对输入新西兰的精液进行牛支原体检测。

IF 1.1 4区 农林科学 Q3 VETERINARY SCIENCES New Zealand veterinary journal Pub Date : 2023-07-01 DOI:10.1080/00480169.2023.2186506
D Jaramillo, J Foxwell, L Burrows, A Snell
{"title":"对输入新西兰的精液进行牛支原体检测。","authors":"D Jaramillo,&nbsp;J Foxwell,&nbsp;L Burrows,&nbsp;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,&nbsp;J Foxwell,&nbsp;L Burrows,&nbsp;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}
引用次数: 0

摘要

目的:评价三种PCR方法检测牛支原体在稀释(扩展)牛精液中的适用性,以及逆转录-PCR (RT-PCR)适应性作为生存能力的代表。材料和方法:比较四种基于商用试剂盒的核酸提取方法,检测未稀释和稀释精液中提取的核酸中PCR抑制剂的存在。比较两种实时荧光定量PCR和一种常规荧光定量PCR检测精液中牛支原体DNA的分析灵敏度、分析特异性和诊断特异性,并与微生物培养进行比较。此外,RT-PCR适用于仅检测RNA,并在活的和非活的牛分枝杆菌上进行测试,以确定其区分两者的能力。结果:稀释精液中未检测到明显的PCR抑制作用。无论精液稀释度如何,除一种方法外,所有DNA提取方法均相同。实时PCR检测的灵敏度估计为45.6 cfu/ 200µL精液吸管(2.2 × 102 cfu/mL)。传统PCR的灵敏度低10倍。实时荧光定量PCR未发现交叉反应,诊断特异性为100 (95% CI = 94.04-100) %。RT-PCR在区分活牛分枝杆菌和非活牛分枝杆菌方面效果较差。从不同处理中提取的RNA杀死牛分枝杆菌的平均定量周期(Cq)值在失活后0-48小时保持不变。结论及临床意义:实时荧光定量PCR适于筛选稀释精液检测牛支原体,防止牛支原体通过输入性精液入侵。实时PCR检测可以互换使用。RT-PCR不能可靠地显示牛分枝杆菌的生存能力。根据这项研究的结果,已经为希望检测牛支原体牛精液的其他实验室制定了一项方案和指南。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
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.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
New Zealand veterinary journal
New Zealand veterinary journal 农林科学-兽医学
CiteScore
3.00
自引率
0.00%
发文量
37
审稿时长
12-24 weeks
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.
期刊最新文献
Comparison of bacterial culture results obtained from three different sampling locations in dogs and cats with chronic nasal disease. Folliculectomy for the treatment of pre-ovulatory follicular stasis in three illegally captured West Coast green geckos (Naultinus tuberculatus) to enable wild rehabilitation. A pilot study to detect the effects of a green-lipped mussel (Perna canaliculus) nutraceutical on working farm dogs with musculoskeletal abnormalities using accelerometry. Pets in the workplace: a scoping review. Ulnocarpal arthrodesis as a new treatment for ectrodactyly in a dog and a cat.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1