Use of an internal control in a nested-PCR assay for Mycoplasma hyopneumoniae detection and quantification in tracheobronchiolar washings from pigs.
We have previously reported a nested PCR assay for the detection of Mycoplasma hyopneumoniae directly in tracheobronchiolar washings from living pigs in field conditions. Here, we describe the construction and use of an internal control to monitor the presence of PCR inhibitors. A PCR modified target DNA was constructed by insertion of a small DNA fragment into the M. hyopneumoniae specific DNA target. We have demonstrated that the internal control failed to be amplified in only three tracheobronchiolar washings samples out of the 362 tested. This control molecule was inserted in a Spiroplasma citri derived plasmid vector and introduced into S. citri cells by electroporation. After a few passages we ensured that the recombinant plasmid became inserted into the genome of S. citri. PCR amplification of the DNA of this transformed S. citri strain using nested PCR primers led to amplification of a 900-bp fragment which can be discriminated from the M. hyopneumoniae PCR product 700 bp. The S. citri transformants with the integrated internal control were added to the tracheobronchiolar washings prior to PCR and used as an internal control to check the efficiency of sample processing, and to demonstrate the presence of inhibitors. Furthermore, we have been able to estimate the number of mycoplasma cells in the tracheobronchiolar washings. Quantitation was performed by comparing the PCR signal intensity of the specific M. hyopneumoniae template with known concentrations of the S. citri competitor. The titer in tracheobronchiolar washings ranged approximatively from 10(4)to 10(8)M. hyopneumoniae cells per ml of clinical specimen. Quantitative PCR can be a useful tool for monitoring the progression of M. hyopneumoniae in the disease process.