What if a PCR test could give us some information regarding the viability of the pathogen targeted? This new publication from the MycoLab led by Dr.s Albert Canturri and Maria Pieters, is sharing the development of a new mRNA-PCR to answer this question.
Methods
Results
- This new PCR targets the M. hyopneumoniae gene mhp165.
- Limit of Detection: 1 bacterial genome copy equivalent per μL was the lowest amount of genome reliably detected. This corresponds to 8 bacterial genome copy equivalents per assay.
- No other Mycoplasma species was detected by the assay.
- The assay was found to be highly repeatable with very low inter and intra-essay variation.
- RNA-based PCR was negative in cells inactivated via formaldehyde addition or autoclaving. In cells kept room temperature or standard incubation conditions, RNA detection remained consistent for up to 20 days (end of the study).
- In the formaldehyde-inactivated culture, Ct values increased over time until 25 min post-inactivation, at which point mRNA was no longer detected (See below).
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.
