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 Mycopla sma hyopneumoniae directly in tracheobronchiolar washings from living pigs in field conditions. Here, we describe the construction and use of an inter nal control to monitor the presence of PCR inhibitors. A PCR modified targe t DNA was constructed by insertion of a small DNA fragment into the M. hyop neumoniae specific DNA target. We have demonstrated that the internal contr ol 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 electro poration. After a few passages we ensured that the recombinant plasmid beca me inserted into the genome of S. citri. PCR amplification of the DNA of th is transformed S. citri strain using nested PCR primers led to amplificatio n of a 900-bp fragment which can be discriminated from the M. hyopneumoniae PCR product 700 bp. The S. citri transformants with the integrated interna l control were added to the tracheobronchiolar washings prior to PCR and us ed 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 washin gs. Quantitation was performed by comparing the PCR signal intensity of the specific M. hyopneumoniae template with known concentrations of the S. cit ri competitor. The titer in tracheobronchiolar washings ranged approximativ ely from 10(4) to 10(8) M. hyopneumoniae cells per mi of clinical specimen. Quantitative PCR can be a useful tool for monitoring the progression of M. hyopneumoniae in the disease process. (C) 2000 Academic Press.