Comparison of DNA extraction procedures for detection of Mycoplasma bovis directly from extended bovine semen straw samples using a commercial M. bovis PCR.

IF 2.3 2区 农林科学 Q1 VETERINARY SCIENCES BMC Veterinary Research Pub Date : 2024-10-26 DOI:10.1186/s12917-024-04333-z
Emma Taylor, Alannah Deeney, Colin Birch, Georgia Mayne, Anne Ridley
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Abstract

Background: Mycoplasma bovis is a global pathogen of cattle but was detected for the first time in New Zealand in 2017, triggering a response under their Biosecurity Act as an "unwanted organism". Following a lengthy eradication campaign, the Ministry of Primary Industries (MPI) now requires all bovine semen destined for export to New Zealand to be screened with an M. bovis-specific real-time PCR (rtPCR) compliant with amended import health standard (IHS) test requirements aimed at preventing the accidental importation of M. bovis. The standard stipulates that semen samples cannot be centrifuged prior to DNA extraction. To comply with these strict requirements, one of the listed tests was validated together with different DNA preparation steps and compared with existing in-house procedures. DNA was extracted from semen straws using the current in-house semi-automated platform procedures for processing culture, tissue and body fluid sample submissions and was compared with the stipulated test requirements. DNA from centrifuged and unspun semen samples spiked with M. bovis was also compared.

Results: The rtPCR had a sensitivity and specificity of 100% (95% confidence interval = 79-100% and 74-100%, respectively) when testing DNA from other Mycoplasma species or bovine semen spiked with the latter, with a high level of repeatability for within- and between- run replicates. The consistent limit of detection was 0.001 pg/µl M. bovis DNA and between 5.3 × 102 and 7.5 × 102 CFU/ml M. bovis when artificially spiked in semen. DNA extracted using the KingFisher Flex was detected with lower Cq values than the Maxwell 16, but the comparable improvements in sensitivity were mainly associated with non-centrifuged samples (p < 0.001). None of the procedures tested impeded the detection sensitivity of M. bovis in the presence of competitor organisms Acholeplasma laidlawii, Mycoplasma bovigenitalium and Ureaplasma diversum, confirming M. bovis specificity of the polC target.

Conclusions: Under the experimental conditions applied, this rtPCR test efficiently detected M. bovis in extended bovine semen straw samples from DNA extracted using both semi-automated extraction platforms, irrespective of prior centrifugation of extended semen.

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使用商用牛支原体 PCR 直接从扩展的牛精液秸秆样本中检测牛支原体的 DNA 提取程序比较。
背景:牛支原体是一种全球性的牛病原体,但 2017 年首次在新西兰被检测到,引发了新西兰《生物安全法》将其作为 "不受欢迎的生物体 "的应对措施。经过漫长的根除运动后,新西兰初级产业部(MPI)现在要求所有出口到新西兰的牛精液都必须使用牛海绵状芽孢杆菌特异性实时 PCR(rtPCR)进行筛查,以符合修订后的进口卫生标准(IHS)检测要求,从而防止牛海绵状芽孢杆菌的意外进口。该标准规定,精液样本在提取 DNA 之前不能离心。为了符合这些严格的要求,我们对所列检测中的一项进行了验证,同时采用了不同的 DNA 制备步骤,并与现有的内部程序进行了比较。我们使用现有的内部半自动化平台程序从精液吸管中提取 DNA,用于处理培养基、组织和体液样本,并与规定的检测要求进行比较。此外,还比较了离心精液样本和未离心精液样本中牛海绵状芽孢杆菌的 DNA:在检测其他支原体或添加了牛支原体的牛精液中的 DNA 时,rtPCR 的灵敏度和特异性均为 100%(95% 置信区间分别为 79-100% 和 74-100%),且在运行内和运行间重复的重复性很高。检测限一致为 0.001 pg/µl 牛支原体 DNA,在精液中人工添加牛支原体时,检测限为 5.3 × 102 至 7.5 × 102 CFU/ml。使用 KingFisher Flex 提取的 DNA 的 Cq 值比 Maxwell 16 低,但灵敏度的提高主要与非离心样本有关(p 结论):在所应用的实验条件下,该 rtPCR 检测法可从使用两种半自动提取平台提取的 DNA 中有效检测出扩展牛精液秸秆样本中的牛海绵状芽孢杆菌,而与扩展精液是否事先离心无关。
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来源期刊
BMC Veterinary Research
BMC Veterinary Research VETERINARY SCIENCES-
CiteScore
4.80
自引率
3.80%
发文量
420
审稿时长
3-6 weeks
期刊介绍: BMC Veterinary Research is an open access, peer-reviewed journal that considers articles on all aspects of veterinary science and medicine, including the epidemiology, diagnosis, prevention and treatment of medical conditions of domestic, companion, farm and wild animals, as well as the biomedical processes that underlie their health.
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