TY - JOUR
T1 - Insight into the intrinsic sensitivity of the PCR assay used to detect CMV infection in amniotic fluid specimens
AU - Avidor, Boaz
AU - Efrat, Gabi
AU - Weinberg, Miriam
AU - Kra-Oz, Zipi
AU - Satinger, Judith
AU - Mitrani-Rosenbaum, Stella
AU - Yaron, Yuval
AU - Shulman, Lester
AU - Tepperberg-Oikawa, Michal
AU - Wolf, Dana
AU - Berger, Stephen A.
AU - Lipitz, Shlomo
AU - Mendelson, Ella
AU - Giladi, Michael
N1 - Funding Information:
This work was partially supported by the Leo Mintz Fund for Medical Research, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. We thank Roche Diagnostics for donating Cobas Amplicor CMV Monitor test kits. The technical assistance of Mr. N. Maisler is greatly appreciated. We thank Rodman Avidor for his faithful support.
PY - 2004/4
Y1 - 2004/4
N2 - Background: PCR detection of human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) is the most sensitive tool for diagnosis of fetal infection, but has sub-optimal sensitivity. It has been suggested that inhibition by AF reduces the sensitivity of this assay, however this assumption has never been thoroughly studied. Several PCR assays have been shown to improve sensitivity, but comparative studies are insufficient to choose the optimal approach. Objectives: To assess the effect of AF inhibition on PCR sensitivity and to determine the most sensitive assay for diagnosing fetal infection. Study design: Plasmid containing HCMV DNA was tested by PCR, in water and in non-infected AF, to assess the inhibitory effect of AF. Twenty-three AF-infected samples were tested by various PCR protocols. AF supernatant, with or without DNA extraction, and AF cells, were assayed by single-round and semi-nested PCR. Viral load was measured in the supernatant by a commercial quantitative PCR kit. Results: The plasmid model demonstrated that single-round PCR was 2000-fold less sensitive in AF compared with water. Semi-nested PCR was only 10-fold less sensitive. Single-round PCR was 30% sensitive in HCMV-infected AF supernatants, and detected viral loads higher than 2.3×106 viral copies/ml. Extraction of DNA from the supernatant increased the sensitivity of this assay to 89% and the detection limit to 5.2×104copies/ml. Semi-nested PCR performed on supernatant, with and without DNA extraction, was 96% and 100% sensitive, respectively, with a detection threshold of 3.8×103copies/ml. Single-round and semi-nested PCR were 89% and 100% sensitive, respectively, in cells. The commercial quantitative PCR assay was 100% sensitive. Conclusions: AF supernatant is inhibitory to PCR. The two most sensitive assays were semi-nested PCR performed on DNA extracted from the supernatant and the commercial quantitative PCR kit. Of these two, the latter is standardized, non-labor-intensive, and allows minimal opportunity for contamination, thereby making it the preferred method for diagnosis.
AB - Background: PCR detection of human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) is the most sensitive tool for diagnosis of fetal infection, but has sub-optimal sensitivity. It has been suggested that inhibition by AF reduces the sensitivity of this assay, however this assumption has never been thoroughly studied. Several PCR assays have been shown to improve sensitivity, but comparative studies are insufficient to choose the optimal approach. Objectives: To assess the effect of AF inhibition on PCR sensitivity and to determine the most sensitive assay for diagnosing fetal infection. Study design: Plasmid containing HCMV DNA was tested by PCR, in water and in non-infected AF, to assess the inhibitory effect of AF. Twenty-three AF-infected samples were tested by various PCR protocols. AF supernatant, with or without DNA extraction, and AF cells, were assayed by single-round and semi-nested PCR. Viral load was measured in the supernatant by a commercial quantitative PCR kit. Results: The plasmid model demonstrated that single-round PCR was 2000-fold less sensitive in AF compared with water. Semi-nested PCR was only 10-fold less sensitive. Single-round PCR was 30% sensitive in HCMV-infected AF supernatants, and detected viral loads higher than 2.3×106 viral copies/ml. Extraction of DNA from the supernatant increased the sensitivity of this assay to 89% and the detection limit to 5.2×104copies/ml. Semi-nested PCR performed on supernatant, with and without DNA extraction, was 96% and 100% sensitive, respectively, with a detection threshold of 3.8×103copies/ml. Single-round and semi-nested PCR were 89% and 100% sensitive, respectively, in cells. The commercial quantitative PCR assay was 100% sensitive. Conclusions: AF supernatant is inhibitory to PCR. The two most sensitive assays were semi-nested PCR performed on DNA extracted from the supernatant and the commercial quantitative PCR kit. Of these two, the latter is standardized, non-labor-intensive, and allows minimal opportunity for contamination, thereby making it the preferred method for diagnosis.
KW - Amniotic fluid
KW - Cytomegalovirus
KW - Polymerase chain reaction (PCR)
UR - http://www.scopus.com/inward/record.url?scp=10744231518&partnerID=8YFLogxK
U2 - 10.1016/S1386-6532(03)00166-5
DO - 10.1016/S1386-6532(03)00166-5
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
AN - SCOPUS:10744231518
SN - 1386-6532
VL - 29
SP - 260
EP - 270
JO - Journal of Clinical Virology
JF - Journal of Clinical Virology
IS - 4
ER -