David R Herndon, Paige C Grossman, Julianne K Hwang, Lindsay M W Piel
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Both swab types were then analyzed via conventional and quantitative PCR. This dataset showed that the use of nasopharyngeal swabs in lieu of nasal swabs resulted in higher sensitivity, reduced inhibition during quantitative PCR, and higher bacterial copy numbers per swab. Moreover, it was demonstrated that diagnostic sensitivity could be further increased during quantitative PCR via ten-fold dilution of the extracted DNA. To confirm these observations in naturally infected animals, we conducted a field study employing a production flock of domestic sheep using both nasal and nasopharyngeal swabbing techniques. Extracted DNA was assessed using the same molecular techniques, where detection of Mycoplasma ovipneumoniae was confirmed by sequencing of either the rpoB or 16S rRNA gene. Similar improvements were observed for nasopharyngeal swabs and template treatment methods within the naturally infected flock.</p><p><strong>Conclusions: </strong>Results demonstrate increased diagnostic sensitivity and specificity when sampling with nasopharyngeal swabs as compared to nasal swabs. Therefore, alternate field-testing strategies employing nasopharyngeal swabs should be considered for diagnosis of the presence of M. ovipneumoniae. Importantly, sample treatment following acquisition was found to affect the sensitivity of quantitative PCR, where dilution of eluted DNA template doubled the calculated sensitivity. This demonstrates that, in addition to anatomical location, the presence of inhibitory components in swab extracts also strongly influences diagnostic performance.</p>","PeriodicalId":9041,"journal":{"name":"BMC Veterinary Research","volume":"20 1","pages":"502"},"PeriodicalIF":2.3000,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11529185/pdf/","citationCount":"0","resultStr":"{\"title\":\"Upper respiratory tract detection of Mycoplasma ovipneumoniae employing nasopharyngeal swabs.\",\"authors\":\"David R Herndon, Paige C Grossman, Julianne K Hwang, Lindsay M W Piel\",\"doi\":\"10.1186/s12917-024-04342-y\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Flock-level prevalence and characterization of Mycoplasma ovipneumoniae is determined almost exclusively using nasal swabbing followed by molecular detection with either quantitative PCR or multi-locus sequence typing. 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To confirm these observations in naturally infected animals, we conducted a field study employing a production flock of domestic sheep using both nasal and nasopharyngeal swabbing techniques. Extracted DNA was assessed using the same molecular techniques, where detection of Mycoplasma ovipneumoniae was confirmed by sequencing of either the rpoB or 16S rRNA gene. Similar improvements were observed for nasopharyngeal swabs and template treatment methods within the naturally infected flock.</p><p><strong>Conclusions: </strong>Results demonstrate increased diagnostic sensitivity and specificity when sampling with nasopharyngeal swabs as compared to nasal swabs. Therefore, alternate field-testing strategies employing nasopharyngeal swabs should be considered for diagnosis of the presence of M. ovipneumoniae. Importantly, sample treatment following acquisition was found to affect the sensitivity of quantitative PCR, where dilution of eluted DNA template doubled the calculated sensitivity. 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引用次数: 0
摘要
背景:羊群中卵型肺炎支原体的流行率和特征描述几乎都是通过鼻腔拭子测定,然后用定量 PCR 或多焦点序列分型进行分子检测。然而,与其他解剖位置相比,鼻腔拭子的诊断性能和效率尚未在羊群中得到确定。本研究的目的是评估鼻咽拭子与鼻拭子检测卵肺炎支原体的诊断能力:结果:在对家养绵羊进行卵肺炎支原体接触控制研究期间,采集了鼻腔和鼻咽拭子。然后通过常规和定量 PCR 对这两种拭子进行分析。该数据集显示,用鼻咽拭子代替鼻拭子可提高灵敏度,减少定量 PCR 的抑制作用,并提高每个拭子的细菌拷贝数。此外,实验还证明,通过将提取的 DNA 稀释 10 倍,可进一步提高定量 PCR 的诊断灵敏度。为了在自然感染的动物身上证实这些观察结果,我们使用鼻拭子和鼻咽拭子技术对家羊生产群进行了实地研究。我们使用相同的分子技术对提取的 DNA 进行了评估,并通过 rpoB 或 16S rRNA 基因测序确认了卵肺炎支原体的检测结果。在自然感染的鸡群中,鼻咽拭子和模板处理方法也有类似的改进:结果表明,与鼻拭子相比,使用鼻咽拭子采样可提高诊断灵敏度和特异性。因此,在诊断是否存在卵翼肺炎霉形体时,应考虑使用鼻咽拭子的其他现场检测策略。重要的是,样本采集后的处理会影响定量 PCR 的灵敏度,稀释洗脱的 DNA 模板会使计算出的灵敏度提高一倍。这表明,除了解剖位置外,拭子提取物中抑制成分的存在也会对诊断效果产生很大影响。
Upper respiratory tract detection of Mycoplasma ovipneumoniae employing nasopharyngeal swabs.
Background: Flock-level prevalence and characterization of Mycoplasma ovipneumoniae is determined almost exclusively using nasal swabbing followed by molecular detection with either quantitative PCR or multi-locus sequence typing. However, the diagnostic performance and efficiency of swabbing the nasal passage compared to other anatomical locations has not been determined within sheep populations. The goal of this research was to assess the diagnostic capability of nasopharyngeal swabs in comparison to nasal swabs for the detection of Mycoplasma ovipneumoniae.
Results: Nasal and nasopharyngeal swabs were collected during a controlled exposure study of domestic sheep with Mycoplasma ovipneumoniae. Both swab types were then analyzed via conventional and quantitative PCR. This dataset showed that the use of nasopharyngeal swabs in lieu of nasal swabs resulted in higher sensitivity, reduced inhibition during quantitative PCR, and higher bacterial copy numbers per swab. Moreover, it was demonstrated that diagnostic sensitivity could be further increased during quantitative PCR via ten-fold dilution of the extracted DNA. To confirm these observations in naturally infected animals, we conducted a field study employing a production flock of domestic sheep using both nasal and nasopharyngeal swabbing techniques. Extracted DNA was assessed using the same molecular techniques, where detection of Mycoplasma ovipneumoniae was confirmed by sequencing of either the rpoB or 16S rRNA gene. Similar improvements were observed for nasopharyngeal swabs and template treatment methods within the naturally infected flock.
Conclusions: Results demonstrate increased diagnostic sensitivity and specificity when sampling with nasopharyngeal swabs as compared to nasal swabs. Therefore, alternate field-testing strategies employing nasopharyngeal swabs should be considered for diagnosis of the presence of M. ovipneumoniae. Importantly, sample treatment following acquisition was found to affect the sensitivity of quantitative PCR, where dilution of eluted DNA template doubled the calculated sensitivity. This demonstrates that, in addition to anatomical location, the presence of inhibitory components in swab extracts also strongly influences diagnostic performance.
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.