Fluorescent Polymerase Chain Reaction Method Research Articles

Detecting Down syndrome with a novel dual-color competitive quantitative fluorescent polymerase chain reaction method

To develop a rapid method for the detection of Down syndrome (DS) using dual-color competitive quantitative fluorescent polymerase chain reaction (DCC-QF-PCR), and to assess its feasibility for the prenatal diagnosis of Down syndrome. DNA was extracted from peripheral blood of 30 DS patients and 60 normal men, common primers for DSCR and USC2 genes and respective TaqMan probes were designed and synthesized. The results of DCC-QF-PCR were compared with those of QF-PCR which measured the ratio between DSCR and GAPDH. Forty-six amniotic fluid samples were assayed with DCC-QF-PCR. The results were compared with that of karyotyping. Monoclone fragments for DSCR and USC2 genes were obtained from direct cloning of PCR products. DCC-QF-PCR was carried out using different DNA ratios of DSCR and USC2 as the template. The dosage ratio between DSCR and USC2 was calculated. The gene dosage ratio of the DS patients was 1.41-1.74, which was significantly higher than that of normal men (0.93-1.15). The dosage ratio range of DSCR and GAPDH by QF-PCR was comparatively greater than that of DSCR and USC2. Three samples were diagnosed as DS, which was in good agreement with that of karyotyping analysis. There was no significant difference between the gene dosage ratio from DCC-QF-PCR and that of predetermined (P>0.05). DCC-QF-PCR is an accurate, rapid, and low cost method, which only requires tiny amount of sample and therefore has broad application in the genetic and prenatal diagnosis.

Detection of Parental Origin and Cell Stage Errors of a Double Nondisjunction in a Fetus by QF-PCR

To investigate parental origins and cell stage errors of a double nondisjunction in a fetus. For the determination of the most common chromosome anomalies, quantitative fluorescent polymerase chain reaction method using short tandem repeat (STR) DNA markers was applied to a fetus with abnormal ultrasonographic findings. Parental origin and cell stage errors of the trisomies were inferred by comparing the inherited STR alleles. Conventional cytogenetic technique was also applied for the confirmation of the aneuploidies. A double nondisjunction including chromosomes 21 and X (48,XXX,+21) was detected prenatally in the fetus. The origin of both chromosomes was maternal, and the errors were in meiosis I for 21 and meiosis II for X. Molecular results were concordant with cytogenetic results. Molecular techniques could be useful for the pre- and postnatal diagnosis of the common aneuploidies and determining its parental origin. This kind of study will improve knowledge about the mechanisms of nondisjunction and enable appropriate and rapid genetic counseling.

Rapid, High-Throughput Detection of Azalea Lace Bug (Hemiptera: Tingidae) Predation by <I>Chrysoperla rufilabris</I> (Neuroptera: Chrysopidae), Using Fluorescent-Polymerase Chain Reaction Primers

Azalea lace bugs, Stephanitis pyrioides (Scott) (Hemiptera: Tingidae), are the most common pest of azaleas (Rhododendron spp.) in nursery production and the landscape. Although pesticides are commonly used to control lace bugs, natural enemies can be a significant source of lace bug mortality. Lacewings (Neuroptera: Chrysopidae) are natural enemies of lace bugs and easily consume them in laboratory studies. Field studies on lacewing biocontrol of azalea lace bugs are underway; however, monitoring lacewing predation in a nursery environment by direct observation is impractical. Here, we describe a fluorescent-polymerase chain reaction method to estimate S. pyrioides consumption based on the gut contents of lacewing predators. Lace bug DNA was detected in fed lacewings up to 32 h after ingestion. More than 80% of the ingested lace bugs were detected using our method with only one false positive result. The assay is both high-throughput and relatively inexpensive, making it a practical approach to documenting lace bug predation in the field.

Detection of ΔF508del Using Quantitative Real-Time PCR, Comparison of the Results Obtained by Fluorescent PCR

Objective: Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder in the Caucasian population. The molecular diagnosis is difficult since there are about 1,000 mutations in the CF transmembrane regulator gene. The ΔF508del is the cause of the CF in 64% of the cases in Hungary. Our aim was to compare two polymerase chain reaction (PCR)-based method for the detection of ΔF508del. Methods: One hundred and sixteen DNA samples isolated from different tissues (84 blood samples, 18 chorionic villus samples and 14 amniotic fluid samples) were involved in the study. Fluorescent PCR with DNA fragment analysis and quantitative real-time PCR with melting curve analysis were performed on the DNA samples for the detection of ΔF508del. Results: Sixty-five healthy normal samples, 43 heterozygous samples, 6 ΔF508del homozygous samples and 2 ΔF508C homozygous samples were detected by using quantitative real-time PCR combined with melting curve analysis. The fluorescent PCR method did not detect the ΔF508C mutation and these two samples were diagnosed as normal healthy ones. Conclusions: The quantitative real-time PCR with melting curve analysis is a reliable and fast method for the detection of ΔF508del. The results are ready in 1 h following the DNA isolation. The applied primer-probe set with melting curve analysis gives additional information for the presence of other mutations in the ΔF508del region.

Large Pathogenic Expansions in the SCA2 and SCA7 Genes Can Be Detected by Fluorescent Repeat-Primed Polymerase Chain Reaction Assay

Large expansions in the SCA2 and SCA7 genes (>100 CAG repeats) have been associated with juvenile and infantile forms of cerebellar ataxias that cannot be detected using standard polymerase chain reaction (PCR). Here, we describe a successful application of the fluorescent short tandem repeat-primed PCR method for accurate identification of these expanded repeats. The test is robust, reliable, and inexpensive and can be used to screen large series of patients, although it cannot give a precise evaluation of the size of the expansion. This test may be of practical value in prenatal diagnoses offered to affected or pre-symptomatic at-risk parents, in which a very large expansion inherited from one of the parents can be missed in the fetus by standard PCR.