Ploufragan-Plouzané Laboratory, Mycoplasmology-Bacteriology Unit, French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Ploufragan, France.
European University of Brittany-Loire, Rennes, France.
J Appl Microbiol. 2018 Aug;125(2):345-355. doi: 10.1111/jam.13770. Epub 2018 May 14.
A new multiplex qPCR, targeting Mycoplasma (M.) hyopneumoniae, M. hyorhinis and M. flocculare, was developed and the relationship between detection of those mycoplasma species and the extent of gross pneumonia-like lesions in slaughtered pigs lungs were investigated.
The multiplex qPCR method targets the p102, p37 and fruA genes and has detection limits of 14, 146 and 16 genome equivalents μl for M. hyopneumoniae, M. hyorhinis and M. flocculare, respectively. In all, 671 lungs were collected and analysed, among them 666 were scored for macroscopic pneumonia and categorized according to the extent of the lesions (no or minor lesions, moderate lesions and extensive lesions). According to results of multiplex qPCR, 59·5% were positive for M. hyopneumoniae, 3·4% for M. hyorhinis and 34·7% for M. flocculare, with on average, 3·1 × 10 , 9·7 × 10 and 5·7 × 10 genome equivalents of mycoplasma ml , respectively. More results showed that no or minor lesions were associated with multiplex qPCR-negative results or qPCR-positive results for M. flocculare. Moderate to extensive lesions were positively correlated with qPCR-positive results for M. hyopneumoniae. Extensive lesions were associated with qPCR-positive results for at least two mycoplasma species (M. hyopneumoniae and M. hyorhinis).
The findings also indicated that M. hyopneumoniae and M. hyorhinis significantly increased the odds for a lung to have macroscopic pneumonia. No relationship was found between the extent of lesions and the mycoplasma genome load.
This new multiplex qPCR appears to be specific, sufficiently sensitive and repeatable. The validation of this method with field samples guarantees its use for field epidemiological investigations, particularly to gain more insight into the aetiology of the porcine respiratory disease complex.
开发了一种新的多重 qPCR，针对支原体（M.）肺炎、M. hyorhinis 和 M. flocculare，研究了检测这些支原体物种与屠宰猪肺中大体肺炎样病变程度之间的关系。
多重 qPCR 方法针对 p102、p37 和 fruA 基因，对 M.肺炎、M. hyorhinis 和 M. flocculare 的检测限分别为 14、146 和 16 基因组当量 μl。总共收集和分析了 671 个肺，其中 666 个肺对宏观肺炎进行评分，并根据病变程度（无或轻度病变、中度病变和广泛病变）进行分类。根据多重 qPCR 的结果，59.5%的猪为 M.肺炎阳性，3.4%的猪为 M. hyorhinis 阳性，34.7%的猪为 M. flocculare 阳性，平均每个肺中支原体的基因组当量分别为 3.1×10 、9.7×10 和 5.7×10 。更多结果表明，无或轻度病变与多重 qPCR 阴性结果或 M. flocculare 的 qPCR 阳性结果相关。中度至广泛病变与 M.肺炎的 qPCR 阳性结果呈正相关。广泛病变与至少两种支原体（M.肺炎和 M. hyorhinis）的 qPCR 阳性结果相关。
研究结果还表明，M.肺炎和 M. hyorhinis 显著增加了肺部出现大体肺炎的几率。病变程度与支原体基因组载量之间没有关系。
这种新的多重 qPCR 似乎具有特异性、足够的灵敏度和可重复性。该方法通过现场样本的验证，保证了其在现场流行病学调查中的使用，特别是可以更深入地了解猪呼吸道疾病综合征的病因。
