le richelieu canada sequence homology search was performed using the blast program of the nih web site samples negative for the appropriate band by pcr were subjected to a first round rt pcr followed by a second round pcr rt pcr reaction mix 50 u03bcl consisted of 5 mm mgcl2 50 mm tris ph 8 0 15 mm nh4 2so4 10 mm b mercaptoethanol 500 u03bcm datp dctp dgtp and dttp 0 025 bsa 1 u03bcm of each outer primer and titan enzyme mix roche molecular biochemicals laval canada and 5 u03bcl nucleic acid sample the reverse transcription step was performed at 42 u00b0 c for 30 m the first and second rounds pcr were performed as described above the precautions addressed elsewhere kwok and higuchi 1989 were followed to minimize the risk of false positive results caused by the carry over of previously amplified dna for example extraction of nucleic acids and preparation of pcr rt pcr mix were performed under a sterile flow bench only aliquoted reagents and filter tips were used and negative controls were incorporated into each run second round pcr nested pcr all amplicon u02dc 450 bp sequences related to bacterial 16s ribosomal rna gene no 450 bp amplicon detected by nested pcr from first round pcr therefore bacteria are in an u201c rna state u201d and are referred to herein as u201c nanoforms u201d 11 samples 12 positive for 450 bp amplicon by nested pcr from first round pcr cell wall deficient pathogenic microorganisms which may be mycoplasma so called l forms or potentially other types are difficult to detect therefore their involvement in.