Quantitative detection of HCMV-DNA from urine in infants by FQ-PCR
HE Rong, LIU Lanqing, L Shengmin, et al. Virology Laboratory, 2nd Clinical Hospital, China Medical University, Shenyang 110004, China
Objective Human cytomegalovirus (HCMV) infection is common, about 60%-100% adults are infected with HCMV. Conventional polymerase chain reaction (PCR) assay can detect HCMV easily but its predictive values for detecting those with symptomatic HCMV infection is low. The present study aimed at evaluation of fluorescent quantitative PCR (FQ-PCR) assay in diagnosis of symptomatic HCMV infection by detecting quantitatively HCMV-DNA from urine in infants. Methods 1. Subject and specimens Urine: samples were collected from two groups of infants;group 1 subjects had symptoms or signs such as jaundice, hepatosplenomegaly etc., and were highly suspected to have HCMV infection. Group 2 consisted of healthy infants. Five to ten ml of urine samples were collected from each subject and stored at -70℃until detection. 2. Conventional PCR: Each PCR tube contained 20 μl solution, and the PCR products were 244bp. 3. FQ-PCR: A dual-labeled probe hydrolysis FQ-PCR method was used. Using dual-labeled probes and 5′-exonuclease , the 5′ terminal nucleotides of the probe were cleft by hydrolysis. It was an in vitro monitoring and real-time detection method. 4. Statistics: Comparison of the mean values of the two groups was done by t-test, and comparison of rates was done by χ 2 test. Results 1. HCMV DNA levels: Among 89 patients suspected of HCMV infection clinically, 52 were positive for HCMV DNA by FQ-PCR; these patients were considered to have symptomatic infection. Among 82 healthy subjects, 30 were positive and were considered to have asymptomatic infection. HCMV-DNA copies in 52 symptomatic infection patients ranged from 3.8 to 9.9 log (mean 5.3±1.4) and in 30 asymptomatic patients ranged from 2.7 to 4.6 log (mean 3.5±0.7). Copies in symptomatic patients were significantly higher than those in the asymptomatic ones (P0.01). 2. Defining of a threshold: The optimal threshold was sought by using a receiver operating characteristic curve. The analysis showed that the level of 10 5 copies/ml urine was the best threshold. When this threshold was used , 25 (48.1%) of 52 symptomatic subjects would have been identified, while none of the asymptomatic subject could be identified. The sensitivity for detecting those with symptomatic disease was 48.1%, and specificity was 100%. The positive and negative predictive values were 100% and 52.6% respectively. 3. Comparison of FQ-PCR and conventional PCR assays: We randomly selected 94 subjects from the two groups (including 56 subjects who were suspected to have HCMV infection clinically and 38 healthy infants) and detected HCMV DNA by using both FQ-PCR and conventional PCR methods. Thirty subjects were positive and 36 negative by both assays. The positive rate of FQ-PCR was 60.6% (57/94), and that of conventional PCR was 33.0% (31/94), the consistency rate of the 2 methods was 70.2% (χ 2=25.30, P0.01). FQ-PCR assay was superior to conventional PCR (χ 2=23.3,P0.01). 4. The sensitivity and specificity of FQ-PCR: In this study, the primer and the probe bound to HCMV-DNA only, but not to HSV-DNA or EBV-DNA. HCMV-DNA was quantified as few as 10-copies/ml urine. Conclusion High levels of HCMV-DNA in urine is closely correlated with clinically apparent disease, and a threshold level of HCMV-DNA in urine could be defined as highly predictive of the occurrence of symptomatic infection. FQ-PCR assay could be useful as a diagnostic measure for symptomatic HCMV infection in clinic.
【Fund】： “九五”国家科技攻关项目 ( 96 90 40 6 0 8)
【CateGory Index】： R446.5
【CateGory Index】： R446.5