Microorganisms associated with genito-urinary tract infections are oft
en difficult to detect due to limitations associated with culture tech
niques. We have applied PCR-based detection of clinical isolates to co
mplex sample matrices. Clinical isolates of Chlamydia trachomatis, Esc
herichia coli, Proteus vulgaris and Pseudomonas aeruginosa were grown,
diluted and used to spike human urine and human semen. The urine and
semen samples were centrifuged and the bacterial pellet was kept. A ly
sis buffer containing proteinase K, 8-methoxypsoralen and lauryl alcoh
ol polyether was exposed to UV light to remove the bacterial DNA in th
e proteinase K, and was added to the bacterial pellet. After digestion
, the proteinase K was destroyed and the lysates were subjected to 35
cycles of 16S rDNA amplification using a hot-starts technique and two
primer pairs specific for eubacterial 16S rDNA: 8FPL-806R and 515FPL-1
3B. After amplification, the amplicons were cloned and sequenced to co
nfirm amplification of the bacteria used to spiked the samples. We wer
e able to detect as few as 10(5) bacteria per mi of urine or semen. Th
irty unspiked semen samples were tested by PCR, and 17 were positive,
including 10 samples negative by routine culture. The amplicons were c
loned and sequenced for four PCR-positive/culture-negative semen sampl
es: the 16S rDNA sequences obtained were mainly from strict anaerobes,
including Prevotella spp. and Peptostreptococcus spp. Some 16S rDNA s
equences were obtained that did not match any other 16S rDNA sequences
available in various nucleic acid databases. Half of 14 unspiked urin
e sample tested were positive by PCR, including four samples negative
by routine culture. Amplicons were cloned and sequenced for one urine
sample showing only Lactobacillus spp. by routine culture, and sequenc
es from Bacteroides ureolyticus, Clostridium spp., Corynebacterium ure
alyticum, Peptostreptococcus spp., and Lactobacillus acidophilus were
found. These methods have great promise for the rapid detection of via
ble, but non-culturable bacteria in semen and urine. We are currently
applying this technique for the detection of bacteria associated with
idiopathic inflammatory conditions.