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At least 919 records · Page 51Linked to original sources
PubMed2006

Development of a quantitative-competitive PCR for quantification of human cytomegalovirus load and comparison with antigenaemia, viraemia and pp67 RNA detection by nucleic acid sequence-based amplification.

AIM: The human cytomegalovirus (HCMV) is an important pathogen in immunocompromised patients, such as transplant recipients. The use of sensitive and rapid diagnostic assays can have a great impact on antiviral prophylaxis and therapy monitoring and diagnosing active disease. Quantification of HCMV DNA may additionally have prognostic value and guide routine management. The aim of this study was to develop a reliable internally-controlled quantitative-competitive PCR (QC-PCR) for the detection and quantification of HCMV DNA viral load in peripheral blood and compare it with other methods: the HCMV pp65 antigenaemia assay in leukocyte fraction, the HCMV viraemia, both routinely employed in our laboratory, and the nucleic acid sequence-based amplification (NASBA) for detection of HCMV pp67-mRNA. METHODS: Quantitative-competitive PCR is a procedure for nucleic acid quantification based on co-amplification of competitive templates, the target DNA and a competitor functioning as internal standard. In particular, a standard curve is generated by amplifying 10(2) to 10(5) copies of target pCMV-435 plasmid with 10(4) copies of competitor pCMV-C plasmid. Clinical samples derived from 40 kidney transplant patients were tested by spiking 10(4) copies of pCMV-C into the PCR mix as internal control, and comparing results with the standard curve. RESULTS: Of the 40 patients studied, 39 (97.5%) were positive for HCMV DNA by QC-PCR. While the correlation between the number of pp65-positive cells and the number of HCMV DNA genome copies/mL and the former and the pp67mRNA-positivity were statistically significant, there was no significant correlation between HCMV DNA viral load assayed by QC-PCR and HCMV viraemia. CONCLUSIONS: The QC-PCR assay could detect from 10(2) to over 10(7) copies of HCMV DNA with a range of linearity between 10(2) and 10(5) genomes.

Adult↗
PubMed2005

A rapid and sensitive assay for quantification of siRNA efficiency and specificity.

RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed against Thymosin beta4 (Tbeta4); determined their effectiveness at silencing Tbeta4 and have both excluded off-target silencing of the Tbeta4 homologue Thymosin beta10 (Tbeta10) and demonstrated partial knockdown of Tbeta10 despite significant (12/23; 52%) sequence mismatch. This assay system is applicable to any RNAi study where there is a risk of targeting homologous genes and to the monitoring of off-target effects at the genome level following microarray expression profiling.

Journal Article↗
PubMed2001

Promoter-specific transcription of the IGF2 gene: a novel rapid, non-radioactive and highly sensitive protocol for mRNA analysis.

The human insulin-like growth factor-II (IGF2) is a regulatory peptide which is critical in normal fetal growth. IGF2 gene transcription is controlled by the usage of four promoters P1-P4 of which promoters P2-P4 are genomically imprinted. Disruption of imprinting and the resulting increase of gene dosage have been shown to be implicated in tumor progression in a variety of human tumors. Due to the need for high amounts of tissue material for conventional methods such as Northern blotting or ribonuclease protection assay (RPA), studies on IGF2 expression have most often been limited to the detection of total IGF2 transcript, though different dysregulatory events can be responsible for the abundance of IGF2 mRNA found in many tumors. We established a highly sensitive competitive RT-PCR assay for the four different transcripts of the IGF2 gene with transcript-specific external RNA competitors in which we take advantage of fluorescence-based quantification on a semiautomated sequencer. The amount of total RNA needed is approximately 100 times lower than the amounts required for Northern blotting or RPA, so that even cytological samples can be analyzed. We applied the assay to a series of eleven hepatoblastomas (HB) in which normal adjacent liver tissue could also be analyzed.

Child, Preschool↗
PubMed2004

Rapid and quantitative detection of mumps virus RNA by one-step real-time RT-PCR.

We developed a new TaqMan-based one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detection and quantification of mumps virus RNA. Oligos targeting the matrix protein gene of mumps virus were designed by using our oligo designing and analyzing software, Oligoware 1.0. Oligos's specificity was tested with 5 strains (4 laboratory isolated and 1 Jeryl Lynn strain) of mumps virus. The suggested TaqMan-based one-step real-time RT-PCR assay correctly detected the 4 laboratory-isolated strains and 1 Jeryl Lynn strain. To confirm the specificity of the TaqMan PCR assay, parainfluenza type 1, 2, 3 strains, sendai virus, and measles virus (vaccine strain) were tested, and no cross-reactivity was observed between mumps and tested strains. In addition, a BLAST (NCBI) search showed no genomic cross-reactivity with other viruses or cells. Testing of the assay's reproducibility was repeated several times, and the same results were achieved. The new assay was able to quantify the concentrations of mumps virus gene ranging from 10(1) to 10(8) copies per reaction sensitively with generated plasmid standards. In addition, it was shown that a significant correlation (R2 = 0.9564) between genome number as determined by one-step real-time RT-PCR and the corresponding number of plaque in paired samples was found with regression analysis. The results of one-step real-time RT-PCR assay also corresponded well to those of nested PCR. We conclude that our one-step real-time RT-PCR assay is a reliable, specific, and sensitive tool for the diagnosis of mumps virus. We consider that these results come from highly conserved primers and probe set that were designed with Oligoware 1.0.

Base Sequence↗
PubMed2006

An efficient fungal RNA-silencing system using the DsRed reporter gene.

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.

Acremonium↗
PubMed2004

Development of quantitative competitive-reverse transcriptase-polymerase chain reaction for detection and quantitation of avian leukosis virus subgroup J.

Infection with avian leukosis virus subgroup J (ALV-J) causes severe economic losses in the broiler industry by increasing mortality, producing tumors, and decreasing weight gain in chickens. The quantitation of ALV-J is difficult because of its failure to produce a cytopathic effect in cell culture systems and the nonspecificity of antigen-capture enzyme-linked immunosorbent assay (ELISA) tests. This study was performed to develop a quantitative competitive-reverse transcriptase-polymerase chain reaction (QC-RT-PCR) method based on coamplification of ALV-J genomic RNA and a known amount of a synthesized RNA competitor. The 369 bp RNA competitor was constructed by restriction enzyme treatment of an ALV-J specific 545 bp PCR product, ligation, transformation into Escherichia coli, and in vitro transcription. The competitor contained the same amplification primer annealing sites and sequence as the original viral RNA, except that it had a 176 bp internal deletion. Coamplified RT-PCR products were visualized by electrophoresis and ethidium bromide staining, and fluorescences were quantified using computer-assisted image analysis. The sensitivity of this new QC-RT-PCR method was 25 fg of viral RNA, and 10-fold dilutions were differentiable. This method allowed absolute and relative quantification of ALV-J RNA copy numbers and was simpler than previously published methods for ALV-J quantification.

Animals↗
PubMed2004

One-step real-time duplex reverse transcription PCRs simultaneously quantify analyte and housekeeping gene mRNAs.

We developed a one-step real-time duplex reverse transcription PCR (RT-PCR) method using the LightCycler platform. This method allows simultaneous reverse transcription and real-time PCR amplification of two mRNAs of specific genes of interest (analyte genes) and mRNA of constantly transcribed genes (housekeeping genes) in a single-tube reaction. Specimen total nucleic acids were used because eukaryotic cDNA is discriminated from genomic DNA using exon-spanning primers and/or fluorescence resonance energy transfer (FRET) probes. Transcripts of murine arginase I and hypoxanthine-phosphoribosyl transferase (HPRT; housekeeping gene) or murine arginase II analyte and porphobilinogen deaminase (PBGD; housekeeping gene) were quantified in such duplex RT-PCRs. Twenty-minute reverse transcription reactions at 55 degrees C followed by 18 high-stringency step-down thermal cycles and 25 relaxed-stringency fluorescence acquisition cycles produced sensitive and accurate RT-PCR results. Fluorescent signal spillover between channels was fully compensated. A matrix of duplex PCRs at variable ratios of target standards yielded equations for factors that correct PCR-specific target ratio-dependent deviations in quantification. The one-step real-time duplex RT-PCRs reliably and accurately determined 10-10,000 copies of each target over a 100,000-fold range of target copy ratios (analyte to housekeeping mRNA = 10(-2.5)-10(2.5)) in a single assay.

Animals↗
PubMed1997

Usefulness of DNA viral load quantification for cytomegalovirus disease monitoring in renal and pancreas/renal transplant recipients.

BACKGROUND: The purpose of this prospective study was to evaluate the usefulness of quantifying DNA-cytomegalovirus (CMV) load for the diagnosis and monitoring of CMV disease among renal and pancreas transplant patients under immunosuppressive drugs. METHODS: A longitudinal study was conducted among 34 consecutive, unselected renal and pancreas/renal transplanted patients in our unit. During the first 3 posttransplant months, weekly monitoring of CMV infection and CMV disease was done, involving the determination of viremia by the shell vial assay, qualitative DNAemia by semi-nested polymerase chain reaction (PCR) and quantitative DNAemia by the hybrid capture system (HCS), a new and original hybridization method (337 samples were collected for each test). Qualitative and quantitative DNAemia results were blinded to physicians and three grades of disease were defined according to CMV related symptom occurrence. RESULTS: PCR was the most sensitive (100%) but the least specific (78%) method for the diagnosis of CMV disease. HCS was specific for CMV genome detection, sensitive and reproducible. Blood DNA levels above 60 pg/ml were predictive of severe or moderate CMV disease (sensitivity, 92%; specificity, 100%). A significant decrease in viral load was observed after ganciclovir administration, and a positive PCR or HCS result at the end of the antiviral treatment was associated with relapse of CMV infection or disease. CONCLUSIONS: It is concluded that quantitative DNAemia detection, with this new commercially available method, can predict disease and may be useful for a rational evaluation of ganciclovir preemptive therapy in such patients.

Adolescent↗
PubMed1999

Quantitation of hepatitis B virus genomic DNA by real-time detection PCR.

Quantitation of hepatitis B virus (HBV) DNA in serum is a useful method for the monitoring of HBV replication. We attempted to develop a quantitative assay system for HBV DNA that is more sensitive, accurate, and reproducible than existing systems. We detected HBV DNA by real-time detection PCR (RTD-PCR) based on Taq Man chemistry. The efficacy of this assay was evaluated by quantitatively measuring sequential levels of synthetic DNA and DNA in clinical serum samples. The detection limit of this system was as few as 10 DNA copies/reaction. A linear standard curve was obtained between 10(1) and 10(8) DNA copies/reaction. The coefficient of variation for both intra- and interexperimental variability indicated remarkable reproducibility. This system detected HBV DNA in 100% of chronic hepatitis B patients tested and never detected HBV DNA in healthy volunteers who were negative for HBV markers. These observations suggest that RTD-PCR is an excellent candidate for a standard HBV quantification method.

Adult↗
PubMed2006

The analysis and quantification of a clonal B cell response in a hyperimmunized anti-D donor.

Healthy volunteers are hyperimmunized with RhD-positive red cells in order to obtain plasma containing high titres of anti-D immunoglobulin, which is used for the prevention of haemolytic disease of the fetus and newborn. We analysed the anti-D immune response in a donor who had been hyperimmunized for 7 years and who showed declining anti-D titres despite re-immunization. A phage display library representing the complete immunorepertoire and a second library representing the IGHV3 superspecies family genes (IGHV3s) repertoire in the donor were constructed and analysed. A clonal Ig-gene rearrangement was quantified in the peripheral blood by limiting dilution polymerase chain reaction (PCR) All RhD-binding phages from both libraries, except one, had heavy chains with IGH-VDJ rearrangements of the same clonal origin, but with different patterns of somatic mutations and joined with different light chains. Limiting dilution PCR performed on mRNA and genomic DNA showed a frequency of 1 clonal B cell in 2000 IgG1/3-positive B cells. We show the presence of clonally related RhD-specific B cells in a hyperimmunized anti-D donor who had declining anti-D titres and who was unresponsive to re-immunization. Furthermore, we found a high frequency of clonal B cells. These results contribute to the understanding of the immune response against RhD in hyperimmunized anti-D donors.

Amino Acid Sequence↗
PubMed1995

Isolation of genomic DNA from the earthworm species Eisenia fetida.

Our interest in detecting genotoxic exposure in earthworms led us to isolate high quality DNA from the Eisenia fetida species. For that, we compared a modification of the conventional phenol-chloroform extraction procedure, usually referred to as the Maniatis procedure, to two commercially available kits reportedly eliminating multiple partitions in phenol and chloroform, namely the Qiagen and Nucleon protocols. From the 260 nm optical density values, the commercial kits extracts hinted toward higher DNA recovery with those procedures. However, the 260/280 nm ratios indicated that the quality of the DNA isolated with the modified Maniatis procedure was purer than that isolated with the commercial kits, the latter being most probably contaminated by proteins and/or RNA. The Maniatis procedure was slightly modified by the introduction of a potassium acetate step for protein precipitation and by shortening the proteinase K treatment from 12-18 h to only 2 h. The higher quality of the DNA isolated by phenol-chloroform extraction was confirmed by quantification with the fluorescent 3,5-diaminobenzoic acid assay. Preliminary results suggest that the modified Maniatis procedure herein described is not only applicable for DNA adducts studies using 32P-postlabelling techniques but is also suitable for DNA extraction from other earthworm species such as Lumbricus terrestris.

Animals↗
PubMed2026

TPMM: three-component posterior mixture model enables robust inverton detection in low-depth metagenomes and suggests potential viral invertons.

SUMMARY: Bacterial phase variation enables reversible, locus-specific phenotypic switching, often driven by DNA inversion (invertons). To identify these events, researchers commonly rely on sequencing reads that provide orientation-specific support. Metagenomic sequencing, which captures total genetic material independent of cultivation, offers a powerful platform for the comprehensive study of invertons. However, computational inverton calling from metagenomic data is difficult at low sequencing depth: hard read-support cutoffs can miss true events, while sequence-only predictors lack read-backed interpretability and uncertainty quantification. To address this, we present TPMM, a three-component posterior mixture model for inverton calling in metagenomic data. TPMM explicitly incorporates sequencing depth to formulate inverton detection as a probabilistic mixture problem. Starting from candidates flanked by inverted repeats, the model classifies the candidates into noise, low-probability, or high-probability inversion signals using read evidence. Finally, TPMM assigns posterior probabilities as soft labels and applies cumulative Bayesian False Discovery Rate control to robustly identify true invertons. On two real gut metagenomic datasets, TPMM agrees well with PhaseFinder at high depth but recovers substantially more invertons under systematic downsampling, demonstrating superior performance in sparse-data regimes. We further examine potential reversible inversion elements in viral genomes and provide supporting analyses, suggesting a broader scope for inversion-mediated regulation. AVAILABILITY: The source code of TPMM is available via: https://github.com/KennyxxD/TPMM.

Metagenomics↗
PubMed1996

An alanine racemase gene as a new index for detecting Escherichia coli in foods.

A gene of alanine racemase, a typical prokaryotic enzyme, was evaluated as a new index for detecting Escherichia coli in foods. An alanine racemase gene fragment containing a non-conserved sequence of the gene was amplified from genomic DNA of E. coli by a polymerase chain reaction, and then labeled with digoxigenin as a probe for detecting E. coli. Food samples and bacteria were each treated as at 25 degrees C for 10 min in 0.1N NaOH containing 0.5% SDS, before being directly spotted on to nylon membranes for DNA hybridization. The probe was specific for E. coli; all 48 strains of E. coli examined, including such pathogenic strains as E. coli O157:H7, showed positive signals, whereas all 59 strains of non-E. coli species, except for one strain (Shigella sonnei), did not show a signal. Various foods inoculated with E. coli K-12 showed positive signals whereas no uninoculated foods showed any signal. Quantification of E. coli cells in the death phase by the hybridization method showed good correlation with that by the plate culture method. The alanine racemase gene could prove useful as an index for detecting E. coli in foods.

Alanine Racemase↗
PubMed2006

Starvation-induced expression of autophagy-related genes in Arabidopsis.

BACKGROUND INFORMATION: Autophagy is a catabolic process for degradation of cytoplasmic components in the vacuolar apparatus. A genome-wide survey recently showed evolutionary conservation among autophagy genes in yeast, mammals and plants. To elucidate the molecular and subcellular machinery responsible for the sequestration and subsequent digestion of intracellular material in plants, we utilized a combination of morphological and molecular methods (confocal laser-scanning microscopy, transmission electron microscopy and real-time PCR respectively). RESULTS: Autophagy in Arabidopsis thaliana suspension-cultured cells was induced by carbon starvation, which triggered an immediate arrest of cell growth together with a rapid degradation of cellular proteins. We followed the onset of these responses and, in this report, provide a clear functional classification for the highly polymorphic autophagosomes by which the cell sequesters and degrades a portion of its own cytoplasm. Quantification of autophagy-related structures shows that cells respond to the stress signal by a rapid and massive, but transient burst of autophagic activity, which adapts to the stress signal. We also monitored the real-time expressions of AtATG3, AtATG4a, AtATG4b, AtATG7 and AtATG8a-AtATG8i genes, which are orthologues of yeast genes involved in the Atg8 ubiquitination-like conjugation pathway and are linked to autophagosome formation. We show that these autophagy-related genes are transiently up-regulated in a co-ordinated manner at the onset of starvation. CONCLUSIONS: Sucrose starvation induces autophagy and up-regulates orthologues of the yeast Atg8 conjugation pathway genes in Arabidopsis cultured cells. The AtATG3, AtATG4a, AtATG4b, AtATG7 and AtATG8a-AtATG8i genes are expressed in successive waves that parallel the biochemical and cytological remodelling that takes place. These genes thus serve as early markers for autophagy in plants.

Arabidopsis↗
PubMed2006

Increased number of neurons expressing androgen receptor in the basolateral amygdala of pathologically aggressive dogs.

The present study aimed to determine the extent to which the androgen receptor (AR) is directly involved in the hormonal modulation of pathological canine aggressive behaviour in the basolateral nuclear group (BNG) of the amygdaloid body. A stereological quantification of AR-positive neurons was performed in the BNGs of normally behaving and aggressive male dogs. The BNG was selected because it is involved in sexual and behavioural activities that are influenced by androgens. In the aggressive dogs the BNG contained a significantly higher number of AR-positive neurons compared with normally behaving dogs suggesting differences in androgen activity within the BNGs of both the groups. However, additional mechanisms are likely to be involved because the AR-negative fraction of BNG neurons was also increased in the aggressive dogs. It was concluded that most of the AR was unliganded because a cytoplasmic staining pattern of AR positivity was observed in the canine BNG neurons. This indicates that genomic androgen actions, which are mediated through the AR are of minor importance in the testosterone modulation of canine aggression within the BNG. Other non-genomic mechanisms through which androgens may exert their action in the BNG are discussed. The aromatase pathway is suggested to be the main mechanism through which testosterone exerts its action within the BNG.

Aggression↗
PubMed2005

Comparison of a real-time reverse transcriptase PCR assay and a culture technique for quantitative assessment of viral load in children naturally infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory infection of children. Understanding RSV pathogenesis and evaluating interventions requires quantitative RSV testing. Previous studies have used the plaque assay technique. Real-time reverse transcriptase PCR (RTrtPCR) offers possible greater sensitivity, stability after freeze/thaw, and lower cost, thus facilitating multicenter studies. We developed RTrtPCR assays based upon the RSV N and F genes. The N-gene assay detected greater RSV quantity and was further evaluated. Standard curves utilized both extractions from RSV culture supernatants of known quantity and cloned purified copies of the target DNA. In vitro, the ratio of RSV subgroup A (RSV-A) genome copies to PFU was 153:1. A total of 462 samples collected quantitatively from 259 children were analyzed in duplicate by RTrtPCR. Results were compared with those of RSV plaque assays performed on fresh aliquots from the same children. Duplicate RTrtPCR results were highly correlated (r2 = 0.9964). The mean viral load from nasal washes obtained on the first study day was 5.75 +/- standard error of the mean 0.09 log PFU equivalents (PFUe)/ml. Viral load by RTrtPCR correlated with plaque assay results (r2 = 0.158; P < 0.0001). Within individuals, upper and lower respiratory tract secretions contained similar viral concentrations. RSV-A-infected children had 1.17 log PFUe higher viral loads than did those with RSV-B (P < 0.0001). RSV quantification by RTrtPCR of the N gene is precise and has significant, though limited, correlation with quantitative culture. The utility of the RTrtPCR quantification technique for clinical studies would be solidified after its correlation with RSV disease severity is established.

Base Sequence↗
PubMed2002

Interstitial deletions at the long arm of chromosome 13 may be as common as monosomies in multiple myeloma. A genotypic study.

BACKGROUND AND OBJECTIVES: Deletions at the long arm of chromosome 13, mostly at the q14, and monosomy of chromosome 13 are described to be common in multiple myeloma (MM). 13q- has been associated with an adverse outcome and it has been proposed as one of the most important prognostic factors for MM patients. Deletions of 13q14 are rare in monoclonal gammopathy of undetermined significance (MGUS) and are thus believed to be associated with the development of the full myeloma phenotype. DESIGN AND METHODS: A genotyping analysis on purified neoplastic plasma cells was performed on 14 consecutive MM cases to determine the minimally deleted region at chromosome 13. Freshly obtained bone marrow was analyzed by flow cytometry in order to establish the percentage of plasma cell infiltration (CD38+BB4+CD56+/-CD19-). Neoplastic enrichment was carried out using BB4 coated immunomagnetic beads. This method allowed us to monitor the enrichment process. DNA obtained from the enriched neoplastic population and DNA from the clean fraction was amplified using combination sets of chromosomes 12 and 13. Amplimers were run on acrylamide gels, analyzed by automatic fluorescence quantification, and their size determined using the software programs Genescan and Genotyper. RESULTS: In 11 patients electropherograms were suggestive of loss of heterozygosity (LOH) for polymorphic markers located at the long arm of chromosome 13. Four patients showed monosomy and 7 had interstitial deletions in the telomeric region. LOH was not evidenced at chromosome 12 in any sample. The minimal region with deletion was defined by the markers D13S159 (13q32.2, centromeric) and D13S1267 (13q32.3, telomeric). A gene with a potentially pathogenic role may be located in this very small region. Three patients did not show LOH at chromosome 13 by genotypic analysis; however, in one of these, proximal deletion at the long arm of chromosome 13 (13q2.1-2.2) was demonstrated by comparative genomic hybridization (CGH). INTERPRETATION AND CONCLUSIONS: These findings suggest that interstitial deletions of the long arm of chromosome 13 may be more common than previously recognized. The methodologic approach reported in this work may simplify LOH analysis in MM patients. The potential uses of genotyping analysis in risk stratification remain to be investigated.

Adult↗
PubMed1995

Detection of human cytomegalovirus DNA: how, when and where?

Although several methods have been utilized for the detection and quantification of human cytomegalovirus (HCMV) DNA, all of them can be divided into three groups: (i) detection of HCMV DNA directly in tissues by in situ hybridization or in situ polymerase chain reaction (PCR); (ii) detection of HCMV DNA in cell or tissue lysates by hybridization with DNA or RNA probes differently labelled-labels were progressively modified in order to provide an increasing sensitivity (hybridization products were revealed by radioactive, colorimetric or chemiluminescent procedures); (iii) detection of HCMV DNA in cell or tissue lysates by qualitative (single-step and nested) and quantitative (semiquantitative, competitive or noncompetitive) PCR. The selection of the methods to be employed depends primarily on the clinical situation which must be evaluated. Clinical samples for HCMV genome detection must vary accordingly.

Blotting, Southern↗