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At least 199 records · Page 11Linked to original sources
PubMed2002

Rapid detection of human immunodeficiency virus type 1 subtype e infection by PCR.

The CRF01_AE (subtype E) strain of human immunodeficiency virus type 1 (HIV-1), originally reported in Thailand, spread rapidly to and showed prevalence in several countries in Southeast Asia, including Taiwan. This strain was also found in other regions of the world. Based on sequence analysis of the vpu gene, a nested PCR assay including an outer primer pair and a subtype E-specific inner primer pair was developed in this study for rapid detection of subtype E viruses. It was tested with 397 HIV-1-positive samples of known subtypes. For these samples, the sensitivity of detection of subtype E viruses was 100% (127 of 127), and the specificity was 97.8% (264 of 270). Although six samples of either subtype A or G showed a positive PCR, most of the cross-reactivity could be reduced by raising the annealing temperature from 54 degrees C to 63 degrees C. When tested with 195 HIV-positive samples of unknown subtypes, the assay had a sensitivity of 98.0% and a specificity of 98.6%. This is a simple, convenient, and sensitive method for rapid detection of subtype E viruses, especially in regions in which viruses of subtypes B and E are predominant.

DNA, Viral↗
PubMed1998

[The relation of neonatal hyaline membrane disease and congenital human cytomegalovirus infection].

To study the relation of neonatal hyaline membrane disease (HMD) and congenital human cytomegalovirus (HCMV) infection, two pairs of primers were synthesized according to the sequence of HCMV conserved region of the immediate--early(IE) gene. The nested polymerase chain reaction (nested-PCR) was used for the detection of HCMV-DNA of the paraffin-embedded lung tissues from neonatal HMD and non-neonatal HMD. 11 of 30 neonatal HMD patients were positive, the positive rate was 36.7%. Specimens of non-neonatal HMD patients were all negative. The difference between the two groups was significant (chi 2 = 5.05, P < 0.025). There is close relationship between neonatal HMD and congenital HCMV infection.

Cytomegalovirus↗
PubMed2004

PCR technique for detecting Toxoplasma gondii in animal amniotic fluid.

The goal of diagnosing congenital toxoplasmosis is early detection of maternofetal transmission, for early treatment to prevent unwanted sequelae. Polymerase chain reaction (PCR) is a method used recently for detecting toxoplasmosis during pregnancy. Amniotic fluid is a the clinical specimen used, since it provides a rapid, simple and safe method to obtain accurate results. The advantages of the PCR technique are high sensitivity, specificity and positive predictive value compared with other laboratory methods. To determine the sensitivity, specificity and lower detection limits in our laboratory, amplification of the B1 gene by nested PCR was performed on Toxoplasma gondii tachyzoites added to animal amniotic fluid samples. From 48 samples, our technique detected T. gondii in 30 out of 41 positive samples, and gave negative results for all the negative samples. The sensitivity for this nested PCR was 73%, the specificity was 100%, and the efficiency of the test was 77.1%. The nested PCR technique is recommended as a diagnostic method for detecting T. gondii in suspected congenital toxoplasmosis animals.

Amniotic Fluid↗
PubMed1994

Integration of traditional and modern methods in the identification of AFB cultures isolated from clinical specimens of patients with skin diseases.

This article reports the identification of 57 AFB cultures isolated from clinical specimens by using traditional methods (TM, including biochemical and cultural methods) and modern ELISA with monoclonal antibody (McAb-ELISA) and nested primer gene amplification assay (NPGAA). The representive AFB culture M. A1, A7, A19, A21 and A22) isolated from human lepromas were identified as new species by TM and it was shown that they were not identical to M. leprae by McAb-ELISA and NPGAA. Among another set of samples (M. S17, S1, S2, S2R, S7, S29), M. S17 was identical to M. scrofulaceum as assessed by TM only, while the others were found to be similar to M. tuberculosis and different from M. leprae using TM and McAb-ELISA, and identical to M. tuberculosis with NPGAA. The authors conclude that TM and MM are very useful for identifying mycobacteria, while MM was much more sensitive and specific than TM. The selection and use of these methods depends on practical need.

Base Sequence↗
PubMed1994

[Rapid identification of mycobacteria by combined method of polymerase chain reaction and the gen-probe DNA hybridization system].

We developed the rapid detection and identification method of mycobacteria, involving amplification of mycobacterial 16S rRNA gene by nested PCR and identification of M. tuberculosis complex or M. avium-intracellulare complex (MAC) by hybridization protection assay (HPA) using the acridinium-ester (AE) labeled DNA probe. The specificity of the nested PCR combined with DNA probe test was excellent in terms of detection of mycobacterial organisms and identification of M. tuberculosis or MAC. The detection limits of the present method were 10 fg DNA for M. tuberculosis, and 100 fg DNA for MAC, respectively. We further investigated on the optimum temperature for hybridization in HPA with AE labeled DNA probe because there was the difference in the mode of DNA-RNA hybridization from that of DNA-DNA hybridization. In our method, the optimum temperature of hybridization was estimated as 55 +/- 1 degrees C. In preliminary experiments on two clinical cases, we practically detected and identified M. tuberculosis and MAC in clinical specimens, such as sputa, by using this newly devised method. We concluded that this method is useful for rapid detection and identification of M. tuberculosis and MAC in clinical specimens.

Adult↗
PubMed1997

Etiology and prognosis of cryptogenic liver cirrhosis: possible contribution of hepatitis B virus.

Three female patients without type B or type C viral hepatitis, alcoholic, metabolic or autoimmune liver disease, were selected from 250 cases with histologically proven liver cirrhosis (M:F = 183:67). All three cases showed at least one positive aspect among three parameters of serum anti-HBc (RPHA, x1), HBV-DNA (gene S, nested PCR) and liver HBs and/or pre-S2 antigen (immunoperoxidase methods). Two cases may suggest a spontaneous disappearance of HBV from sera. Another case may suggest a contribution of mutant HBV which can not be detected by the routine tests. These HBV-related cirrhotic patients have done well clinically and have not been associated with hepatocellular carcinoma during the period from 6 to 12 years of follow-up when compared with 59.6% and 65.4% prevalence of hepatocarcinogenesis in type B and type C hepatitis-associated cirrhosis during the observation period of six and seven years on average, respectively.

Aged↗
PubMed2000

Isolation and characterization of mutants for the vesicular acetylcholine transporter gene in Drosophila melanogaster.

The Drosophila vesicular acetylcholine transporter gene (Vacht) is nested within the first intron of the choline acetyltransferase gene (Cha). To isolate Vacht mutants, we performed an F(2) genetic screen and identified mutations that failed to complement Df(3R)Cha(5), a deletion lacking Cha and the surrounding genes. Of these mutations, three mapped to a small genomic region where Cha resides. Complementation tests with a Cha mutant allele and rescue experiments using a transgenic Vacht minigene have revealed that two of these three mutations are nonconditional lethal alleles of Vacht (Vacht(1) and Vacht(2) ). The other is a new temperature-sensitive allele of Cha (Cha(ts3) ). Newly isolated Vacht mutants were used to reexamine the existing Cha mutations. We found that all deficiencies uncovering Cha also lack Vacht function, reflecting the nested organization of the two genes. The effective lethal phase for Vacht(1) is the embryonic stage, whereas that for Vacht(2) is the larval stage. Viable first-instar larvae homozygous for Vacht(2) showed reduced motility. Adult flies heterozygous for Vacht mutations were found to have defective responses in the dorsal longitudinal muscles following high-frequency brain stimulation. Since cholinergic synapses have been shown to be involved in the giant fiber pathway that mediates this response, the result suggested that reduction in the Vacht activity to 50% causes an abnormality in cholinergic transmission when stressed by a high-frequency stimulus.

Alleles↗
PubMed2000

Borrelia burgdorferi-associated cutaneous marginal zone lymphoma: a clinicopathological study of two cases illustrating the temporal progression of B. burgdorferi-associated B-cell proliferation in the skin.

AIMS: A relationship between Borrelia burgdorferi and primary cutaneous B-cell lymphoma (PCBCL) has recently been confirmed following demonstration of the organism in lesional skin of patients with PCBCL. We report herein two cases of B. burgdorferi-associated PCBCL which strengthen this association by demonstrating the organism in cutaneous B-cell infiltrates present at sites in which PCBCL subsequently developed. METHODS AND RESULTS: All studies were performed on formalin-fixed paraffin-embedded tissues. These were examined by routine light microscopy and immunohistochemically by a standard streptavidin-biotin-complex technique. Genotypic studies were also undertaken using semi-nested polymerase chain reaction (PCR) for immunoglobulin heavy chain gene rearrangement, and nested PCR for B. burgdorferi flagellin gene. Both patients presented with erythematous skin lesions, biopsy of which showed dense perivascular infiltrates comprising small T-lymphocytes and collections of B-blasts. Primary cutaneous marginal zone lymphoma (MZL) developed subsequently in both cases at the same site. PCR for B. burgdorferi flagellin gene was positive in the perivascular lymphocytic infiltrates and the succeeding lymphomas in both patients. CONCLUSIONS: These results show that, at least in some instances, PCBCL arises from chronically stimulated lymphoid tissue acquired in the skin in response to B. burgdorferi infection. This may have significant therapeutic implications and warrant further studies on the extent of this association.

Adult↗
PubMed1996

A nested polymerase chain reaction for the detection of Borrelia burgdorferi sensu lato based on a multiple sequence analysis of the hbb gene.

A highly sensitive nested polymerase chain reaction method was designed for the detection of a wide spectrum of strains from Borrelia burgdorferi sensu lato. This technique allows the detection of as little as 3 fg of total genomic DNA extracted and purified from pure cultures of the organism, this amount corresponds to less than 10 organisms. Two sets of primers homologous to conserved spots in the coding region of the hbb gene, encoding a conserved histone-like protein, were constructed. These were based on a multiple sequence alignment of 39 strains representing all the genomic groups described in B. burgdorferi sensu lato.

Base Sequence↗
PubMed2002

Can the reverse transcriptase-polymerase chain reaction for prostate specific antigen and prostate specific membrane antigen improve staging and predict biochemical recurrence?

OBJECTIVE: To evaluate the perioperative gene-specific primed nested reverse transcription-polymerase chain reaction (RT-PCR) for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) for staging patients undergoing radical prostatectomy and predicting biochemical recurrence. PATIENTS AND METHODS: In 80 consecutive patients undergoing radical prostatectomy for prostate cancer, blood samples were drawn before, during and 1 and 7 days after removing the prostate. After buffy coat and mRNA extraction, gene-specific primed nested RT-PCR was performed for PSA, PSMA and glyceraldehyde-3-phosphate dehydrogenase mRNA, and Southern blot analysis of the PCR reaction. RESULTS: The sensitivity of gene-specific RT-PCR to detect tumour cells was comparable with random primed RT-PCR. In the 80 patients the stage distribution was pT1 in two (2.5%), pT2 in 30 (37.5%) and > or = pT3 in 48 (60%); the nodal status was pN0 in 57 (71%), pN1 in 11 (14%) and pN2 in 12 (15%). The gene-specific RT-PCR reaction for PSA and PSMA was positive in no patients with pT1, 11 (37%) with pT2 and 23 (48%) with stage > or = pT3 disease. The result for PSA was positive in 12 (52%) and for PSMA in 11 (48%) of those with positive nodal status. Neither gene-specific RT-PCR for PSA or PSMA was able to predict organ-confined disease (P > 0.5). After a median (range) follow-up of 37 (11-67) months a biochemical recurrence was predicted in 65% of patients by preoperative RT-PCR for both PSA and PSMA, with a sensitivity, specificity, positive and negative predictive value of 58%, 80%, 87% and 47%, respectively; the assay after surgery predicted a recurrence in 73%, with respective values of 68%, 84%, 84% and 57%. CONCLUSIONS: Gene-specific primed nested RT-PCR for PSA and PSMA is a sensitive and simple assay; it might add substantial information for tumour staging in individual patients. RT-PCR before surgery allows the prediction of recurrence in 65% of cases and after surgery in 73%.

Blotting, Southern↗
PubMed2005

Reassessing the Dlx code: the genetic regulation of branchial arch skeletal pattern and development.

The branchial arches are meristic vertebrate structures, being metameric both between each other within the rostrocaudal series along the ventrocephalic surface of the embryonic head and within each individual arch: thus, just as each branchial arch must acquire a unique identity along the rostrocaudal axis, each structure within the proximodistal axis of an arch must also acquire a unique identity. It is believed that regional specification of metameric structures is controlled by the nested expression of related genes resulting in a regional code, a principal that is though to be demonstrated by the regulation of rostrocaudal axis development in animals exerted by the nested HOM-C/Hox homeobox genes. The nested expression pattern of the Dlx genes within the murine branchial arch ectomesenchyme has more recently led to the proposal of a Dlx code for the regional specification along the proximodistal axis of the branchial arches (i.e. it establishes intra-arch identity). This review re-examines this hypothesis, and presents new work on an allelic series of Dlx loss-of-function mouse mutants that includes various combinations of Dlx1, Dlx2, Dlx3, Dlx5 and Dlx6. Although we confirm fundamental aspects of the hypothesis, we further report a number of novel findings. First, contrary to initial reports, Dlx1, Dlx2 and Dlx1/2 heterozygotes exhibit alterations of branchial arch structures and Dlx2-/- and Dlx1/2-/- mutants have slight alterations of structures derived from the distal portions of their branchial arches. Second, we present evidence for a role for murine Dlx3 in the development of the branchial arches. Third, analysis of compound Dlx mutants reveals four grades of mandibular arch transformations and that the genetic interactions of cis first-order (e.g. Dlx5 and Dlx6), trans second-order (e.g. Dlx5 and Dlx2) and trans third-order paralogues (e.g. Dlx5 and Dlx1) result in significant and distinct morphological differences in mandibular arch development. We conclude by integrating functions of the Dlx genes within the context of a hypothesized general mechanism for the establishment of pattern and polarity in the first branchial arch of gnathostomes that includes regionally secreted growth factors such as Fgf8 and Bmp and other transcription factors such as Msx1, and is consistent both with the structure of the conserved gnathostome jaw bauplan and the elaboration of this bauplan to meet organismal end-point designs.

Animals↗
PubMed1998

Nested expression and sequential downregulation of the Xenopus caudal genes along the anterior-posterior axis.

Expression of the Xenopus Xcad-1 and Xcad-2 genes initiates during early gastrulation exhibiting a dorsoventral asymmetry in their domains of transcription. At mid-gastrulation the ventral preference becomes stronger and the caudal genes take up a posterior localization in their expression, which they will maintain until their downregulation along the dorsal midline. Comparison of the three Xenopus caudal genes revealed a temporal and spatial nested set of expression patterns. The transcription of the caudal genes is sequentially downregulated with the one expressed most caudally (Xcad-2) being shut down first, this sequence is most evident along the dorsal midline. This pattern of expression suggests a role for the caudal genes as posterior determinants along the anteroposterior axis. In chicken, mouse, man and Xenopus three members of the caudal family have been identified in the genome. Even though in Xenopus the Xcad-3 gene has been previously described, in order to obtain a better insight on the role of the caudal genes a comparative study of all three frog genes was performed.

Animals↗
PubMed2003

Gene-expression analysis of single cells-nested polymerase chain reaction after laser microdissection.

AIM: The structural and functional characteristics of cells are dependent on the specific gene expression profile. The ability to study and compare gene expression at the cellular level will therefore provide valuable insights into cell physiology and pathophysiology. METHODS: Individual cells were isolated from frozen colon tissue sections using laser microdissection. DNA as well as RNA were extracted, and total RNA was reversely transcribed to complementary DNA (cDNA). Both DNA and cDNA were analyzed by nested polymerase chain reaction (PCR). The quality of isolated DNA and RNA was satisfactory. RESULTS: Single cells were successfully microdissected using an ultraviolet laser micromanipulator. Nested PCR amplification products of DNA and cDNA of single cells could clearly be visualized by agarose gel electrophoresis. CONCLUSION: The combined use of laser microdissection and nested-PCR provides an opportunity to analyze gene expression in single cells. This method allows the analysis and identification of specific genes which are involved in physiological and pathophysiological processes in a complex of variable cell phenotypes.

Colon↗
PubMed1998

Genetic subtyping and epidemiological study of feline immunodeficiency virus by nested polymerase chain reaction-restriction fragment length polymorphism analysis of the gag gene.

Genetic subtyping of feline immunodeficiency virus (FIV) was carried out by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. A 329-base pair fragment in the FIV gag gene was amplified by nested PCR, then digested with restriction enzymes, HindIII, PvuII and BamHI. Using these restriction enzymes, FIV isolates belonging to subtypes A, B and D, which had been classified on the basis of the env gene V3-V5 sequence, could be discriminated. Genetic subtypes of FIV prevalent in Japan were investigated using the gag-nested PCR-RFLP analysis. Of 88 FIV-infected cats, PCR products of 70 cats showed a subtype B RFLP pattern (digestion only with PvuII), those of nine cats had a subtype D RFLP pattern (digestion only with BamHI), and those of seven cats had a subtype A RFLP pattern (digestion only with HindIII). The PCR products of the remaining two cats had subtype A and B RFLP patterns (digestion with both HindIII and PvuII). The digestion pattern of the gag-nested PCR-RFLP analysis was unchanged after in vivo passages of the virus. These results suggest that the gag-nested PCR-RFLP analysis is useful as a simple method for FIV genetic subtyping.

Animals↗
PubMed2003

Influence of RNA secondary structure on HEV gene amplification using reverse-transcription and nested polymerase chain reaction.

BACKGROUND: Single-stranded RNA has the potential to form secondary structures that may result in intrastrand misalignment of repeats and may be responsible for DNA mutation. Two amplicons obtained from amplification of hepatitis E virus (HEV) gene by reverse transcription and nested polymerase chain reaction (RT-nPCR) were of unexpected size and had the same misalignment. They did not contain the target region between the internal priming sites but contained two fragments flanking the target region joined by a 12-base sequence instead. OBJECTIVES: To determine whether the unexpected amplicons obtained were due to secondary structures present in the HEV RNA. STUDY DESIGN: HEV RNA sequences were obtained from the GenBank database and the software DNASIS was used to predict the presence of secondary structures within the amplification target regions. The free energy barriers of the secondary structures, which indicate their stability, were also calculated. Conventional RT-nPCR protocol was subsequently modified to eliminate RNA secondary structures. RESULTS: An extensive stem-loop structure was predicted to exist between the two internal priming sites of the HEV RNA by the DNASIS software. Its free energy barrier was found to be significant and might have resulted in the deletion of the target region located between the internal priming sites. Increased temperature and addition of dimethyl sulphoxide (DMSO) in the reverse transcription step gave the expected amplicon after the nested polymerase chain reaction. CONCLUSION: Spontaneous secondary structure formation can influence the outcome of RNA gene amplification and should be considered an important factor when designing primers and adopting protocols for RNA gene amplification.

Base Sequence↗