MULTIPLEX PCR FOR THE DETECTION OF MYCOPLASMA-FERMENTANS, M-HOMINIS AND M-PENETRANS IN CELL-CULTURES AND BLOOD-SAMPLES OF PATIENTS WITH CHRONIC-FATIGUE-SYNDROME

A multiplex polymerase chain reaction (PCR) was initially developed to detect the presence of mycoplasma genus DNA sequences in cell culture s and to differentiate between three human pathogenic mycoplasma speci es simultaneously. The assay in turn, proved to be a useful tool for t he detection of mycoplasma infection in human DNA samples. One set of oligonucleotide primers which are specific for a highly conserved regi on among all members of the genus mycoplasma along with three other pr imer sets which are specific for Mycoplasma fermentans, Mycoplasma hom inis and Mycoplasma penetrans species were used in this assay. The sen sitivity of detection was determined by infecting peripheral blood mon onuclear cells (PBMC) of healthy individuals with known bacterial copy numbers from each species, extracting the DNA, and subjecting 1 mu g of DNA from each sample to 40 cycles of amplification. By using agaros e gel electrophoresis the detection level was determined to be 7, 7, 9 and 15 mycoplasma cells per mu g of human genomic DNA for M. genus, M . fermentans, M. hominis and M. penetrans, respectively. The assay was applied to DNA extracted from the PBMCs of individuals suffering from chronic fatigue syndrome (CFS) (n=100) as determined by the Center fo r Dlisease Control (CDC) criteria, and compared to healthy individuals (n=100). The percentage of M. genus infection was found to be 52% in CFS patients and only 15% in healthy individuals. Mycoplasma fermentan s, M. hominis and M. penetrans were detected in 32, 9 and 6% of the CF S patients while they were detected in 8, 3 and 2% of the healthy cont rol subjects, respectively. This assay provides a rapid and cost effic ient procedure to screen either cell cultures or clinical samples for the presence of three potentially pathogenic species of mycoplasma wit h a high level of sensitivity and specificity. (C) 1998 Academic Press .