{"title":"对输入新西兰的精液进行牛支原体检测。","authors":"D Jaramillo, J Foxwell, L Burrows, A Snell","doi":"10.1080/00480169.2023.2186506","DOIUrl":null,"url":null,"abstract":"<p><strong>Aims: </strong>To evaluate the fitness of three PCR assays for the detection of <i>Mycoplasma bovis</i> in dilute (extended) bovine semen, and a reverse transcriptase-PCR (RT-PCR) adaptation as a proxy for viability.</p><p><strong>Materials and methods: </strong>Four commercial kit-based methods for nucleic acid extraction were compared to test for the presence of PCR inhibitors in nucleic acid extracted from undiluted and diluted semen. Then, analytical sensitivity, analytical specificity, and diagnostic specificity of two real-time PCR and one conventional PCR were evaluated for the detection of <i>M. bovis</i> DNA in semen and compared against microbial culture. Furthermore, an RT-PCR was adapted to detect RNA only and tested on viable and non-viable <i>M. bovis</i> to establish its ability to discriminate between the two.</p><p><strong>Results: </strong>No significant PCR inhibition was detected from the dilute semen. All DNA extraction methods except one were equivalent, regardless of semen dilution. The analytical sensitivity of the real-time PCR assays was estimated as 45.6 cfu per 200 µL semen straw (2.2 × 10<sup>2</sup> cfu/mL). The conventional PCR was 10 times less sensitive. No cross-reactivity was observed for the real-time PCR for any of the bacteria tested and the diagnostic specificity was estimated as 100 (95% CI = 94.04-100) %. The RT-PCR was poor in distinguishing between viable and non-viable <i>M. bovis</i>. The mean quantification cycle (Cq) values for RNA extracted from different treatments to kill <i>M. bovis</i> remained unchanged 0-48 hours after inactivation.</p><p><strong>Conclusion and clinical relevance: </strong>The real-time PCR were fit for the purpose of screening dilute semen for the detection of <i>M. bovis</i> to prevent incursion via importation of infected semen. The real-time PCR assays can be used interchangeably. The RT-PCR could not reliably indicate the viability of <i>M. bovis.</i> Based on the results from this study, a protocol and guidelines have been produced for laboratories elsewhere that wish to test bovine semen for <i>M. bovis</i>.</p>","PeriodicalId":19322,"journal":{"name":"New Zealand veterinary journal","volume":null,"pages":null},"PeriodicalIF":1.1000,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"<i>Mycoplasma bovis</i> testing for the screening of semen imported into New Zealand.\",\"authors\":\"D Jaramillo, J Foxwell, L Burrows, A Snell\",\"doi\":\"10.1080/00480169.2023.2186506\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Aims: </strong>To evaluate the fitness of three PCR assays for the detection of <i>Mycoplasma bovis</i> in dilute (extended) bovine semen, and a reverse transcriptase-PCR (RT-PCR) adaptation as a proxy for viability.</p><p><strong>Materials and methods: </strong>Four commercial kit-based methods for nucleic acid extraction were compared to test for the presence of PCR inhibitors in nucleic acid extracted from undiluted and diluted semen. Then, analytical sensitivity, analytical specificity, and diagnostic specificity of two real-time PCR and one conventional PCR were evaluated for the detection of <i>M. bovis</i> DNA in semen and compared against microbial culture. Furthermore, an RT-PCR was adapted to detect RNA only and tested on viable and non-viable <i>M. bovis</i> to establish its ability to discriminate between the two.</p><p><strong>Results: </strong>No significant PCR inhibition was detected from the dilute semen. All DNA extraction methods except one were equivalent, regardless of semen dilution. The analytical sensitivity of the real-time PCR assays was estimated as 45.6 cfu per 200 µL semen straw (2.2 × 10<sup>2</sup> cfu/mL). The conventional PCR was 10 times less sensitive. No cross-reactivity was observed for the real-time PCR for any of the bacteria tested and the diagnostic specificity was estimated as 100 (95% CI = 94.04-100) %. The RT-PCR was poor in distinguishing between viable and non-viable <i>M. bovis</i>. The mean quantification cycle (Cq) values for RNA extracted from different treatments to kill <i>M. bovis</i> remained unchanged 0-48 hours after inactivation.</p><p><strong>Conclusion and clinical relevance: </strong>The real-time PCR were fit for the purpose of screening dilute semen for the detection of <i>M. bovis</i> to prevent incursion via importation of infected semen. The real-time PCR assays can be used interchangeably. The RT-PCR could not reliably indicate the viability of <i>M. bovis.</i> Based on the results from this study, a protocol and guidelines have been produced for laboratories elsewhere that wish to test bovine semen for <i>M. bovis</i>.</p>\",\"PeriodicalId\":19322,\"journal\":{\"name\":\"New Zealand veterinary journal\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.1000,\"publicationDate\":\"2023-07-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"New Zealand veterinary journal\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://doi.org/10.1080/00480169.2023.2186506\",\"RegionNum\":4,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"VETERINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"New Zealand veterinary journal","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1080/00480169.2023.2186506","RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"VETERINARY SCIENCES","Score":null,"Total":0}
Mycoplasma bovis testing for the screening of semen imported into New Zealand.
Aims: To evaluate the fitness of three PCR assays for the detection of Mycoplasma bovis in dilute (extended) bovine semen, and a reverse transcriptase-PCR (RT-PCR) adaptation as a proxy for viability.
Materials and methods: Four commercial kit-based methods for nucleic acid extraction were compared to test for the presence of PCR inhibitors in nucleic acid extracted from undiluted and diluted semen. Then, analytical sensitivity, analytical specificity, and diagnostic specificity of two real-time PCR and one conventional PCR were evaluated for the detection of M. bovis DNA in semen and compared against microbial culture. Furthermore, an RT-PCR was adapted to detect RNA only and tested on viable and non-viable M. bovis to establish its ability to discriminate between the two.
Results: No significant PCR inhibition was detected from the dilute semen. All DNA extraction methods except one were equivalent, regardless of semen dilution. The analytical sensitivity of the real-time PCR assays was estimated as 45.6 cfu per 200 µL semen straw (2.2 × 102 cfu/mL). The conventional PCR was 10 times less sensitive. No cross-reactivity was observed for the real-time PCR for any of the bacteria tested and the diagnostic specificity was estimated as 100 (95% CI = 94.04-100) %. The RT-PCR was poor in distinguishing between viable and non-viable M. bovis. The mean quantification cycle (Cq) values for RNA extracted from different treatments to kill M. bovis remained unchanged 0-48 hours after inactivation.
Conclusion and clinical relevance: The real-time PCR were fit for the purpose of screening dilute semen for the detection of M. bovis to prevent incursion via importation of infected semen. The real-time PCR assays can be used interchangeably. The RT-PCR could not reliably indicate the viability of M. bovis. Based on the results from this study, a protocol and guidelines have been produced for laboratories elsewhere that wish to test bovine semen for M. bovis.
期刊介绍:
The New Zealand Veterinary Journal (NZVJ) is an international journal publishing high quality peer-reviewed articles covering all aspects of veterinary science, including clinical practice, animal welfare and animal health.
The NZVJ publishes original research findings, clinical communications (including novel case reports and case series), rapid communications, correspondence and review articles, originating from New Zealand and internationally.
Topics should be relevant to, but not limited to, New Zealand veterinary and animal science communities, and include the disciplines of infectious disease, medicine, surgery and the health, management and welfare of production and companion animals, horses and New Zealand wildlife.
All submissions are expected to meet the highest ethical and welfare standards, as detailed in the Journal’s instructions for authors.