Abstract：
Mycoplasma hyopneumoniae detection in clinical specimens is accomplished by PCR targeting bacterial DNA. However, the high stability of DNA and the lack of relationship between bacterial viability and DNA detection by PCR can lead to diagnostic interpretation issues. Bacterial messenger RNA is rapidly degraded after cell death, and consequently, assays targeting mRNA detection can be used for the exclusive detection of viable bacterial cells. Therefore, this study aimed at developing a PCR-based assay for the detection of M. hyopneumoniae mRNA and at validating its applicability to differentiate viable from inert bacteria. Development of the RNA-based PCR encompassed studies to determine its analytical sensitivity, specificity, and repeatability, as well as its diagnostic accuracy. Comparisons between DNA and mRNA detection for the same target gene were performed to evaluate the ability of the RNA-based PCR to detect exclusively viable M. hyopneumoniae after bacterial inactivation using various methods. The RNA-based PCR was also compared to the DNA-based PCR as a tool to monitor the growth of M. hyopneumoniae in vitro. Under the conditions of this study, the developed RNA-based PCR assay detected only viable or very recently inactivated M. hyopneumoniae, while the DNA-based PCR consistently detected cells irrespective of their viability status. Changes in growth activity over time were only observable via RNA-based PCR. This viability PCR assay could be directly applied to evaluate the clearance of M. hyopneumoniae or to determine the viability of the bacterium at late stages of eradication programs.
摘要：
临床标本中猪肺炎支原体的检测是通过PCR靶向细菌DNA来完成的。然而,DNA的高稳定性以及细菌活力与PCR检测DNA之间缺乏关系可导致诊断解释问题。细菌信使RNA在细胞死亡后迅速降解，因此，靶向mRNA检测的测定可用于唯一检测活细菌细胞。因此,这项研究旨在开发一种基于PCR的检测猪肺炎支原体mRNA的方法，并验证其在区分活细菌和惰性细菌方面的适用性。基于RNA的PCR的发展包括确定其分析灵敏度的研究，特异性，和可重复性，以及它的诊断准确性。进行相同靶基因的DNA和mRNA检测之间的比较,以评估基于RNA的PCR在使用各种方法灭活细菌后检测唯一存活的猪肺炎支原体的能力。还将基于RNA的PCR与基于DNA的PCR进行比较,作为监测猪肺炎支原体体外生长的工具。在本研究的条件下,开发的基于RNA的PCR测定法仅检测到活的或最近灭活的猪肺炎支原体，而基于DNA的PCR始终如一地检测细胞，无论它们的生存力状态如何。生长活性随时间的变化仅可通过基于RNA的PCR观察到。该生存力PCR测定可直接用于评估猪肺炎支原体的清除或在根除程序的后期确定细菌的生存力。
