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...
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American Society for Microbiology
2025-05-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.03591-23 |
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| author | Ziqing Liu Shouchang Zhou Chenchen Meng Doudou Ren Weizhen Xiong Guanhui Liu Jinpeng Xu |
| author_facet | Ziqing Liu Shouchang Zhou Chenchen Meng Doudou Ren Weizhen Xiong Guanhui Liu Jinpeng Xu |
| author_sort | Ziqing Liu |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-ef4f4120c1a249d8972d37f17e4a3210 |
| institution | DOAJ |
| issn | 2165-0497 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj-art-ef4f4120c1a249d8972d37f17e4a32102025-08-20T03:11:32ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972025-05-0113510.1128/spectrum.03591-23A TaqMan-MGB real-time PCR for discriminating between MS-H-live vaccine and field Mycoplasma synoviae strainsZiqing Liu0Shouchang Zhou1Chenchen Meng2Doudou Ren3Weizhen Xiong4Guanhui Liu5Jinpeng Xu6School of Life Science and Food Engineering, Hebei University of Engineering, Hebei, ChinaHuayu Agricultural Science and Technology Co., Ltd., Hebei, ChinaHuayu Agricultural Science and Technology Co., Ltd., Hebei, ChinaSchool of Life Science and Food Engineering, Hebei University of Engineering, Hebei, ChinaSchool of Life Science and Food Engineering, Hebei University of Engineering, Hebei, ChinaSchool of Life Science and Food Engineering, Hebei University of Engineering, Hebei, ChinaSchool of Life Science and Food Engineering, Hebei University of Engineering, Hebei, ChinaABSTRACT 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.https://journals.asm.org/doi/10.1128/spectrum.03591-23Mycoplasma synoviaeMS-H vaccinereal-time PCRDIVAhlyC gene |
| spellingShingle | Ziqing Liu Shouchang Zhou Chenchen Meng Doudou Ren Weizhen Xiong Guanhui Liu Jinpeng Xu A TaqMan-MGB real-time PCR for discriminating between MS-H-live vaccine and field Mycoplasma synoviae strains Microbiology Spectrum Mycoplasma synoviae MS-H vaccine real-time PCR DIVA hlyC gene |
| title | A TaqMan-MGB real-time PCR for discriminating between MS-H-live vaccine and field Mycoplasma synoviae strains |
| title_full | A TaqMan-MGB real-time PCR for discriminating between MS-H-live vaccine and field Mycoplasma synoviae strains |
| title_fullStr | A TaqMan-MGB real-time PCR for discriminating between MS-H-live vaccine and field Mycoplasma synoviae strains |
| title_full_unstemmed | A TaqMan-MGB real-time PCR for discriminating between MS-H-live vaccine and field Mycoplasma synoviae strains |
| title_short | A TaqMan-MGB real-time PCR for discriminating between MS-H-live vaccine and field Mycoplasma synoviae strains |
| title_sort | taqman mgb real time pcr for discriminating between ms h live vaccine and field mycoplasma synoviae strains |
| topic | Mycoplasma synoviae MS-H vaccine real-time PCR DIVA hlyC gene |
| url | https://journals.asm.org/doi/10.1128/spectrum.03591-23 |
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