Semi-quantitative Polymerase Chain Reaction Technique Research Articles

Impact of Host Plant Resistance on the Tritrophic Interactions Between Wheat Genotypes,Schizaphis graminum(Homoptera: Aphididae), andCoccinella septempunctata(Coleoptera: Coccinellidae) Using Molecular Methods

The objective of the current study was to evaluate the impact of wheat resistance on digestibility of Schizaphis graminum (Rondani) in the Coccinella septempunctata L. gut. Four wheat genotypes including an ancient wheat species (Einkorn) and three Iranian wheat genotypes (Azadi, Ommid, and Moghan2) were used. Einkorn was found to be a highly resistant species, while Azadi, Ommid, and Moghan2 were characterized as resistant, semiresistant, and susceptible genotypes, respectively, based on some biological parameters of S. graminum on these plants. Adult C. septempunctata starved for 24 h were fed one S. graminum reared on the selected wheat genotypes. A semiquantitative polymerase chain reaction technique using specific primers for the gene cytochrome oxidase II (COII) of S. graminum showed a significant effect of plant resistance on S. graminum DNA disappearance time. The DNA half-lives of S. graminum fed on Einkorn, Azadi, Ommid, and Moghan2 in C. septempunctata gut was 3.3, 3.6, 4.8, and 6.7 h, respectively. The findings suggest that digestibility of S. graminum fed on resistant plant genotypes is higher than those fed on susceptible plants because of the lighter weights and smaller sizes of the first. This phenomenon could result in faster ingestion of S. graminum by C. septempunctata on these resistant plants. It is also possible that some metabolites found differentially in Einkorn and Azadi genotypes may have affected S. graminum quality as a food for C. septempunctata.

Alteration of intestinal intraepithelial lymphocytes after massive small bowel resection

Background. Intraepithelial lymphocytes (IEL) comprise the inner most layer of the gut immune system, and play a critical role in protecting the host from enteric organisms. Massive small bowel resection (MSBR) is one such clinical condition where patients are at particularly high risk for the development of such enteric infectious complications. Because of this, we hypothesized that the IEL may change significantly after the formation of a MSBR. To address this, a mouse model of MSBR was created and the acute phenotypic and functional characteristics of the IEL were studied. Materials and methods. Mice underwent a 70% mid-small bowel resection. After 7 days, IEL were isolated and analyzed for phenotypic changes by flow cytometry. IEL cytokine expression was performed with semiquantitative polymerase chain reaction techniques. To assess the functional significance of these changes, IEL proliferative response was assessed in vitro. Results. MSBR led to significant decreases in specific IEL subpopulations: CD 44+ (used as a marker of cell maturity); CD 8αβ+ (marker of thymic derivation), and CD 69+ (marker of T cell activation). Compared with controls, IEL TNF-α mRNA expression increased 84%, while IL-2 and IL-10 mRNA expression decreased by 69 and 72%, respectively. Spontaneous proliferation of IEL in the MSBR group was significantly higher than controls, however, proliferation failed to increase with T cell stimulation. Conclusion. These changes suggest a shift to a more immature and possibly less activated cell population. It is possible that such alterations may play an important role in the increase in enterically derived infections in patients with MSBR.

Singlet Oxygen Mediates the UVA-induced Generation of the Photoaging-associated Mitochondrial Common Deletion

Mutations of mitochondrial (mt) DNA accumulate during normal aging. The most frequent mutation is a 4,977-base pair deletion also called the common deletion, which is increased in photoaged skin. Oxidative stress may play a major role in the generation of large scale mtDNA deletions, but direct proof for this has been elusive. We therefore assessed whether the common deletion can be generated in vitro through UV irradiation and whether reactive oxygen species are involved in this process. Normal human fibroblasts were repetitively exposed to sublethal doses of UVA radiation and assayed for the common deletion employing a semiquantitative polymerase chain reaction technique. There was a time/dose-dependent generation of the common deletion, attributable to the generation of singlet oxygen, since the common deletion was diminished when irradiating in the presence of singlet oxygen quenchers, but increased when enhancing singlet oxygen half-life by deuterium oxide. The induction of the common deletion by UVA irradiation was mimicked by treatment of unirradiated cells with singlet oxygen produced by the thermodecomposition of an endoperoxide. These studies provide evidence for the involvement of reactive oxygen species in the generation of aging-associated mtDNA lesions in human cells and indicate a previously unrecognized role of singlet oxygen in photoaging of human skin.

Development and Application of an Improved Semiquantitative Technique for Detecting Low-Level Cryptosporidium parvum Infections in Mouse Tissue Using Polymerase Chain Reaction

An improved semiquantitative technique was developed for measuring low infectious doses of Cryptosporidium parvum in neonatal mice using polymerase chain reaction (PCR). Separate litters of neonatal mice were inoculated with 0, 10(2), 10(3), or 10(4) C. parvum oocysts and killed 96 hr postinfection. A segment of the ileum or the entire whole intestine was then removed from subgroups of mice in each litter and total DNA was extracted using standard procedures. By employing a CP15/60-based semiquantitative PCR technique, C. parvum DNA was detected in mice infected with as few as 10(2) oocysts. DNA isolated from the ileum of infected mice produced a more intense PCR signal than DNA isolated from the whole intestine. This technique was used to study the intracellular development of C. parvum sporozoites that had been exposed in the oocyst stage to either 0, 15, 20, 25, or 30 kRad gamma-irradiation. A CP15/60 PCR signal was observed in ileum tissue from mice infected with 0-kRad- or 15-kRad-irradiated C. parvum oocysts. A very slight PCR signal was generated by PCR on ileum tissue DNA from mice infected with 20-kRad-irradiated oocysts, whereas no signal was observed in PCR on intestinal DNA from mice infected with oocysts exposed to higher radiation doses.

A semi-quantitative method for measuring Cryptosporidiumparvum infection using polymerase chain reaction

A semi-quantitative method for measuring Cryptosporidium parvum infection in mice using polymerase chain reaction (PCR) was developed for evaluating therapeutic reagents against cryptosporidiosis. A competitor molecule composed of CP15/60 primer-binding sequences flanking an unrelated sequence was synthesized and used in PCR to amplify competitor and target CP15/60 sequences. For estimating relative intestinal parasite numbers, seven-day-old BALB/C mice were infected with 0, 10 2, 10 3, or 10 4 C. parvum oocysts. At 24, 48, 72, and 96 h post-infection, three mice per group were killed and the intestine from each mouse was processed for DNA. Semi-quantitative PCR using serial dilutions of competitor molecule and a constant amount of mouse tissue DNA showed that challenge doses of 10 3 and 10 4 oocysts could be easily detected at both 72 and 96 h post-infection. CP15/60-specific PCR products were not observed at earlier timepoints (24 and 48 h) nor at any timepoint with the 0 or 10 2 challenge doses. The semi-quantitative PCR technique proved to be more sensitive, rapid, and cost-effective compared to conventional histological scoring of cryptosporidial stages in tissue sections from C. parvum-infected mice. A rapid method for extracting DNA from infected mouse intestine was developed and, when used in the semi-quantitative PCR, proved to be as reproducible as conventional DNA extraction methods.

A frequent mutation/polymorphism in tumor suppressor gene INK4B (MTS-2) in papillary and medullary thyroid cancer

Structural genetic changes of tumor suppressor genes MTS-1/INK4A and MTS-2/INK4B were demonstrated in a variety of human cancers but not in thyroid cancer until now. Because MTS-2 encodes the tumor suppressor p15, a protein related to the transforming growth factor-beta inhibition of many epithelial cells such as thyrocytes, we investigated MTS-1 and MTS-2 genes in 87 thyroid cancers (29 papillary, 26 follicular, 31 medullary, and 1 anaplastic), 8 goiters, and 38 control DNAs by using a semiquantitative polymerase chain reaction technique. We failed to demonstrate homozygous deletions of MTS-1 and MTS-2 in thyroid tumors, but we demonstrated a highly frequent base pair exchange of the MTS-2 gene 27 codons upstream the 5' end of exon 2. This genetic change formerly described as polymorphism was found to a lesser degree (15%), in control DNA when compared with papillary thyroid cancer and medullary thyroid cancer (35% and 32%, respectively), and it paralleled a higher prevalence of extensive lymph node metastases in thyroid cancer (p < 0.01). In addition, we could demonstrate that genetic changes at site 27 upstream the 5' end of exon 2 were harbored as somatic mutations in 2 of 10 thyroid cancers with simultaneously investigated corresponding control tissue. We conclude that base pair exchange at this site most likely has biologic importance for the tumor suppressor p15 and may contribute to tumorigenesis and lymphatic spread of differentiated and medullary thyroid cancer.

Analysis of the peripheral T-cell receptor V beta repertoire in newly diagnosed patients with type I diabetes.

Type I diabetes is an autoimmune disease characterised by a marked activation of peripheral T cells around the time of clinical diagnosis. Studies of T-cell antigen receptor V beta (TCRBV) gene usage in type I diabetes have been conflicting. Using a semi-quantitative polymerase chain reaction technique and flow cytometry we have investigated the TCRBV gene usage of 13 newly diagnosed patients with type I diabetes and 11 normal healthy controls. No preferential TCRBV gene usage was found between patients and controls even after matching for HLA-DR3 and/or -DR4. In addition, no significant differences in TCRBV gene usage were found between sequential samples taken over a period of up to 7 months following diagnosis. These results suggest that the TCR repertoire of these patients is heterogeneous and it is unlikely that a single 'pathogenic' T-cell clone is dominant at the clinical onset of the disease.

Polymerase chain reaction detection of human cytomegalovirus in over 2000 blood specimens correlated with virus isolation and related to urinary virus excretion

The polymerase chain reaction (PCR) as applied to human cytomegalovirus (HCMV) detection should provide a valuable tool for rapid, reliable diagnosis of infection, thereby allowing prompt treatment. However, to date the high sensitivity of this technique and the lack of semi-quantitative interpretation have hindered establishing its validity for diagnosing systemic infection. We describe a rapid, simple, semi-quantative PCR technique for HCMV detection. The validity of the technique was tested objectively by analyzing over 2000 leukocytes specimens by PCR and comparing the results with virus isolation from urine and blood in concomitant samples in the absence of any clinical data. It could thus be established that this technique had a sensitivity and specificity of 97%. When the PCR signal corresponded to ⩾ 8000 genome equivalents for 10 4 leukocytes, the predictive value for viremia was 86%. This semi-quantitative PCR technique should allow rapid diagnosis of systemic infection and provide a reliable means of monitoring clearance of CMV from blood during drug therapy.

Definition of human papillomavirus type 16 DNA levels in low and high grade cervical lesions by a simple polymerase chain reaction technique.

Human papillomavirus type 16 (HPV 16) is associated with a high proportion of cervical cancers and pre-cancers but has also been reported by some workers to be widely distributed in the normal population. Using a semi-quantitative polymerase chain reaction technique (PCR) operated with carefully regulated sensitivity we have established two distinct levels of HPV 16 DNA which distinguish between high and low grade cervical lesions. The potential use of such an approach in the understanding and management of HPV related cervical disease is discussed.