2.2 Detection of urine HCMV-DNA
This technique is based on TaqMan PCR- Fluorescence probe technology,
1ml of urine specimen was centrifuged at 12000 rpm for 5 min. Urine
supernatant was discarded, and the pellet was collected and mixed with
50 μl of DNA extraction solution. Putting the10 μl of the above mixture,
10 μl of the treated negative-positive control,and 10 μl × 4 of the
quantitative reference substance were respectively added into each
individual tube with the 40μl PCR mixture (Quantitative Diagnostic Kit
for HCMV-DNA, Hunan, China) and centrifuged for 10s, PCR was performed
using the 7500 real-time PCR system (Applied Biosystem, CA, USA ) Under
the following conditions: one cycle of 5℃for 2 min and 94℃ for 5 min;
followed by 45 cycles of 94℃ for 15 s and 57℃ for 31 s; one cycle of 25℃
for 10 s. The fluorescence signal emitted by fluorescein-labeled probe
is collected by PCR instrument, and the amplification curve and Ct value
are judged. HCMV DNA ≥ 4.0 for 102 copies/ml of
specimen was regarded as positive.