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M Zerbini

Publications and source records attributed to M Zerbini.

At least 55 records · Page 3Linked to original sources
PubMed1997

Co-localization of two different viral genomes in the same sample by double-chemiluminescence in situ hybridization.

A double-chemiluminescence in situ hybridization has been developed that combines the advantages of chemiluminescence with the detection of two different viral DNAs, i.e., herpes simplex virus (HSV) DNA and cytomegalovirus (CMV) DNA, in infected cells in the same specimen. For the simultaneous detection of these two different viral DNAs, we used a biotinylated HSV DNA probe, which can be visualized by a streptavidin-horseradish peroxidase (HRP) complex amplified with biotinyl tyramide. This probe was followed by the use of a luminol-based chemiluminescent substrate for HRP and a digoxigenin-labeled CMV DNA probe visualized by antidigoxigenin Fab fragments conjugated with alkaline phosphatase (AP). This is followed by the detection with a dioxetane phosphate derivate as chemiluminescent substrate for AP. Since the final product of both chemiluminescent reactions was light emission, sequential images for the two hybridizations were taken and analyzed using a high-performance luminograph connected to an optical microscope and to a personal computer for image analysis. Positive signals for the presence of both HSV DNA and CMV DNA were noticed in infected cells in the same specimen with a sharp localization, absence of cross reactions and absence of background.

Cell Line↗
PubMed1996

Chemiluminescent immunoperoxidase assay for the dot blot hybridization detection of Parvovirus B19 DNA using a low light imaging device.

A new synthesized stable trifluoro-substituted acridan (2',3',6'-trifluorophenyl 10-methylacridan-9-carboxylate known as Lumigen PS-3) has been applied as chemiluminescent substrate of the horseradish peroxidase (HRP) enzyme (neutral isoenzyme C) in a dot blot hybridization assay for the detection of B19 Parvovirus DNA. The dot blot hybridization assay uses digoxigenin-labeled DNA probes which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with HRP. The results obtained using PS-3 reagent or the luminol-based enhanced chemiluminescence detection system (ECL Amersham and Renaissance DuPont NEN kits) were compared. A high-performance, low-intensity-light imaging luminograph apparatus to collect light emission was used. The detection systems using the different chemiluminescent substrates gave sensitive and reproducible results for calibration graphs, with high precision (relative standard deviation 5-18%). With the chemiluminescent assay it was possible to detect 0.5, 1, or 2 pg of target homologous DNA, using PS-3, ECL, or Renaissance (RE) reagents, respectively, while colorimetry had a detection limit of 5 pg. When clinical samples were analyzed the positive reference sera and the PCR-positive products gave light emissions with values higher than background at 2 sigma level, while the negative samples gave a signal comparable to the background noise for all chemiluminescent reagents. The PS-3 reagent detected one more dilution (1/256) than ECL and RE (1/128) of positive reference sera.

DNA, Viral↗
PubMed1996

Comparative evaluation of virological and serological methods in prenatal diagnosis of parvovirus B19 fetal hydrops.

Human parvovirus B19 infection in pregnancy represents a potential hazard to the fetus since fetal loss or fetal hydrops can occur. The risk of fetal loss due to transplacental B19 transmission has been evaluated in several studies using different diagnostic methods on maternal and fetal specimens. We analyzed the diagnostic value of virological and serological techniques on maternal serum, fetal cord blood, and amniotic fluid specimens obtained at the time of clinical diagnosis of fetal hydrops in 18 cases of B19 fetal hydrops. B19 DNA was detected by nested PCR, dot blot hybridization, and in situ hybridization assay. Anti-B19 immunoglobulin M and G antibodies were detected by immunoassays using recombinant B19 antigens. Our data suggest that for maternal sera, virological and serological methods have a complementary role in diagnosis, while for fetal specimens the in situ detection of B19 DNA in fetal cord blood is the most sensitive diagnostic system.

Adult↗
PubMed1996

Detection of parvovirus B19 DNA in bone marrow cells by chemiluminescence in situ hybridization.

A chemiluminescence in situ hybridization method was developed for the search of B19 parvovirus DNA in bone marrow cells, employing digoxigenin-labeled B19 DNA probes, immunoenzymatically detected with a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. The light emitted from the in situ-hybridized probe was analyzed and measured by a high-performance luminograph connected to an optical microscope and to a personal computer for the quantification of the photon fluxes from the single cells and for image analysis. The chemiluminescence in situ hybridization was applied to bone marrow cell smears of patients with aplastic crisis or hypoplastic anemia, who had been previously tested by in situ hybridization with colorimetric detection, dot blot hybridization, and nested PCR. The chemiluminescent assay provided an objective estimation of the data, proved specific, and showed an increased sensitivity in detecting B19 DNA compared with in situ hybridization with colorimetric detection.

Anemia, Aplastic↗
PubMed1996

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA.

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.

Cell Line↗
PubMed1995

Persistent B19 parvovirus infections in haemophilic HIV-1 infected patients.

B19 infection can be acquired by transmission with blood factors in patients with congenital bleeding disorders, requiring clotting factor concentrates. In immunodeficient patients, the failure of immunity to clear B19 virus may produce persistent infections. The presence of B19 DNA in blood samples from seven haemophilic patients with concomitant HIV-1 infection was studied over a period of three-to-four years. Dot blot hybridization assays with DNA and RNA probes were used to detect medium high viremias, and polymerase chain reaction (PCR) to detect very low viremic titres. Three patients were negative for B19 DNA in all the blood samples, while four patients were persistently positive for B19 DNA. Viral persistence, which in one patient was detected throughout the study period (40 months), occurred at low titre in all four positive patients with some recurrent increases in viral titre. In the four patients persistently positive for B19 DNA, acute or chronic clinical symptoms and signs that could be associated with B19 were not noted when virus was present at low titre (B19 DNA detectable only by PCR). When patients had a higher viral titre (B19 DNA detectable by dot blot hybridization) acute manifestations (aplastic crisis, Fifth disease, fevers, pneumonitis) were found.

Adolescent↗
PubMed1995

Extent of sequence variability in a genomic region coding for capsid proteins of B19 parvovirus.

Genomic variability in human parvovirus B19 was analysed by direct partial sequencing of different isolates collected in Italy between 1989 and 1994. DNA was purified from viremic serum samples and the region of viral genome coding for structural proteins was amplified by polymerase chain reaction and partially sequenced (nt. 2400-3400). Data were compared to reference isolates Wi (U.K., 1973) and Au (U.S.A., 1982). The average relative distance between isolates was estimated at the low level of 0.61%, comparable with the distance to reference isolates. Out of 22 nucleotide substitutions found, 9 resulted in amino acid changes. From our results, this region of human parvovirus B19 genome appears to be stably conserved.

Amino Acid Sequence↗
PubMed1995

Automated detection of digoxigenin-labelled B19 parvovirus amplicons by a capture hybridization assay.

An automated method to identify B19 amplicons, directly labelled with digoxigenin during amplification reaction was developed. The labelled amplicons were hybridized with a biotinylated B19 oligo-probe and captured on commercially available test tubes coated with streptavidin. The hybridized amplicons labelled with digoxigenin were detected using anti-digoxigenin Fab fragments conjugated to peroxidase and the colourimetric reaction automatically evaluated as an immunoenzymatic assay. Fifty serum samples were tested by the assay and the results were in accordance with those obtained by Southern blot analysis of amplified products. Due to the high sensitivity, specificity and reproducibility shown, the assay seems to be a practical and reliable test for the diagnosis of B19 infection and can be easily adapted to identify any digoxigenin-labelled amplified product of viral genomes.

Bacterial Proteins↗
PubMed1995

Parvovirus B19 clearance from peripheral blood after acute infection.

The clearance of B19 parvovirus from peripheral blood was followed after acute infection to determine how long the virus is present in blood, even at low titer. The presence of B19 DNA in serum was investigated with dot blot hybridization during an epidemic. Fourteen patients positive for B19 DNA were followed for up to 1 year for its presence in samples taken monthly, using both dot blot hybridization and nested polymerase chain reaction (PCR). All patients examined showed medium to high viremic titers (detectable with hybridization assay) only at the beginning of observation; later the virus titer decreased and was detectable only by nested PCR. Of the 14 patients followed, 13 were positive for B19 DNA by nested PCR for 2-6 months; 1 patient showed a persistent infection associated with chronic arthritis and was positive for B19 DNA for 1 year without clearance of the virus.

Acute Disease↗
PubMed1994

Active or recent parvovirus B19 infection in children with Kawasaki disease.

Because parvovirus B19 occasionally causes some of the features typical of Kawasaki disease, we investigated B19 involvement in 15 children with Kawasaki disease. Active or recent B19 infection, as shown by B19-DNAaemia, positive B19-specific IgM antibodies, or both, was diagnosed in 10 patients (67%). A high frequency of all major criteria for diagnosis of Kawasaki disease (60%), anaemia (60%), coronary aneurysms (30%), and arthropathy (30%) was found in children with B19-associated Kawasaki disease. Thus B19 may have a pathogenic role in the development of Kawasaki disease, with other predisposing factors.

Antibodies, Viral↗
PubMed1994

Rapid diagnosis of cytomegalovirus encephalitis in patients with AIDS using in situ hybridisation.

AIMS: To evaluate the presence of cytomegalovirus (CMV) DNA in the cerebrospinal fluid of patients with AIDS and suspected viral encephalitis using an in situ hybridisation assay with digoxigenin labelled CMV DNA probes. METHODS: The presence of CMV DNA was evaluated in cerebrospinal fluid cells of 10 patients with AIDS using in situ hybridisation. The positivity of CMV DNA was confirmed by the presence of CMV induced antigens in the same specimens. The presence of CMV DNA and CMV induced antigens was also analysed in peripheral blood leucocytes. The time required to perform the in situ hybridisation assay was about eight hours. RESULTS: The in situ hybridisation assay was sensitive, specific, and provided good resolution. Six patients proved positive for the presence of CMV DNA in CSF cells and all six also proved positive for CMV DNA in blood leucocytes. Of the six CMV positive patients, five were treated with specific antiviral drugs: of these, one died during the treatment while four clinically recovered after one month of treatment. CONCLUSIONS: The in situ hybridisation assay using digoxigenin labelled CMV DNA probes can be used as a valuable diagnostic test for the detection of CMV DNA in the cerebrospinal fluid cells of patients with suspected CMV encephalitis and can therefore prompt adequate antiviral therapeutic intervention.

AIDS-Related Opportunistic Infections↗
PubMed1993

Evaluation of strand-specific RNA probes visualized by colorimetric and chemiluminescent reactions for the detection of B19 parvovirus DNA.

A dot-blot hybridization assay was developed to detect B19 DNA using strand-specific RNA probes labelled with digoxigenin. The sensitivity of the assays was evaluated either using 'plus' and 'minus' sense RNA probes in two different hybridization assays, or in two successive reactions of the same assay. The hybridized probes were revealed immunoenzymatically using anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The enzyme was visualized by colorimetric reaction. Since 'minus' sense RNA probe gave the best results in the dot-blot procedures, we increased the sensitivity of the hybridization assay visualizing the 'minus' sense digoxigenin-labelled RNA probe by chemiluminescent reaction. In these experimental conditions up to 20 fg of target B19 DNA could be visualized. In the search for B19 DNA, 4656 serum samples were analyzed by chemiluminescent reaction of 'minus' sense digoxigenin-labelled RNA probe and for comparison with the digoxigenin-labelled DNA probe. Positive results were confirmed by Southern blotting. Out of 4656 serum samples analyzed, 4648 gave negative results, 1 resulted positive to all the hybridization assays, 6 only using RNA probe and 1 only by DNA probe.

Adult↗