Modified Polymerase Chain Reaction Research Articles

The apolipoprotein B R3500Q gene mutation in Spanish subjects with a clinical diagnosis of familial hypercholesterolemia

Familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB) are autosomal codominant diseases characterized by elevated LDL cholesterol levels and premature coronary artery disease. Mutations of the LDL-receptor and apolipoprotein B genes, which affect the binding domains of their protein products, are the causal defects. Securing the diagnosis of these conditions by molecular assays is important because it mandates early intervention for coronary risk reduction. DNA screening for apolipoprotein B R3500Q gene mutation was performed in 913 unrelated Spanish individuals with a clinical diagnosis of FH using a modified polymerase chain reaction protocol and restriction enzyme genotyping. Thirteen FDB heterozygotes were identified (frequency of 1.4% in subjects with a clinical diagnosis of FH). The prevalence of hypercholesterolemic subjects with FDB in the general Spanish population was estimated to be as low as 2.8×10 −5 (95% CI, −3.1×10 −4 to 3.7×10 −4). The ancestors of 11 out of 13 FDB carriers were from Galicia, a region of Celtic ancestry in Northwestern Spain. As the series included 100 unrelated subjects of Galician ancestry, FDB appears to be an important genetic cause of hypercholesterolemia in this region. All the R3500Q mutations were found on the same allele, assigned to haplotype XbaI−/MspI+/EcoRI−/3HVR48, suggesting that the mutant alleles are identical by descent in people from Spain, as observed in other Caucasian populations. In conclusion, the R3500Q mutation of the apolipoprotein B gene, a common cause of FH in central Europe, is infrequent in the general Spanish population, but it is common in Galicia.

Phenotypic and Molecular Characterization of Isolates of Ornithobacterium rhinotracheale from Peru

Twenty-five isolates of Ornithobacterium rhinotracheale were examined by agar gel precipitation, immunoperoxidase assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot analysis, and a polymerase chain reaction. All of the isolates were identified as serotype A. Protein profiles of whole cell extracts were similar for all the isolates, and a polypeptide with a molecular weight of 33 kD was a major component, being present in all the isolates. In the main, proteins of 33, 42, 52, and 66 kD were recognized in immunoblots with sera from chickens naturally exposed to O. rhinotracheale. A modified polymerase chain reaction assay identified O. rhinotracheale DNA from all the isolates and tracheal swabs, producing amplicons of 784 bp, and distinguished O. rhinotracheale from bacterial agents causing similar clinical signs.

Results on single cell PCR for Huntington's gene and WAVE™ product analysis for preimplantation genetic diagnosis

Triple repeat base pair amplification is the basis for a number of prevalent genetic diseases such as Huntington's, Fragile X, Myotonic Dystrophy and others. We have chosen to investigate the use of PCR to amplify a portion of the Huntington's gene in single cells in order to develop a clinical test system for preimplantation genetic diagnosis (PGD). Amplification of CAG triple repeat sequences poses difficulties due to resistance of GC melting for amplification. Special PCR modifications are necessary to carry out the amplification of GC rich areas found in most triple base pair expansions. We have used a modified polymerase chain reaction (PCR) protocol to amplify the expanded repeat sequence of the Huntington's gene with satisfactory efficiency. Detection of the amplified expanded CAG repeats is shown to be possible using both agarose gel electrophoresis and high definition denaturing high pressure liquid (DHPLC) chromatography. The incidence of allele dropout (ADO) is documented.

Laboratory and field evaluation of polymerase chain reaction-based forensic DNA profiling for use in identification of human blood meal sources of Aedes aegypti (Diptera: Culicidae).

We modified polymerase chain reaction (PCR)-based forensic DNA profiling for field studies on the feeding behavior of Aedes aegypti, the principal mosquito vector of dengue virus. Human DNA was extracted from oral swabs of human subjects and from blood-engorged mosquitoes, DNA was quantified by slot blot, and alleles at variable number tandem repeats and three short tandem repeats loci were amplified by PCR. Alleles were separated electrophoretically and then visualized by silver staining. A custom software program was written to match DNA fingerprints of potential human hosts to allelic profiles detected in engorged mosquitoes, and to calculate error rates for identification of human hosts of single and multiple-host blood meals. At 29 degrees C in the laboratory, human DNA recovered from mosquito blood meals declined an average of 67% 8 h after feeding and 90% after 24 h. We obtained complete allelic profiles from seven of 10 mosquitoes collected after 24 h. In a field trial, complete DNA profiles were obtained successfully for 43 people living in a rural village in south central Thailand and for 20 of 100 Ae. aegypti that contained blood and were collected in those peoples' homes. Blood imbibed from more than one person was detected in 45% (9 of 20) of the meals. Sixty-five percent of the meals contained blood from nonresidents of the house in which the mosquito was collected or from people who were not profiled; data consistent with the hypothesis that human movement is important for the spread of dengue virus within and among communities. When using alleles at four loci, all of the Thais and nine members spanning three generations of a Chinese-American family had unique allelic profiles. Error rates from classifying possible multiple-host meals as single-host meals were low (1-8%), with the highest error associated with closely related people. Results from our laboratory and field studies indicated that DNA profiling can be used to study the details and epidemiological implications of Ae. aegypti blood-feeding behavior.

MODIFIED POLYMERASE CHAIN REACTION AND SELECTIVE MEDIA METHODS FOR DETECTION OF HELICOBACTER PYLORI IN PORK LOIN CHOPS1

Abstract Selective media, and a modified polymerase chain reaction (PCR) were evaluated for use in the detection and enumeration of Helicobacter pylori in pork loin chops. The chops were treated with tap water, or H. pylori‐ATCC 43504, packaged in polyvinyl chloride (PVC) film (7 days), or vacuum packaged (24 days), stored at 2C, and analyzed for survival of H. pylori. Modifying the amplification cycles and annealing temperature of the PCR resulted in detection of H. Pylori on inoculated chops. Helicobacter pylori survived for 5, and 10 days on the PVC, and vacuum packaged chops, respectively. Skirrow agar was the most effective medium, when compared to blood agar, and egg yolk emulsion agar.

Significance of Linkage Disequilibrium between Mutation C282Y and a MseI Polymorphism in Population Screening and DNA Diagnosis of Hemochromatosis

ABSTRACT: An increasing number of studies demonstrate a lack of phenotypic expression in subjects found to be homozygous for the common hereditary hemochromatosis (HH) mutation, C282Y. In this study the impact of possible overestimation of C282Y homozygosity, as a consequence of a MseI polymorphism identified in intron 4 of the HFE gene, was investigated in South African subjects. Utilization of a modified polymerase chain reaction (PCR)-based assay highlighted the potential implications with respect to genotype/phenotype correlation studies, particularly in the general population. Mistyping rather than lack of disease association provides a plausible explanation for the phenomenon of C282Y homozygosity without iron overload. Reassessment of C282Y mutation status in such cases may result in justified population screening in HH.

Tick-borne borrelioses pathogen identification in Ixodes ticks (Acarina, Ixodidae) collected in St. Petersburg and Kaliningrad Baltic regions of Russia.

Two isolated Baltic seashore populations of Ixodes ticks were studied as vectors of different Borrelia genospecies in Russia by using darkfield microscopy and modified polymerase chain reaction (PCR). In the Kalinigrad region (Kurish Spit, forests near the settlements of Lesnoye and Rybachy), 788 Ixodes ricinus (L.) adults and nymphs were collected by flagging and studied by darkfield microscopy during 1995-1996. There were 88 darkfield microscopy positive specimens (11.2%) of which 69 were also analyzed by PCR. Borrelia afzelii and B. garinii were found individually and together in ticks. In this region, on the Kurish Spit, 7 patients with tick borrelioses were observed: 2 in the Russian part of Spit and 5 in the Lithuanian part. A significant difference was found between Borrelia prevalence during the spring and fall peaks of tick abundance. Specimens that were darkfield microscopy positive prevailed in the fall (25.15%) in comparison with the spring peak (7.3%). The number of specimens with identified genospecies prevailed in the spring: 22 out of 35 versus 4 out of 31 in the fall. Among 29 PCR positive I. ricinus, 21 contained B. afzelii, 3 had B. garinii, and 2 had dual infection. In 1995, only B. afzelii infected specimens were observed. In the vicinity of St. Petersburg (the seashore of the northern Gulf of Finland, in forests near Lisy Nos, Morskaja) during 1992-1996, 31 patients with a tick-borne borrelioses were registered. We collected 487 Ixodes persulcatus Schulze by flagging and studied them by darkfield microscopy in 1995-1996 of which 144 ticks (29.6%) were darkfield microscopy positive. Sixty darkfield-positive specimens were analyzed by PCR, and in 88.3% of cases genospecies were identified. B. afzelii and B. garinii were identified individually and together in ticks. In 1995, I. persulcatus with dual infection prevailed with 11 out of 21 (52.4% positive), whereas in 1996, most I. persulcatus ticks contained B. garinii (81.2%). Dual infection was observed in 4 of 32 (12.5%) ticks. Dual infections in I. persulcatus females increased within the seasonal peak of tick activity as was observed in 1995 and in 1996. Many patients not only had erythema migrans, but also exhibited early neurological symptoms that coincided with the number of tick vectors that had dual infections in June, indicating that these patients were bitten by female ticks that had dual infections. A significant difference existed between levels of infection in I. ricinus and I. persulcatus, with all 3 types of Borrelia infection observed 2 times more often in I. persulcatus than in I. ricinus and dual infection occurred in I. persulcatus 3.7 times more often. It appeared that I. persulcatus is a much more dangerous vector of tick-borne borrelioses than I. ricinus.

K-ras mutation and loss of heterozygosity of chromosome 17p and survival in colorectal cancer.

The development of colorectal cancer (CRC) is thought to be a multistage process involving alterations to several types of genes, including oncogenes and tumour suppressor genes. This study examined the associations between allelic deletions of chromosome 17p in the region of the p53 gene and K-ras gene mutation and survival among CRC patients. Resected specimens from 233 patients were examined. Point mutation of codon 12 of K-ras was assessed using a modified polymerase chain reaction method. Allelic deletion of 17p was demonstrated by loss of heterozygosity (LOH) with the marker Mfd144. Fifty-seven tumours (24%) showed somatic point mutation of codon 12 of K-ras and 86 tumours (37%) showed LOH of Mfd144. There were 107 tumours (46%) with either K-ras mutation or LOH and 18 tumours (8%) with both. Compared with patients with neither alteration, significantly poorer survival was experienced only by those with both alterations (P = 0.015). However, when this variable was introduced into a multivariate analysis controlling for the patient's age and tumour stage, it failed to show a statistically significant independent effect on survival. Point mutation of K-ras and LOH of Mfd144 in CRC does not add to the prognostic information already available from clinicopathological staging.

Direct amplification of unknown genes and fragments by Uneven polymerase chain reaction

Gene cloning is a time-consuming task for molecular biologists, because it often takes weeks or months to construct, screen and finally clone a gene from a DNA library. Thus, more effective methods are needed for gene cloning. This paper describes a modified polymerase chain reaction (PCR) cycling condition, Uneven PCR, to generate specific unknown fragments or genes directly from total DNA instead of cloning fragments from DNA libraries. The essence of the method is to use two different annealing temperatures in consecutive PCR cycles to effectively amplify the target products while inhibiting the synthesis of non-specific products. Under favorable conditions, a desired DNA fragment or gene in the size range up to approximately 4 kb can be obtained and ready for cloning within a day or two.

Identification of Reproducible PCR-RAPD Markers that Enable the Differentiation of Closely Related Six-Rowed Malting Barley (Hordeum VulgareL.) Cultivars

Varietal purity of barley seed and malt lots is important to the malting and brewing industries. This study was conducted to see if a modified polymerase chain reaction (PCR) technique using random DNA primers (PCR-RAPD) could generate repeatable polymorphisms among the closely related six-rowed malting cultivars Morex, Robust, Stander, Excel, and elite breeding line M77. From a total of 80, 30 random 10-mer primers were selected based on their ability to generate reproducible polymorphisms between the cultivars Steptoe and Morex. Bulked DNA isolated from the leaves of 10–12 plants of each cultivar was amplified using the Stoffel fragment of recombinant Thermus aquaticus DNA polymerase and one primer per PCR. Nine of the 30 primers detected repeatable polymorphisms among the malting lines. Each cultivar or line could be distinguished from another with the exception of Stander and M77. The results of six primers were reproduced in a second laboratory. It was concluded that this PCR-RAPD technique could be useful for distinguishing the four six-rowed malting barley cultivars. A faster version of the technique may be required for practical cultivar identification by the barley processing industry.

DNA amplification fingerprinting (DAF) applied to the investigation ofAcinetobacter baumanii isolated from intensive care unit patients

Acinetobacter are important emerging nosocomial pathogens. In this paper thirteen Acinetobacter baumanii from intensive care patients were isolated. These were initially typed using the API-20 NE biotyping system and antibiogram analysis. Results obtained using these methods failed to convincingly characterise the organisms. In this report a method to characterise these Acinetobacter baumanii isolates is described, which utilises a modified polymerase chain reaction (PCR), capable of generating genomic fingerprints, known as DNA amplification fingerprinting (DAF). Purified chromosomal DNA of cultured clinical isolates of Acinetobacter baumanii were subjected to DAF using the M13 universal sequencing primer. Polymorphic DNA bands produced, were visualised after agarose gel electrophoresis and ethidium bromide staining. Results demonstrated that six of the thirteen clinical isolates represented one group and a second group of two isolates displayed identical fingerprint patterns. The remaining four organisms were all unique. This genotype based method is rapid, simple, reproducible and may have potential as a means of specifically typing Acinetobacter spp. allowing the route(s) of nosocomial transmission to be identified and to assess the efficiency of instituted infection control measures.

A Negative Regulatory Element of the Macrophage-Specific Human Mannose Receptor Gene Represses Its Expression in Nonmyeloid Cells

We have cloned the putative promoter of the human mannose receptor gene using the ligation-mediated polymerase chain reaction. This modified polymerase chain reaction method depends upon the ligation of restricted genomic DNA fragments to a sequence of DNA containing a generic primer site. Approximately 400 bp of genomic DNA sequence immediately upstream from the 5′ end of the lectin gene was amplified with this strategy. Primer-extended reverse transcription identified several 5′ ends of the mannose receptor mRNA corresponding to differential use of initiation transcription sites. DNA sequence analysis of the 5′ flanking sequence of the mannose receptor gene indicated the presence of a TATA box and various putative binding sites for several transcription activators. The insertion of the sequence into a plasmid containing a promoterless luciferase reporter gene reveals a promoter activity with a high cell type specificity, efficient expression upon transfection into macrophage type cells, and lack of efficiency upon transfection into nonmyeloid cells. A series of deletion mutants reveals that this cell-type-specific promoter activity is mediated by a negative regulatory element located at the 5′ end of the isolated promoter.

Diagnosis of Chlamydia pneumoniae infection in patients with community-acquired pneumonia by polymerase chain reaction enzyme immunoassay.

We conducted a prospective study of 385 patients who had community-acquired pneumonia with use of a modified polymerase chain reaction (PCR) assay that detects amplified DNA by enzyme immunoassay (EIA). We used PCR-EIA to improve detection of Chlamydia pneumoniae infection and to differentiate C. pneumoniae infection from other chlamydial infections. Cultures of throat swab specimens from four patients yielded Chlamydia species (C. pneumoniae, one patient; Chlamydia species, two patients; and C. psittaci, one patient). C. pneumoniae was repeatedly detected by PCR-EIA for thirteen (3.4%) of these 385 patients. Six of these 13 patients were infected with the human immunodeficiency virus. Ten (76.9%) of the patients who were positive by PCR-EIA had IgG titers of > or = 1:16, and two (15.4%) of the 13 patients had IgG titers of < 1:16; no sera was available in one case. Other pathogens were recovered in eight (61.5%) of the 13 cases in which C. pneumoniae was detected by PCR-EIA. In addition, for 46 (11.9%) of the 385 patients the titers of antibody were considered diagnostic of C. pneumoniae infection; however, as 36 of the 46 patients were infected with the human immunodeficiency virus (which may have affected their serological response to C. pneumoniae), interpretation of these titers was problematic. As PCR-EIA was more sensitive than was culture for detecting C. pneumoniae infection in this study, this method may be a valuable tool for the prompt diagnosis of this infection.

Cytotoxicity of Simvastatin to Pancreatic Adenocarcinoma Cells Containing Mutant ras Gene

Simvastatin (SV), a 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitor, inhibits the synthesis of mevalonic acid. The dose‐dependent (0.1–100 μg/ml) cytotoxicity of SV towards human (MIAPaCa‐2, Panc‐1, HPC‐1, HPC‐3, HPC‐4, PK‐1, PK‐9) and hamster (T2) pancreatic carcinoma cell lines was determined by MTT assay. At up to 20 μg/ml of SV, the effect was reversible and was restored by 60 μg/ml mevalonic acid. Point mutation of Ki‐ras at codon 12 in each cell line was detected by means of the modified polymerase chain reaction. The concentration of SV necessary to achieve 50% cytotoxicity was about 10 μg/ml, and at this concentration of SV, DNA synthesis assayed in terms of [3H]thymidine uptake, isoprenylation of p21ras examined by Western blotting and cell progression from G1 to S phase of the cell cycle analyzed by flow cytometry were all inhibited. Isoprenylation inhibitors of p21ras, such as SV, are expected to be useful for the treatment of pancreatic cancer.

Identification of Chicken Liver Glucose Transporter

A cDNA, Ch-GT2, encoding a glucose transporter was cloned by screening a chicken liver λgt11 cDNA library and by modified polymerase chain reaction (PCR) techniques. The encoded protein is highly homologous to rat and human GLUT 2. Within the predicted amino acid sequence, there are 12 putative transmembrane helices with a relatively large exofacial hydrophilic loop between transmembrane segments 1 and 2, which is characteristic of mammalian GLUT 2. Expression of Ch-GT2 gene in E. coli results in sixfold increase in the uptake of 2-deoxy-D-glucose uptake, and this increment can be reduced by incubation with phloretin. Thus, Ch-GT2 is a bona fide chicken liver glucose transporter. A deletion mutant generated by PCR was expressed in bacteria and shown to be functional, indicating that the N-terminal seven amino acid residues of Ch-GT2 is not essential for transport of glucose. From Northern blot analysis, Ch-GT2 is prominently expressed in chicken liver with a transcript of 4.0 kb, but not in chicken brain and heart, suggesting that distinct types of glucose transporters are present in various chicken tissues.

Conservation of the Segment 4 Gene Sequence and of a Leucine Zipper Motif in VP4 among Five US Bluetongue Viruses

Full-length cDNA copies of the segment 4 (M1) genes of US Bluetongue viruses serotypeo-2, -10, -11, -13, and -17 were selectively amplified using genomic double-stranded RNA segments from purified BTV virions as templates and a modified polymerase chain reaction (Clamp-R). They were then cloned into pUC 19 plasmids and both strands of several clones were sequenced. The length of all five segment 4 genes is 1981 nucleotides, which is 30 nucleotides shorter than that of the BTV serotype- 10 reported by Y. Yu, A. Fakusho, and P. Roy ( Nucleic Acids Res. 15, 7206 (1987)). The 5′-and 3′-noncoding regions of all five segment 4 genes are identical among all serotypes. The plus sense strand of the BTV segment 4 gene, which encodes the VP4 protein, possesses a single long open reading frame with an initiation codon (ATG) at nucleotides 9-1l and a stop codon (TAA) at nucleotides 1941-1943. This open reading frame encodes for a protein of 644 amino acid residues with a predicted molecular weight of about 75 kDa and a pl of +7 to +7.9. A potential leucine zipper motif was detected near the carboxyl terminus of the deduced VP4 amino acid sequence. The phylogenetic analysis using the sequences of these five cognate segment 4 genes is consistent with the results of our previous phylogenetic studies of cognate genome segments 5, 6, 8, 9, and 10. Serotype-10, -11, -13, and -17 are closely related and serotype-2 is the most distantly related among the five US BTV serotypes.

Detection of in vivo expression of interleukin-10 using a semi-quantitative polymerase chain reaction method in Schistosoma mansoni infected mice

A modified polymerase chain reaction (PCR) assay for analysis of cytokine gene expression from reverse-transcribed (R/T) RNA obtained from small numbers of cells is described in detail. This method employs a previously described dot-blot format and utilizes a target specific radioactive oligonucleotide probe which hybridizes to the PCR amplified product, thus increasing both specificity and sensitivity. This obviates the need for repeated electrophoresis gels and easily accomodates large experiments (e.g., numerous samples or kinetic studies), using small amounts of RNA from low cell numbers. Manipulation of many samples is further enhanced with the use of a PCR thermocycler, which like the dot-blot apparatus is designed in a 96-well format. We describe the use of the house-keeping enzyme hypoxanthine phosphoribosyltransferase (HPRT) as an internal standard, which is especially suitable since its range of detectability of expression is similar to that of the cytokines under test. This enables one to obtain an accurate measure of losses or degradation of RNA, as well as controlling for efficiency of the R/T and PCR reactions. These reactions are further controlled by inclusion of a standard curve consisting of a titration of a known amount of RNA from a cell line expressing the cytokine under test. As well as controlling for the R/T-PCR, this standard curve also enables one to obtain a semi-quantitative measure of cytokine expression by different cell populations during an immune response. We show that this method can be used successfuly for studying differential expression of IL-10 in different microenvironments during infection of mice with Schistosoma mansoni.

High-Sequence Conservation among the United States Bluetongue Viruses Cognate M2 Genes Which Encode the Nonstructural NS1 Tubule Protein

Full-length cDNA copies of the M2 gene of BTV-2, -11, and -13 serotypes obtained by a modified polymerase chain reaction (Clamp-R) were cloned into pUC19 plasmid. The entire nucleotide sequences of each M2 gene were determined and compared with BTV-10 and BTV-17, thus completing the sequencing of these cognate M2 gene segments from all five U.S. BTV serotypes. Each M2 segment contained 1769 nucleotides, a single open reading frame (ORF) with an initiation codon at nucleotides 35-37, and a termination codon at nucleotides 1691-1693. This ORF can encode the 552-amino-acid NS1 protein (64 KDa) which has an isoelectric point of 7. Analyses of the nucleotide and deduced amino acid sequences of the five U.S. BTV serotypes indicated that the most recently isolated BTV-2 serotype was more distantly related than BTV-10, -11, -13, or -17. Analyses of the evolutionary relatedness of the cognate M2 genes by codon positions indicate that the rate of mismatch accumulations in the first and second base codon positions are less than 4%. However, the mismatch accumulations in the third base codon position are quite evident (23%) when BTV-2 serotype was compared with the other U.S. BTV serotypes. This suggests that BTV-2 has separated from the other four U.S. serotypes long before they themselves diverged. These data also indicate that the five U.S. BTV serotypes were apparently derived from two distinct gene pools that reflected geographic distribution in North America.

Evolutionary analyses of five US bluetongue viruses using the cognate S2 genes

Full-length cDNA copies of S2 genes (segment 8), coding for non-structural protein 2 (NS2) of bluetongue virus serotypes 2, 11, 13 and 17 were selectively synthesized by a modified polymerase chain reaction (Clamp-R) and cloned into the PstI site of the pUC19 plasmid. Each of these S2 cognate genes was 1125 nucleotides in length with an initiation and a termination codon at nucleotides 20–22 and 1082–1084, respectively, resulting in a long open reading frame capable of coding for a protein of 354 amino acids. The deduced amino acid sequence of NS2 protein had a high concentration of lysine and contained a relatively low number of tryptophan and histidine residues. There was a highly conserved hydrophilic region at the carboxyl termini of predicted NS2 proteins in all five BTV serotypes, even though the amino acid sequence in this region in BTV-2 was more variable than in the other four serotypes. There was significant sequence homology of the cognate S2 genes at both the nucleotide and the amino acid levels. Phylogenetic analyses using the S2 gene sequences indicated that BTV-10, -11, -13 and -17 were more closely related and BTV-2 was the most distantly related serotype among the five US bluetongue viruses.

Quantification of human cytomegalovirus DNA using the polymerase chain reaction.

The important goal of developing quantitative assays for viral nucleic acids in clinical samples has been achieved for human cytomegalovirus (HCMV) by using a modified polymerase chain reaction (PCR). A control PCR target sequence was constructed by PCR mutagenesis to allow the post-amplification quantification of HCMV DNA. The control region was identical to a naturally occurring sequence within the glycoprotein B (gB) coding part of the virus genome, except that a unique restriction site, introduced by the aforementioned mutagenesis step, allowed post-amplification differentiation of control/non-control target amplified product. This technique was initially validated using known amounts of cloned control/non-control target DNA, and was found to be sufficiently sensitive to allow the quantification of a range of 10 to 10(6) genome equivalents of virus. The method was applied to urine samples of congenitally infected infants for which infectious virus titres were available. The results obtained demonstrated that the number of infectious virions determined by conventional cell culture represented a small proportion of the HCMV genome present in the samples, as assessed by the quantitative PCR methodology.