Fluorescent Polymerase Chain Reaction Amplification Research Articles

Using short tandem repeat analysis for choriocarcinoma diagnosis: a case series

BackgroundChoriocarcinoma is a highly aggressive, malignant trophoblastic neoplasm that can be gestational or non-gestational in origin. Accurate discrimination between these two subtypes, the causative pregnancy type, and the pregnancy-to-treatment interval for gestational choriocarcinoma are vital for clinical management.MethodsFifteen choriocarcinomas were genotyped using multiplex fluorescent polymerase chain reaction amplification of 15 short tandem repeat (STR) loci and the amelogenin locus (XY determination). Genotype patterns at each locus from tumoral and maternal tissues were compared, and any prior or concurrent mole/placenta was also compared when available. According to STR results showing the presence or absence of the paternal chromosomal complement, the gestational or non-gestational origin of the tumor and the nature of the causative pregnancy was identified.ResultsFourteen tumors were gestational. Of these, seven were androgenetic/homozygous XX, and two were androgenetic/heterozygous XX, indicating that the causative pregnancies were molar pregnancies. Among the nine molar pregnancies, five were of the occult type. A menopausal patient developed a tumor from a mole that occurred seven years ago, identified by the genetically identical allele from the tumor and prior mole. One tumor originating from a previous mole was interrupted by term delivery. Two tumors found eight weeks postpartum were identified as originating from a prior occult mole. A pelvic choriocarcinoma was separated from a genetically distinct third trimester intrauterine placenta. Five gestational tumors were biparental: 2 XX, 3 XY. Of three ovarian tumors, two were confirmed gestational (1 androgenetic/homozygous XX; 1 biparental XY), and one was an ovarian tumor (XX) with a complete match of the genotype for all 15 loci, therefore ascertaining its non-gestational origin.ConclusionGestational choriocarcinoma can originate in an androgenetic or biparental manner. The majority are androgenetic/homozygous XX, while a large number of them might be occult molar pregnancies. The origin of ectopic androgenetic choriocarcinoma with concurrent intrauterine placenta might be from either dispermic twin gestation (mole and coexistent nonmolar fetus) or an antecedent molar pregnancy. Choriocarcinoma shortly postpartum might not be associated with the last placenta. STR analysis can be useful in distinguishing gestational choriocarcinoma from non-gestational, as well as the causative pregnancy, and serve as a helpful examination tool for guiding clinical management.

Selective detection of viable seed-borne Acidovorax citrulli by real-time PCR with propidium monoazide.

In recent years, use of the DNA-intercalating dye propidium monoazide (PMA) in real-time PCR has been reported as a novel method to detect viable bacteria in different types of samples, such as food, environmental, and microbiological samples. In this study, viable cells of Acidovorax citrulli, the causal agent of bacterial seedling blight and fruit blotch, were selectively detected and differentiated from dead cells by real-time fluorescent polymerase chain reaction amplification after the bacterial solution was treated with the DNA-binding dye PMA. The primers and TaqMan probe were based on the A. citrulli genome (Aave_1909, Gene ID: 4669443) and were highly specific for A. citrulli. The detection threshold of this assay was 103 colony-forming units per mL (CFU/mL) in pure cell suspensions containing viable and dead cells and infected watermelon seeds. Application of this assay enables the selective detection of viable cells of A. citrulli and facilitates monitoring of the pathogen in watermelon and melon seeds.

Clonality assessment of adenomatoid tumor supports its neoplastic nature

Adenomatoid tumor is a relatively rare disease that predominantly involves male and female internal genital tracts. Although its clinical and pathologic features are well characterized, there is still controversy regarding its nature as a true neoplasm or a variant of mesothelial hyperplasia of a reactive nature. We sought to resolve this debate by investigating the clonality of uterine adenomatoid tumor from 13 female cases. The mesothelial cells and surrounding normal myometrium were precisely harvested using laser capture microdissection, and genomic DNA was extracted for clonal analysis by assessing the patterns of X-chromosome inactivation. Fluorescent polymerase chain reaction amplification of a highly polymorphic short tandem repeat of the human androgen receptor (HUMARA) gene with and without methylation-sensitive restriction endonuclease HpaII digestion was performed on DNA extracted from mesothelial cells, using normal myometrium and male blood sample as controls. Of the 13 cases successfully amplified, all 10 informative cases showed concordant nonrandom X-chromosome inactivation pattern consistent with monoclonality. In comparison, surrounding normal myometrium showed a polyclonal pattern of X-chromosome inactivation, and male blood sample failed to be amplified after HpaII treatment. Our results demonstrate that adenomatoid tumor is a monoclonal disease favoring a neoplastic process. This neoplastic rather than reactive nature probably accounts for its frequently observed infiltrative growth pattern and the occurrence of diffuse adenomatoid tumor, especially when host immunity is compromised. Additional studies with larger sample sizes will be needed to conclusively prove our conclusion.

Are there fetal stem cells in the maternal brain?

Fetal cells can enter maternal blood during pregnancy but whether they can also cross the blood-brain barrier to enter the maternal brain remains poorly understood. Previous results suggest that fetal cells are summoned to repair damage to the mother's brain. If this is confirmed, it would open up new and safer avenues of treatment for brain damage caused by strokes and neural diseases. In this study, we aimed to investigate whether a baby's stem cells can enter the maternal brain during pregnancy. Deceased patients who had at least one male offspring and no history of abortion and blood transfusion were included in this study. DNA was extracted from brain tissue samples of deceased women using standard phenol-chloroform extraction and ethanol precipitation methods. Genomic DNA was screened by quantitative fluorescent-polymerase chain reaction amplification together with short tandem repeat markers specific to the Y chromosome, and 13, 18, 21 and X. Any foreign DNA residues that could be used to interpret the presence of fetal stem cells in the maternal brain were monitored. Results indicated that fetal stem cells can not cross the blood-brain barrier to enter the maternal brain.

Relative quantitation of cell-free fetal DNA in maternal plasma using autosomal DNA markers

BackgroundCell-free fetal DNA in maternal plasma presents interest as it can possibly serve as a pregnancy associated biomarker. For its quantitation, we investigate the use of fluorescent polymerase chain reaction (PCR) amplification of short tandem repeats (STRs) in pregnancies irrespective of sex. MethodsArtificially chimeric DNA samples were prepared so as to simulate cell-free fetal DNA in maternal plasma at different proportions. The samples were tested with fluorescent PCR amplification of informative STRs and the percentage of the population simulating fetal DNA was plotted against a relative ratio of the alleles detected in each sample. The graphs were used for the quantitation of cell-free fetal DNA in 50 maternal plasma samples. ResultsDetection of at least one paternally inherited fetal STR was possible in 46/50 pregnancies leading to the estimation of the relative percentage of cell-free fetal DNA. Four pregnancies failed to reveal any fetal allele which may reflect undetectable levels (<1%) of fetal DNA. Among pregnancies fetal DNA ranged from 0 to 20% (mean value of 7%). ConclusionOur system can readily estimate the relative levels of cell-free fetal DNA in maternal circulation, normal gestations of either sex and can be exploited for estimating possible variations of this analyte between normal and pathological pregnancies.

Establishment of real-time fluorescent PCR assay to detectStreptococcus suisserotype 2

AbstractA real-time fluorescent polymerase chain reaction (PCR) assay was established to detectStreptococcus suisserotype 2. Primers and Taqman probe were designed according tocps2I(capsular polysaccharide 2I) gene using bio-software Primer Express2.0 and Oligo6.0. An 81 bp DNA fragment was amplified fromS. suisserotype 2 genomic DNA, and the PCR product was cloned into pMD18-T vector and confirmed by DNA sequencing. The real-time fluorescent PCR amplification curve on a Lightcycler® showed that the method is accurate and specific forS. suisserotype 2 amplification, whereas reference bacteriaS. suis,Escherichia coli,Salmonellasp.,Staphylococcus aureus,Shigellasp.,Listeria monocytogenesstrains and a blank control were all negative. Tenfold serial dilutions ofS. suisserotype 2 were used to measure the sensitivity of real-time fluorescent PCR: ten copies of bacteria could be detected in one PCR reaction and only 30 min were required for a single test. To examine the stability of the real-time fluorescent PCR, the positive control was detected at two different times. The threshold cycle (Ct) values showed no statistical differences (P&gt;0.05). Thus, this method was stable and repeatable. These results indicate that this real-time fluorescent PCR technique could be applied for epidemic supervision in entry–exit inspection and quarantine.

Detection of maternal deoxyribonucleic acid in umbilical cord plasma by using fluorescent polymerase chain reaction amplification of short tandem repeat sequences

Objective: Umbilical cord blood is a source of hematopoietic stem cells for transplantation. Although the first clinical applications have been encouraging, concern has been raised about contamination of umbilical blood by maternal cells, which might constitute a theoretical risk of graft-versus-host disease. The aim of this study was to assess the frequency of maternal deoxyribonucleic acid (DNA) contamination in umbilical cord plasma by using fluorescent polymerase chain reaction amplification of highly polymorphic short tandem repeat DNA markers. Study Design: Fifty-seven mother/child pairs were tested for the presence of maternal DNA sequences in cord plasma. After delivery, cord blood samples were collected via gravity. Maternal specific alleles were detected by using polymerase chain reaction amplification of 9 highly polymorphic short tandem repeat markers (D21S11, D21S1411, D21S1412, D18S386, D18S535, MBP-A, MBP-B, D13S631, and D13S634). Results: All 57 mother-child pairs were informative for the identification of uniquely maternal alleles in at least 2 of 9 different short tandem repeat markers used per case. Uniquely maternal DNA sequences were found in 43 of 57 (75%) cord plasma samples. Conclusion: The results of our study demonstrate that maternal DNA is present in the majority of umbilical cord blood plasma samples. The technique described herein might have application in the screening of umbilical cord blood samples for the presence of contaminating maternal genetic material. (Am J Obstet Gynecol 2002;186:117-20.)

Female fetal cells in maternal blood: use of DNA polymorphisms to prove origin.

The nucleated erythrocyte (NRBC) is one of the target fetal cell types for noninvasive genetic diagnosis using maternal peripheral blood. However, it is now known that pregnancy can stimulate the production of maternal NRBCs. When isolating female gamma-positive NRBCs, fluorescence in situ hybridization (FISH) analysis may show two X chromosome signals per nucleus, and therefore it cannot be conclusively determined whether the isolated cells are fetal or maternal in origin. The purpose of this study was to develop a means of verifying that a female cell is fetal on the basis of polymorphic short tandem repeat markers. Peripheral blood samples were obtained from women who had just undergone termination of pregnancy. Nucleated candidate fetal cells were isolated by flow-sorting using antibody to the gamma-chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed using X and Y chromosome specific probes. Female gamma-positive cells and leukocytes were micromanipulated separately and subjected to fluorescent polymerase chain reaction amplification of chromosome 21 and/or 18 STR markers (D21S11, D21S1411, D21S1412, and D18S535). In all ten cases analyzed, the gamma-positive female candidate fetal cells were determined to be fetal in origin by the presence of shared and nonshared DNA polymorphisms when compared with maternal leukocytes. These results show that genetic analysis can be performed on all fetal NRBCs, including female fetal cells that cannot be distinguished from maternal cells based on FISH analysis alone.

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats.

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats

Comparative semi-automated analysis of (CAG) repeats in the Huntington disease gene: use of internal standards

Huntington disease (HD) belongs to the group of neurodegenerative disorders characterized by unstable expanded trinucleotide repeats. In the case of HD, the expansion of a CAG repeat occurs in the IT15 gene. The detection of the expanded CAG repeats has usually involved the electrophoretic separation of polymerase chain reaction (PCR) amplification products using conventional agarose and acrylamide gel electrophoresis. We have undertaken the comparative analysis of sizing CAG repeats of the IT15 gene using radioactive and fluorescent PCR amplification, and the subsequent separation of these products by slab gel and capillary electrophoresis. The assays have been performed on both cloned and sequenced CAG repeats, as well as genomic DNA from HD patients with a wide range of repeat lengths. The mobility of the CAG repeat amplification products of the IT15 gene is greater using capillary electrophoresis compared to slab gel electrophoresis. The analysis of 40 DNA samples from HD patients indicates that the mobility difference increases with the length of the repeat. However, we have devised an allele ladder for sizing the CAG repeats. This ladder provides a mandatory internal calibration system for diagnostic purposes and enables the confident use of either capillary or slab gel electrophoresis for sizing HD alleles.

Prenatal detection of trisomy 13 from amniotic fluid by quantitative fluorescent polymerase chain reaction

Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis from amniotic fluid. However, this requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnant women. An alternative method, which is rapid, inexpensive and suitable for diagnosing trisomies, even from single fetal cells, is the fluorescent polymerase chain reaction (PCR) using polymorphic small tandem repeats (STRs). In this paper, we present the method of rapid prenatal detection of trisomy 13 from amniotic fluid using fluorescent PCR and two highly polymorphic STRs (D13S258 and D13S631). The results obtained by quantitative fluorescent PCR amplification of fetal DNA were concordant with amniocyte karyotyping results in all cases. Two cases of trisomy 13 were detected from 212 amniotic fluids and the results obtained from D13S631 and D13S258 amplification are presented. In the first trisomy 13 case, a triallelic pattern was detected by both markers, and in the second case, D13 markers showed a characteristic 2:1 dosage allele ratio, both of which demonstrate trisomy 13 status. All other heterozygous disomic samples showed an allele intensity ratio of 1:1.

Rapid detection of trisomies 21 and 18 and sexing by quantitative fluorescent multiplex PCR.

Aneuploidies involving chromosomes 21, 18, 13, X and Y account for over 95% of all chromosomal abnormalities in live-born infants. Prenatal diagnosis of these disorders is usually accomplished by cytogenetic analysis of amniotic or chorionic cells but this is a lengthy procedure requiring great technical expertise. In this paper, we assess the diagnostic value of using a quantitative fluorescent polymerase chain reaction (PCR) suitable for the simultaneous and rapid diagnosis of trisomies 21 and 18 together with the detection of DNA sequences derived from the X and Y chromosomes. Samples of DNA, extracted from amniotic fluid, fetal blood or tissues, and peripheral blood from normal adults were investigated by quantitative fluorescent PCR amplification of polymorphic small tandem repeats (STRs) specific for two loci on each of chromosomes 21 and 18. Quantitative analysis of the amplification products allowed the diagnosis of trisomies 21 and 18, while sexing was performed simultaneously using PCR amplification of DNA sequences derived from the chromosomes X and Y. These results indicate the advantages of using two sets of STR markers for the detection of chromosome 21 trisomies and confirmed the usefulness of quantitative fluorescent multiplex PCR for the rapid prenatal diagnosis of selected chromosomal abnormalities.