Development of a nested PCR assay for detection of feline infectious peritonitis virus in clinical specimens.

A diagnostic test for feline infectious peritonitis virus (FIPV) infection based on a nested PCR (nPCR) assay was developed and tested with FIPV, feline enteric coronavirus (FECV), canine coronavirus (CCV), and transmissible gastroenteritis virus (TGEV) and clinical fluid samples from cats with effusive feline infectious peritonitis (FIP). The target sequence for the assay is in the S1 region of the peplomer protein E2 gene. A vaccine strain of FIPV and two wild-type FIPV strains tested positive, but FECV, TGEV, and CCV tested negative. Preliminary tests with 12 cats with clinical evidence of effusive FIP and 11 cats with an illness associated with effusions, but attributed to other causes, were performed. Eleven of the 12 cats with effusive FIP tested positive, while 1 was negative. Ten of the 11 cats ill from other causes tested negative, while 1 was positive. On the basis of clinical laboratory and histopathologic criteria, the preliminary sensitivity and specificity of the assay were 91.6 and 94%, respectively.

Detection of the hepatitis B virus major pre-core mutation by the amplification refractory mutation system technique.

HBeAg/anti-HBe seroconversion is associated, in some patients affected by type B chronic active hepatitis (CAH), with the occurrence of HBV pre-core mutants characterized by a common G-A change at codon 83. Since this mutation has important clinical correlations, we tried to develop a fast and reliable PCR test based on the amplification refractory mutation system (ARMS) technique, which has been successfully used to identify point mutations associated with genetic diseases. Following this approached, we analysed HBV particles isolated from 7 patients with anti-HBe CAH and previously characterized by DNA sequencing. Sera containing only wild type or mutant HBV DNA or a combination of both showed a discrete amplification product only in the presence of the specific wild type or mutant upstream primer. These results confirm the efficacy of the ARMS technique in detecting in a rapid and specific fashion the most common and clinically relevant HBV pre-core mutation.

Applicability of DNA isolated from syncytiotrophoblast vesicles to gene amplification and molecular analysis.

Maternal contamination of fetal DNA represents a major problem when highly sensitive molecular techniques are used in the prenatal diagnosis of genetic diseases. For this reason, we have studied the possibility of using DNA isolated from syncytiotrophoblast vesicles as a target of gene amplification (PCR). Three PCR systems were selected which included a repetitive 149 bp fragment of the Y chromosome, the VNTR locus D1S80, and a portion of the beta-globin gene. The results of these experiments indicate that DNA isolated from syncytiotrophoblast vesicles is free of maternal contamination and is suitable for gene amplification and DNA analysis.

Prenatal paternity testing by DNA analysis.

Prenatal paternity testing was evaluated by DNA analysis in chorionic villus biopsies obtained during the 7th-22nd weeks of gestation. Using highly polymorphic variable number of tandem repeats (VNTR) probes, we analysed four cases consisting of mother/child/alleged father trios. In all cases, we were able to detect maternal and paternal alleles and could establish or exclude paternity. The application of DNA analysis represents a new important diagnostic aid for all cases that require a prenatal identification of paternity.

[Detection of varicella virus with a DNA probe in fetal blood and amniotic fluid]. / Nachweis des Varizellenvirus mit der DNA-Sonde im fetalen Blut und im Fruchtwasser.

In our Centre for Prenatal Diagnosis, we undertook a punctuation of the umbilical cord on a pregnant woman infected with varicella, complicated by viral encephalitis, to diagnose a foetal viraemia. In cooperation with the Toma Laboratory and Clonit Ltd., who have long been working on the development of specific viral genome probes, we succeeded in proving, that the foetus had contracted varicella.

Hepatitis B virus transcripts and surface antigen in human peripheral blood lymphocytes.

The presence of hepatitis B virus (HBV) genome, transcripts, and antigens (HBsAg, HBcAg, HBeAg) was examined in peripheral blood lymphocytes (PBL) from 12 patients with HBsAg-positive (B) chronic active hepatitis (CAH) and 8 normal donors by Southern and Northern blot techniques and enzyme-linked immunoassays (ELISA). HBV DNA was detected in 5 patients with B-CAH as extrachromosomal, full-length monomers of 3.2 kb. In 3 of these patients Northern blot analysis revealed the presence of the 3.6-3.8 kb RNA species, which were accompanied in one case by the HBsAg-specific 2.4 kb transcript. An ELISA performed on cell lysate obtained from this patient showed low but detectable amounts of HBsAg as compared to control PBL incubated with up to 50 micrograms/ml of the viral antigen. Serum HBV DNA was found in 3 patients with B-CAH, whereas all individuals positive for cellular HBV DNA had circulating HBeAg. These data indicate that lymphocytes from some patients with hepatitis B can harbor a transcriptionally and translationally active HBV genome.

Chromosomal location by in situ hybridization of the human Sau3A family of DNA repeats.

The Sau3A family is a human, clustered, highly repetitive, GC-rich DNA family. In situ hybridization studies with a plasmid carrying a Sau3A monomer as a probe have shown that Sau3A sequences are preferentially concentrated in the heterochromatic regions of human acrocentric chromosomes (D and G groups, both in pericentromeric regions and in cytological satellites) and in pericentromeric heterochromatin of chromosome 1. The same chromosomal locations were observed by using as probes two recombinant phages which carry Sau3A-positive genomic sectors. The two sectors differ for the relative proportions of monomer and multiples of Sau3A repeats, which show different extents of homology to the cloned monomer, and for the presence, in one of the two, of a small amount of an unrelated repeat (alphoid DNA). The similarity of the results obtained with the three probes suggests that heterogeneous Sau3A repeats share the same chromosomal localizations and that the two analyzed genomic sectors may not contain significant amounts of repetitive DNAs other than the Sau3A family. A comparison between the chromosomal locations of Sau3A and EcoRI families of repeats has confirmed that each family is characterized by specific chromosomal locations and that single heterochromatic regions may contain both.