Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Mosaicism and accuracy of prenatal cytogenetic diagnoses after chorionic villus sampling and placental biopsies

Discrepant chromosome findings in placenta and fetus (false negative and false positive) after chorionic villus sampling (CVS) are mainly due to confined mosaicism. Non-mosaic normal or abnormal chromosome counts after direct preparation and culture nearly always correctly reflect the fetal chromosome constitution. False-negative results have almost exclusively been restricted to cytotrophoblast cells not representing a fetal chromosome abnormality. Diagnosis of placental mosaicism definitely requires an adequate follow-up by amniocentesis, fetal blood sampling, or sonography before a pregnancy is terminated. When direct preparations and cultured cells are used for cytogenetic diagnoses and placental mosaicism is not taken as proof for a chromosomal abnormality in the fetus, CVS is an accurate diagnostic tool.     

First-trimester prenatal diagnosis in quintuplets: A practical approach using step-by-step embryo reduction

A multiple pregnancy of high rank may occur in a couple at risk for a Mendelian disorder. Prenatal diagnosis is hampered by the difficulty of (1) obtaining chorionic villi from each zygote arid (2) unequivocally relating each sample to the corresponding embryo. The calculation of the genetic risk according to the number of zygotes led us to propose a diagnostic strategy based on embryo reduction, a technique initially designed to improve the perinatal outcome of multiple pregnancies with normal embryos. We report a case in which this approach allowed rational use of first-trimester chorionic villus sampling in a quintuplet pregnancy at risk for non-ketotic hyperglycinaemia, resulting in the selective birth of unaffected twins.     

Prenatal diagnosis of 21-hydroxylase deficiency by rflp analysis of the 21-hydroxylase,complement C4, and HLA class II genes

In 19 pregnancies at risk for 21-hydroxylase deficiency (21OHD) in 18 families with at lea one affected child, prenatal diagnosis was performed by RFLP analysis using the enzymi Taq I and EcoRI and the DNA probes specific for the 21 OH genes, the closely linke complement C4 genes and the highly polymorphic HLA class II genes DRB, DQB, and DPI For fetal DNA analysis either chorionic villi or cultivated amniotic cells were used. In all 1 cases, a clear prenatal diagnosis was possible either with the 21OH probe alone or in mo cases, by combining the results of the different closely linked loci.     

Optimization of Methods for Detecting Hepatitis A Virus in Food

Hepatitis A virus (HAV) is currently recognized as an important human food borne pathogen, and it is one of the most resistant enteric RNA viruses, is highly infectious, and may lead to widespread outbreaks. The aim of this study was to optimize the methods to detect HAV from artificially contaminated food. To this end, strawberry and lettuce were experimentally contaminated with HAV suspension containing 6 × 10⁶ copies/ml. After contamination, HAV persistence and washing procedure were evaluated at 0, 1, 3, 7, and 9 days of storage. Five elution buffers (PBS (pH 7.4)/0.1% Tween80; 50 mM glycine/3% (wt/vol) beef extract (pH 9.5); PBS (pH 7, 4); 25 mM glycine/0.1 Tween80; and 1 M sodium bicarbonate) were used to elute the virus, and qualitative and quantitative PCR were used for HAV detection. HAV was detected by qualitative and quantitative PCR using any of the five elution buffers, but PBS was the most effective. Even after washing, HAV was detected up to 9 days after contamination by quantitative PCR. Quantitative PCR was more sensitive than qualitative PCR since samples containing viral load lower than 1.4 × 10³ copies/ml could not be detected by qualitative PCR. Quantitative PCR can be used for rapid detection of food borne viruses and will help in the monitoring and control of food borne disease.