Specific Oligonucleotide Primers Research Articles

Rapid PCR-based delineation of the porcine nodular worms,Oesophagostomum dentatumandO. quadrispinulatum

At some stages of development, it is impossible to identify the porcine nodular wormsOesophagostomum dentatumandO. quadrispinulatumto the species level using morphological parameters. A molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed to overcome this limitation. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR procedures were developed for the specific amplification of DNA ofO. dentatumorO. quadrispinulatum, which are now used routinely to monitor the purity of larval cultures and to confirm the identity of larvae derived from the intestine or faeces. The application of specific PCR has major implications for studying the population biology of nodular worms in the pig model.

Development of a sensitive nested PCR method for the specific detection ofMycoplasma mycoidessubsp.mycoidesSC

A specific and sensitive test for the detection ofMycoplasma mycoidessubsp.mycoidessmall colony type (SC), the aetiological agent of contagious bovine pleuropneumonia (CBPP) was developed using two nested PCR reactions. The PCR reactions are based on the nucleotide sequence of lipoprotein P72 ofM. mycoidessubsp.mycoidesSC. The two specific oligonucleotide primer pairs were chosen to match those sequence segments of the P72 gene which differ most from the gene of the closely related lipoprotein P67 ofMycoplasmasp. bovine group 7 (strain PG50). The nested PCR reacted with all of the 34 different strains ofM. mycoidessubsp.mycoidesSC analysed, and gave no amplification product with any of the closely related mycoplasmas tested, showing its high specificity. In bronchial lavage fluid experimentally contaminated withM. mycoidessubsp.mycoidesSC, the assay was able to detect as few as two viable cells per ml using a simple lysis procedure prior to the amplification step. With clinical samples, the sensitivity of the nested PCR was about 104–105higher than that of single PCR amplifications performed under the same conditions. The assay was also successfully used to detectM. mycoidessubsp.mycoidesSC in bronchial lavage fluid of experimentally infected cattle and proved to be more sensitive than classical culture methods.

Rapid characterization of human chromosomes in hybrid cell lines by primed in situ (PRINS) labeling.

The primed in situ (PRINS) labeling technique allows a rapid and specific labeling of human chromosomes in situ. This method is based on annealing of specific oligonucleotide primers and subsequent primer extension by a Taq DNA polymerase. We have developed a PRINS protocole for the cytogenetic analysis of somatic hybrid cell lines. Painting of human chromosomes is performed using Alu specific primers. Individual human chromosomes are identified using chromosome-specific alpha-satellite primers. The method was successfully tested to 3 different human-hamster hybrid cell lines. This approach provides an interesting alternative to classical cytogenetic and in situ hybridization techniques for the characterization of the human content of hybrid cell lines.

β-Adrenergic receptor subtype gene expression in timed-pregnant rat myometrium

OBJECTIVE: Classic radioligand binding techniques have suggested that β 1- and β 2-adrenergic receptor subtype proteins are expressed in myometrial tissue; however, to date these observations have not been confirmed at the level of the messenger ribonucleic acid for these clinically important membrane receptors. The studies described in this report sought to use quantitative reverse transcriptase–polymerase chain reaction techniques to confirm expression of messenger ribonucleic acid for the β 1- and β 2-adrenergic receptors in myometrial tissue and to determine whether messenger ribonucleic acid expression for these two adrenergic receptors is modulated during pregnancy. STUDY DESIGN: For these studies total cellular ribonucleic acid was isolated from myometrial tissue obtained from timed-pregnant Sprague-Dawley rats by the guanidium thiocyanate–phenol–chloroform extraction technique; formaldehyde-agarose gels then confirmed isolation of intact ribonucleic acid. Random hexamer primers and reverse transcriptase were used to synthesize complementary deoxyribonucleic acid. Subsequently, polymerase chain reaction was performed with subtype specific 20-mer sense and antisense oligonucleotide primers specific for the rat β 1- and β 2-adrenergic receptors. Inclusion of internal standard deoxyribonucleic acid sequences allowed quantification of the reverse transcriptase–polymerase chain reaction results. RESULTS: By use of total cellular ribonucleic acid isolated from myometrial tissue, reverse transcriptase–polymerase chain reaction generated the expected 328 bp product for the β 1-receptor and the expected 559 bp product for the β 2-receptor along with internal standard deoxyribonucleic acid sequences for both. The identity of the β 1- and β 2-adrenergic receptor polymerase chain reaction products was confirmed on the basis of restriction endonuclease digestions producing the expected deoxyribonucleic acid fragments and by Southern blots using β 1- and β 2-adrenergic receptor–specific complementary deoxyribonucleic acid probes. The reverse transcriptase–polymerase chain reaction studies confirmed a gradual decline in β l-receptor messenger ribonucleic acid and stable expression of β 2-receptor messenger ribonucleic acid during the second half of gestation in pregnant rat myometrial tissue. CONCLUSIONS: In summary, these studies have confirmed, at the messenger ribonucleic acid level, expression of the β 1- and β 2-adrenergic receptor subtypes in timed-pregnant rat myometrial tissue.(Am J Obstet Gynecol 1997;176:349-57.)

Conserved nucleotide sequence of NS5A in hepatitis G virus or GB virus C in Japan and China

A semi-nested PCR method was designed for detecting the NS5A region of hepatitis G virus (HGV) or GB virus C (GBV-C) using specific oligonucleotide primers. Patients, 14 of 69, suffering from hepatic diseases in Japan and 5 of 21 in China showed strong positive signals for HGV or GBV-C by this method. The PCR products were directly sequenced. The nucleotide sequences identified differed from one another by 0–5%, from HGV by 3–9% and GBV-C by 2–7%, the percentages of the latter two being compared with the sequences reported previously. Amino acid sequences were the same or had only one alteration compared with HGV and two to three alterations compared with GBV-C. These results indicate that the nucleotide sequence of NS5A of HGV (or GBV-C) is more conserved than that of NS3 of GBV-C (or HGV) which has been reported to show variations of 0–23%.

5494795 Specific oligonucleotide primers for detection of pathogenic Campylobacter bacteria by polymerase chain reaction : Guerry Patricia; Trust Trevor J Rockville, MD, United States assigned to The United States of America as represented by the Secretary of the Navy

5494795 Specific oligonucleotide primers for detection of pathogenic Campylobacter bacteria by polymerase chain reaction : Guerry Patricia; Trust Trevor J Rockville, MD, United States assigned to The United States of America as represented by the Secretary of the Navy

Differential gene expression of transforming growth factors alpha and beta, epidermal growth factor, keratinocyte growth factor and their receptors in fetal and adult human prostatic tissues and cancer cell lines

Objectives Recent studies have shown that growth factors may play a role in the etiology of benign prostatic hyperplasia (BPH) and prostatic carcinoma. Several growth factors have been reported to be expressed by prostatic tissues, but these growth factors have never been examined in human fetal prostate and compared with adult prostates and cancer cell lines. The present study was designed to investigate the messenger ribonucleic acid (mRNA) expression of transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, TGFbeta3, keratinocyte growth factor (KGF), epidermal growth factor (EGF), EGF receptor (EGF-R), and KGF receptor (KGF-R) in human fetal and adult prostatic tissues and cancer cell lines by reverse-transcriptasepolymerase chain reaction (RT-PCR) using specific oligonucleotide primers. Methods Total RNA was extracted from human fetal and adult prostates (BPH tissues) and cancer cell lines. The gene expression of these growth factors and their receptors was determined by RT-PCR using specific oligonucleotide primers. Conclusions These results suggest that the differential gene expression for various growth factors and their receptors in human fetal and adult prostatic tissues and cancer cell lines may be important in understanding the role of these factors in the pathophysiology of prostatic diseases.

Expression of mRNA encoding intestinal type alkaline phosphatase in rat liver and its increase by fat-feeding.

The presence of types of alkaline phosphatase (ALP) other than the tissue non-specific type enzyme in rat liver and its increase by fat feeding are known. In order to examine expression of intestinal type ALP in liver, specific oligonucleotide primers corresponding to two types of mRNAs of rat intestinal ALP (RTIN-1 and -2) were designed and amplified by means of the reverse transcriptase-polymerase chain reaction (RT-PCR). It was found that RTIN-1 mRNA was expressed only in the intestine but not in the liver, while RTIN-2 mRNA was expressed both in the intestine and in the liver. By fat feeding, expression of RTIN-1 mRNA increased in the intestine and that of RTIN-2 mRNA increased both in the intestine and in the liver. Thus, it was concluded that rat liver expressed one of the intestinal type ALP (RTIN-2) which was enhanced by fat feeding.

Inflammatory cytokine mRNA in human gestational tissues: Implications for term and preterm labor

To determine if inflammatory cytokine mRNA in gestational tissues is present only in the setting of infection-associated preterm labor or under several other clinical conditions. Human gestational tissues were collected from 51 women experiencing 1) term cesarean delivery without labor, 2) normal term vaginal delivery, 3) preterm cesarean delivery without labor, 4) preterm vaginal delivery without chorioamnionitis, and 5) preterm vaginal delivery with concomitant chorioamnionitis. Decidua, chorion, and amnion were isolated, total RNA from each tissue was extracted, and the presence of inflammatory cytokine mRNA was determined by polymerase chain reaction. Interleukin (IL)-1 beta, IL-6, IL-8, and tumor necrosis factor-alpha mRNA was detected using specific oligonucleotide primers. Interleukin-1 beta mRNA was rarely found in tissues preterm without labor but was readily detected in both maternal and fetal tissues after labor, regardless of gestational age. Interleukin-6 mRNA was rare in tissues from the nonlaboring patient but was found in almost all tissues after labor. Interleukin-8 mRNA was detected in all tissues at term, both in nonlaboring and laboring patients. Tumor necrosis factor-alpha mRNA was detected in only 20-50% of tissues after labor, and was rarely detected in the absence of labor. Inflammatory cytokine mRNA is commonly expressed in human gestational tissues after normal labor and preterm labor with or without associated intrauterine infection. There was no difference in the pattern of expression of mRNA inflammatory cytokine in women who did or did not have clinically evident intrauterine infection.

PCR‐based assay for detecting IB‐genes for low molecular weight glutenin subunits related to gluten quality properties in durum wheat

A PCR assay has been developed for selecting low molecular weight (LMW) glutenin genes, located on chromosome 1B, and related to durum wheat quality characteristics. Most durum wheat lines possessing good technological properties can be identified on the basis of the presence of specific 1B LMW glutenin components that have been designated LMW‐2 glutenin subunits. On the basis of nucleotide sequences corresponding to LMW glutenin genes, a series of specific oligonucleotide primers were prepared and used in PCR analysis. A pair of these primers gave a single amplification product which can distinguish between durum wheat lines possessing LMW‐2 glutenin subunits and lines possessing other 1B‐LMW allelic variants. Because the proposed PCR analysis can be carried out using part of the endosperm of a single seed, it represents a helpful approach for speeding up the selection of genotypes possessing LMW‐2 glutenin subunits. This part of primers could also be very useful in genome mapping analysis and for testing the purity of wheat flour stock.

Specific oligonucleotide primers for the identification of Pseudomonas syringaepv. pisiyield one of two possible DNA fragments by PCR amplification: evidence for phylogenetic divergence

Two unique DNA fragments, generated by RAPD-PCR, were used as probes against dot-blots of representative isolates of the seven races of Pseudomonas syringaepv. pisi. DNA from each isolate hybridized only to one of the two probes. Fragments identified from isolates 1691 (race 7) and 203 (race 2), were cloned into pUC18 and sequenced. The resulting sequences were used to design two pairs of oligonucleotide primers which when used in PCR reactions with P. syringaepv. pisicells gave specific amplification products (either a 272 bp or a 132 bp fragment) corresponding to the original cloned fragments. When all four primers were used in combination with specific DNA amplification reactions in 51 isolates of P. syringaepv. pisithey produced one of the two PCR bands. No bands were detected in a range of closely related P. syringaepathovars following PCR amplification. These results suggest that P. syringaepv. pisican be unambiguously identified using specific oligonucleotide primers and that isolates can be classified into two phylogenetic groups, I and II.

Human placenta and fetal membranes express human urocortin mRNA and peptide.

Recently a new member of the corticotropin-releasing factor (CRF) family has been cloned and named urocortin. It has been localized in rat brain and to human chromosome 2. The present study investigated whether human placenta and related tissues express mRNA and immunoreactive urocortin. Using specific oligonucleotide primers, reverse transcriptase-polymerase chain reaction experiments were performed on total RNA isolated from human placenta and decidua collected both at early stage of gestation (8-11 weeks) and at term (39-40 weeks). In addition, experiments were also done on specimens of amnion and chorion collected at term. Independently from the gestational age, placental and decidual cells expressed urocortin mRNA, with a 145 bp DNA band corresponding to the expected length. The expression of urocortin mRNA was also found in amnion and chorion. Using specific antiserum and an immunoperoxidase technique, immunoreactive urocortin was then localized in syncytiotrophoblast cells and in some extent in cytotrophoblast cells of placental villi at term, as well as in fetal membranes and maternal decidua. The present findings revealed that human placenta and gestational related tissues express human urocortin gene and localize immunoreactive urocortin, supporting the concept that these tissues are capable of expressing a large number of neuroendocrine peptides.

Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

PCR-mediated detection of the chemolithotrophic bacterium Thiobacillus cuprinus using 23S rDNA- and 16S/23S intergenic spacer region-targeted oligonucleotide primers

Bioleaching is carried out by chemolithotrophic microorganisms, most of them belonging to the genera Thiobacillus and Leptospirillum. The role of the mixotrophic species T. cuprinus in this process is controversial, since its ecological study applying classical detection techniques to natural or industrial environments is very difficult. For this reason, we have developed an alternative method based on PCR-mediated detection using specific oligonucleotide primers that target variable regions of the 23S rRNA coding gene and of the 16S/23S intergenic spacer region. Specificity and sensitivity of PCR amplifications performed with both kinds of primers were studied.

PCR-mediated detection of the chemolithotrophic bacterium Thiobacillus cuprinus using 23S rDNA- and 16S/23S intergenic spacer region-targeted oligonucleotide primers.

Bioleaching is carried out by chemolithotrophic microorganisms, most of them belonging to the genera Thiobacillus and Leptospirillum. The role of the mixotrophic species T. cuprinus in this process is controversial, since its ecological study applying classical detection techniques to natural or industrial environments is very difficult. For this reason, we have developed an alternative method based on PCR-mediated detection using specific oligonucleotide primers that target variable regions of the 23S rRNA coding gene and of the 16S/23S intergenic spacer region. Specificity and sensitivity of PCR amplifications performed with both kinds of primers were studied.

Structural characterization and fine chromosomal mapping of the human P2Y1 purinergic receptor gene (P2RY1).

Using P2Y1 specific oligonucleotide primers in a Polymerase Chain Reaction on human genomic DNA, we have amplified a region encoding the P2Y1 receptor Restriction analysis and Southern hybridization of the PCR product revealed that the entire open reading frame of the human P2Y1 receptor is coded by an intronless gene. We have previously localized the P2Y1 receptor gene to human chromosome 3. The gene was further localized to a region of chromosome 3 using a subchromosomal hybrid panel containing different segments of chromosome 3. Based on the specific PCR product obtained and its Southern hybridization to the human P2Y1 receptor cRNA, the P2Y1 receptor gene was mapped to human chromosome 3q25.

Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their gpd (glyceraldehyde-3-phosphate dehydrogenase) genes.

Degenerated oligonucleotide primers designed to flank an approximately 1.2-kb fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from ascomycetes and basidiomycetes were used to amplify the corresponding gpd fragments from several species of the ectomycorrhizal fungal taxa Boletus, Amanita, and Lactarius. Those from B. edulis, A. muscaria, and L. deterrimus were cloned and sequenced. The respective nucleotide sequences of these gene fragments showed a moderate degree of similarity (72 to 76%) in the protein-encoding regions and only a low degree of similarity in the introns (56 to 66%). Introns, where present, occurred at conserved positions, but the respective positions and numbers of introns in a given taxon varied. The amplified fragment from a given taxon could be distinguished from that of others by both restriction nuclease cleavage analysis and Southern hybridization. A procedure for labeling DNA probes with fluorescein-12-dUTP by PCR was developed. These probes were used in a nonradioactive hybridization assay, with which the gene could be detected in 2 ng of chromosomal DNA of L. deterrimus on slot blots. Taxon-specific amplification was achieved by the design of specific oligonucleotide primers. The application of the gpd gene for the identification of mycorrhizal fungi under field conditions was demonstrated, with Picea abies (spruce) mycorrhizal roots harvested from a northern alpine forest area as well as from a plant-breeding nursery. The interference by inhibitory substances, which sometimes occurred in the DNA extracted from the root-fungus mixture, could be overcome by using very diluted concentrations of template DNA for a first round of PCR amplification followed by a second round with nested oligonucleotide primers. We conclude that gpd can be used to detect ectomycorrhizal fungi during symbiotic interaction.

The isolation of peste des petits ruminants virus from Northern India

The aetiological agent responsible for an epizootic of a rinderpest-like disease afflicting sheep and goats in three states of northern India was confirmed as peste des petits ruminants virus. To differentiate the virus from rinderpest a number of diagnostic tests were used, including immunocapture ELISA, specific oligonucleotide primers in a reverse transcriptase polymerase chain reaction, immunofluorescence with virus specific monoclonal antibodies and virus isolation. The virulence profile of one isolated in cattle sheep and goats was established. Infected animals developed specific antibody responses and excreted specific antigen in their lachrymal secretions.

The complete nucleotide sequence of yam mosaic virus (Ivory Coast isolate) genomic RNA.

The complete nucleotidic sequence of the yam mosaic virus (YMV) RNA was determined following the cloning of partial segments of the genome by reverse transcription and polymerase chain reactions (RT-PCR) using degenerate and/or specific oligonucleotide primers. YMV genomic RNA is 9,608 nucleotides in length and contains one open reading frame (ORF) encoding a polyprotein of 3,103 amino acids (aa) with a calculated Mr of 350,915. The 5' leader sequence of YMV RNA preceding the ORF is 134 nucleotides (nt) long while the 3' untranslated region (UTR) is 165 nt excluding the poly(A) tail. A computer algorithm predicted that the 3'UTR forms four stem loop structures which form a cloverleaf-like secondary structure. These structures apparently share some homologies with those observed in the 3'UTR of the potato virus Y-NL1 strain. Seven potential recognition sites for the NIa protease were found: one putative cleavage site for the P1 proteinase and one for the HC proteinase. The organization of the YMV genome is therefore similar to the other members of the genus Potyvirus based upon conserved sequence motifs common amongst members of this group. Despite its similarity with the other potyviruses in these conserved regions, YMV appears to be a distinct potyvirus species based upon a comparison of its sequence with those of other potyviruses.

Expression and function of membrane regulators of complement on rat astrocytes in culture.

Human astrocytes express CD59, decay accelerating factor and membrane cofactor protein to restrict the damaging effect of complement (C) activation on their cell surface. 5I2 antigen (5I2 Ag) is the functional analogue of the latter two proteins in rats. We here demonstrate the surface expression on rat astrocytes of CD59 and 5I2 Ag and use sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting to confirm their identity and to quantify expression. Rat CD59 (MW 20,000) was expressed at 720 x 10(3) molecules per cell and 5I2 Ag (MW 58,000 and 64,000) at 625 x 10(3) molecules per cell. Reverse transcription-polymerase chain reaction using specific oligonucleotide primers demonstrated expression of mRNA for each protein. Twenty-four-hour stimulation with inflammatory cytokines (interferon-gamma, tumour necrosis factor-alpha, interleukins-1 beta, -2 and -6) or phorbol myristate acetate had no significant effect on the level of expression of either protein as determined by Western blotting. Lysis caused by classical pathway activation of C in human or rat serum was enhanced by blocking the function of CD59 and 5I2 Ag on rat astrocytes with monoclonal antibodies.