX; and green line: VIC. (A) The result of pMD19-T/MS-H is positive. (B) The result of pMD19-T/XL3 is positive. (C) The result of pMD19-T/MS-H and pMD19-T/XL3 is positive.

Fig 4

Sequence comparison of the MS-H strain (GenBank accession no. KX168666), XL12 (GenBank accession no. OR371720), XL13 (GenBank accession no. OR371721), and XL3 (GenBank accession no. OP850572). XL12, C-type field MS isolate. XL13, L-type field MS isolate.

Fig 5

Sequence of primers and probes of qPCR. The forward primer sequence of the hlyC gene is denoted as hlyC-F (highlighted in red), and the reverse primer sequence is designated as hlyC-R (highlighted in purple), while the probe region for identifying MS-H and its re-isolates is labeled as hlyC-vP (highlighted in blue), while the probe area for detecting field strains is denoted by hlyC-fP (highlighted in green). The R of red is a concatenated base (C/T).