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.     

Vegetation changes in Conservation Reserve Program lands in interior Alaska

Over 14 million hectares of erosion prone cropland in the United States has been converted into grasslands through the Conservation Reserve Program (CRP) administered by the United States Department of Agriculture, however, studies of the effects of CRP enrollment on plant communities and subsequent plant succession are largely lacking. In Delta Junction, Alaska plant communities in CRP fields are transitioning from grasslands to shrub dominated plant communities, which are resulting in compliance problems with program regulations that state “fields must be maintained in a condition that permits easy conversion to cropland”. To determine plant succession and how previous land management and soils might influence the transition, we measured plant populations in 20 CRP fields throughout Delta Junction using modified-Whittaker plots. These data were combined with data on current management practices, previous farming history, soils, soil properties, diversity indices, and time since land was cleared and analyzed with nonmetric multidimensional scaling ordination to determine factors that influence plant succession. Time in the CRP was the only factor consistently influencing plant succession. As time in the CRP increased, the planted introduced grasses brome grass (Bromus inermis) and red fescue (Festuca rubra) and the native pteridophyte (Equisetum arvense) decreased, whereas a native grass (Calamigrostis canadensis), five native forb, two native shrub, and three native tree species increased. Plant diversity increased at a rate of more than 2 species per 1000 m² per year. Regression analyses of plant species and plant groups using time in the CRP as the dependent variable resulted in the identification of outlier CRP fields with significantly more or less than expected covers of vegetation. All fields with these outliers had reasonable explanations for the differences in cover that were unrelated to the overall rate of plant succession. Current management practices will result in incompliant fields and different management practices that result in woody vegetation control is key to maintaining CRP fields in compliance.     

THE CLIMATO-ECOLOGICAL FUNDAMENTALS OF THE “MANMADE COMMUNITIES” OF TROPICAL PERENNIAL CROPS ON HAINAN ISLAND, CHINA

<正> According to the leading national classification of the climate of China, given by B. Huang (revised version 1986), Hainan falls into the tropical climatic realm, subdivided into a "peripheral tropical" type for the northern part and a "middle tropical" type for the southern part of the island. While all-year-round frost-free conditions define the tropics in general, the 20t-isotherm for the coldest month represents the boundary between the peripheral tropical and middle tropical types. Compared to the tropical climate in general, Hainan experiences distinctly lower winter temperatures which thus result in a larger annual temperature variation. In terms of precipitation, Hainan records a long rainy season through the southwest monsoon and typhoon seasons from May until October while winter precipitation is comparably lower. Representing two agroclimatic hazards, strong typhoon winds and cold waves seriously affect Hainan. Although to be defined as "marginal tropica-1", the climatic conditions on Hainan