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PubMed1991

A novel approach to the rapid isolation and nucleotide sequencing of genomic clones.

In this study, a genomic library subdivided into fractions was rapidly screened by a Southern detection technique. Deletion libraries were obtained from recovered genomic clones by single random cuts with nuclease S1. These deletion libraries proved useful for localizing genes in the inserts and yielded, after size fractionation, nested deletions suitable for nucleotide sequencing. An heterologous vector (pDB21) carried the insert used as probe for all hybridizations involved in the process of genomic clones isolation and characterization.

Base Sequence↗

PubMed2005

[Detecting the API2-MALT1 fusion gene and its significance in Hashimoto's thyroiditis and thyroid lymphomas].

OBJECTIVE: To investigate the expression of API2-MALT1 fusion gene mRNA in Hashimoto's thyroiditis, thyroid MALT lymphoma and DLBCL, as well as the correlation between the expression and the pathogenesis and prognosis of two types of thyroid lymphoma. METHODS: Eight cases of Hashimoto's thyroiditis, 12 cases of MALT lymphoma and 3 cases of DLBCL in thyroid were selected and retrieved. API2-MALT1 transcripts were investigated in all cases by reverse transcript touchdown-PCR and nested PCR. The effect of API2-MALT1 fusion gene on the prognosis of thyroid lymphomas was assessed by analyzing the clinicopathological features and the follow-up findings. RESULTS: The variant 1446/1123 transcript was detected in 2 of thyroid lymphomas (1 MALT lymphoma, 1 DLBCL) but not in 8 cases of Hashimoto's thyroiditis and 5 cases of chronic lymphadenitis. Both of API2-MALT1-positive cases were accompanied with Hashimoto's thyroiditis, showing stage I E and no involvement of lymph node. CONCLUSION: The incidence of API2-MALT1 transcript in thyroid lymphomas was low; however, the correlation between API2-MALT1 fusion gene and the progression from Hashimoto's thyroiditis to lymphoma could not be excluded; the thyroid lymphoma with API2-MALT1 transcript seemed to develop on a more indolent clinical course.

Adult↗

PubMed2004

CYP1B1 and CYP19 gene polymorphisms and breast cancer incidence: no association in the ARIC study.

We conducted a nested case control study of 178 incident breast cancer cases and 356 controls in the Atherosclerosis Risk in Communities study. We evaluated the association between breast cancer and Val432Leu polymorphism in the CYP1B1 gene and the tetranucleotide repeats in intron 4 of the CYP19 gene. After adjustment for height, age at menopause, age at menarche, BMI, HRT, and alcohol intake, carriers of the Val/Leu or Val/Val genotype had a 1.45 fold (95% CI 0.85-2.47) greater odds of breast cancer than Leu/Leu carriers. There was no association of the breast cancer with any individual CYP19 allele. Compared to individuals homozygous with the 167 allele, odds ratios were close to 1.0 for the 167 heterozygous genotype and for the remaining tetranucleotide repeats combined. Our data shows no association between breast cancer and the Leu432Val polymorphism of the CYP1B1 gene or the tetranucleotide repeats of the CYP19 gene.

Aromatase↗

PubMed2004

Epizootiologic survey for Babesia microti among small wild mammals in northeastern Eurasia and a geographic diversity in the beta-tubulin gene sequences.

We previously reported that small wild rodents in Japan harbor two types of novel Babesia microti-like parasites (designated as Hobetsu and Kobe types), but not the type commonly found in the northeastern United States (U.S. type) where human babesiosis is endemic. To determine whether these new types of parasites are distributed in places surrounding Japan, an epizootiologic survey was undertaken in three geographically distant areas in northeastern Eurasia; South Korea, Vladivostok in Russia, and Xinjiang in China. Blood samples were collected from a total of 387 animals comprising 24 species. DNAs extracted from the samples were tested by nested PCR targeting babesial nuclear small-subunit rRNA gene (rDNA), which revealed that small rodents harboring B. microti exist in all three survey areas. Sequence analysis showed that all PCR-positive samples had rDNA sequences virtually identical to that of U.S.-type B. microti. However, when beta-tubulin gene sequences were compared, evident geographic variations were seen. By use of primers specific for each of the beta-tubulin genes of Kobe-, Hobetsu-, and U.S.-type parasites, a type-specific PCR was developed. Parasite with Hobetsu- or Kobe-type sequence was not detected from any of the three survey areas. These findings suggest that U.S.-type B. microti is widely distributed among small wild mammals in temperate zones of not only North America, but also Eurasia, whereas that Hobetsu- and Kobe-type parasites may be uniquely distributed in Japan.

Animals↗

PubMed2005

Molecular epidemiology of tick-borne encephalitis virus in Ixodes ricinus ticks in Lithuania.

In Lithuania, 171-645 serologically confirmed cases of tick-borne encephalitis occurred annually [Mickiene et al. (2001): Eur J Clin Microbiol Infect Dis 20:886-888] in 1993-1999, and the tick-borne encephalitis virus (TBEV) seroprevalence in the general population was found previously to be 3.0% [Juceviciene et al. (2002): J Clin Virol 25:23-27]. To assess the risk for TBEV virus infection in Lithuania and to characterize the agent a panel of 3,234 ticks combined into 436 pools [Juceviciene et al., 2005] were tested for presence of TBEV RNA by a nested RT-PCR targeting at the NS5 gene. Six pools were confirmed positive and the prevalence of the infected ticks was 0.2% (if one tick per pool [Juceviciene et al., 2005] was considered positive) and the proportion of positive tick pools was 1.4%. The prevalence of the infected ticks in the Panevezys, Siauliai, and Radviliskis regions (in central Lithuania) was 0.1%, 0.4%, and 1.7% corresponding with a higher TBE disease burden in these regions. The 252-nucleotide NS5-region amplicons, and a longer sequence (737 nucleotides) obtained from one sample from the PrM-E gene region, were sequenced. Phylogenetic analysis of the latter showed that all western type TBEV PrM-E sequences, including the Lithuanian strains, were monophyletic, showed no clustering and had very little variation. The NS5 sequences, although identical within one locality, did not show any mutations common to strains from the two Lithuanian regions, nor could any geographical clustering be found among western type TBEV strains from other areas.

Animals↗

PubMed2000

Genetic dissection of tomato bushy stunt virus p19-protein-mediated host-dependent symptom induction and systemic invasion.

The plus-sense single-stranded RNA of tomato bushy stunt virus (TBSV) encodes a 19-kDa protein, which is translated from a 3' proximal open reading frame (p19) that is entirely nested within the cell-to-cell movement gene (p22). Expression of the cytosolic p19-protein induces either a systemic lethal collapse in Nicotiana benthamiana and N. clevelandii, or necrotic local lesions on resistant N. tabacum. In spinach, the p19-protein is required at high abundance for efficient systemic invasion. This study aimed to determine whether these seemingly different host-dependent biological activities are governed by the same or separate regions on the 172 amino acid p19-protein. For this purpose, codons for charged amino acids predicted to be exposed on the surface of the polypeptide and presumably available for host-specific interactions, were targeted for mutagenesis. A total of 12 mutants were generated, which had no deficiencies in replication or cell-to-cell movement, and substitution of amino acids at the extreme N-terminal end or within the carboxyl 70 amino acids failed to cause a noticeable biological effect on plants. However, mutations dispersed between positions 43 and 85 on the N-terminal half prevented the onset of a systemic lethal necrosis on N. benthamiana and N. clevelandii. With one exception, the same mutants elicited mostly chlorotic, rather than necrotic, local lesions on N. tabacum. Mutations in the central region, which substituted Arg with Gly at positions 72 or 75-78, impaired the ability of TBSV to systemically invade spinach plants. However, substitution with Ala instead of Gly at position 72 had minimal effects on systemic spread in spinach, suggesting the possible influence of protein structure effects. The implications are that regions on the N-terminal portion of the p19-protein mediate interactions in a host-dependent manner and that a central region is required for all activities either by a direct effect of the amino acids or through maintenance of structural integrity.

Mutagenesis, Site-Directed↗

PubMed2005

Polymerase chain reaction for detection of Toxoplasma gondii in human biological samples.

Using the polymerase chain reaction (PCR), Toxoplasma gondii from gene TGR1E with primers TGR1E-1, TGR1E-2 (standard PCR), and from B1 gene with primers TM1, TM2, TM3 (hemi-nested PCR) was detected in biological samples from 347 individuals (441 biological materials). Of the total of 441 biological materials, T. gondii DNA was detected in 5.2 %; it was positive in the following samples: blood (n = 6), blood from newborns (2), biopsies (2) and samples of progenitor cells (2) (from candidates for bone marrow transplantation). DNA of T. gondii was also revealed in 11 samples (8.3 %) of 120 cases of pregnant women during prenatal examinations. A positive result in the blood was also found in two cases of newborn babies from mothers who were infected in later pregnancy. The positive PCR examination was confirmed by serological methods (ELISA and complement fixation test). Agreement of PCR results and the detection of antibodies against toxoplasma was found in 83.3 %. Rapid PCR examination for the confirmation of acute parasitemia T. gondii is particularly important for the patients in whom the infection may cause serious consequences (e.g., for fetus in pregnant women or for patients suffering from imunosuppression).

Adult↗

PubMed2001

Distribution of Nitrogen-Fixing Microorganisms along the Neuse River Estuary, North Carolina.

Nitrogen fixation genes (nifH) were amplified and sequenced from DNA extracted from surface water samples collected from six stations along the length of the Neuse River Estuary, North Carolina, in order to determine the distribution of nitrogen-fixing organisms in the transition from fresh- to saltwater. Nitrogenase genes were detected in all samples by a nested polymerase chain reaction method, and the amplification products from the upriver, midriver, and downriver stations were cloned, sequenced, and used for phylogenetic analysis. The composition of nifH clone libraries from upriver, midriver, and downriver stations (each composed of 14 randomly selected clones) were very diverse (samples from upriver and midriver stations were composed of 14 unique sequences, downriver station composed of 7 unique sequences) and differed among the stations. Some phylotypes were found at more than one station, but were usually found in the upriver and midriver stations or in the midriver and downriver stations, indicating that the phylotypes were probably transported along the river. Cyanobacterial nifH were not found at the most upriver site, but were a large fraction of sequences (50%) recovered from the downriver station, where nitrate concentration was an order of magnitude lower and salinity was higher. In contrast, g proteobacteria nifH sequences were much more common at the midriver and upriver sites (58% and 64%, respectively), compared to the downriver site (14%). Results indicate that substantially different nitrogen-fixing assemblages are present along the river, reflecting differential watershed hydrological inputs, sedimentation, and environmental selection pressures, along the salinity gradient.

Journal Article↗

PubMed2001

Transgenic live-bearing fish and crustaceans produced by transforming immature gonads with replication-defective pantropic retroviral vectors.

Transgenic animals have been routinely produced by microinjecting or electroporating naked DNA into 1-cell-stage embryos or unfertilized eggs. However, these techniques are inapplicable to live-bearing fish and many crustacean species for which unfertilized or newly fertilized eggs are not readily obtainable. In the present study, replication-defective pantropic retroviral vectors carrying a reporter gene (neo(R) or beta-gal) were used to directly transform the immature ovary or testis of a live-bearing fish (Poeciliopsis lucida) and crayfish (Procambarus clarkii). The fraction of the progeny derived from these treated individuals shown to contain the neo(R) reporter gene by an assay based on polymerase chain reaction (PCR) was significant. The PCR-positive individuals were crossed with nontransgenic individuals, and about 50% of the resulting progeny carried the transgene, suggesting that the F(1) animals are germline transgenic. Integration of the transgenes was confirmed by detecting the junction fragments of the genomic DNA associated with transgene constructs. Expression of reporter genes was detected by a reverse transcription-nested PCR assay. These results showed that transgenic live-bearing fish and crustaceans could be easily produced by directly transforming the immature gonads with replication-defective pantropic retroviral vectors.

Journal Article↗

PubMed2005

The "spanning protocol": a new DNA extraction method for efficient single-cell genetic diagnosis.

PURPOSE: We evaluated methods of preparation of DNA from single cells for amplification and preimplantation genetic diagnosis (PGD), including our "spanning protocol." METHODS: Dystrophin gene exons 45 and 51 were amplified by nested polymerase chain reaction (PCR) from a single lymphocyte or blastomere. Amplification efficiencies were compared between DNA extraction by (A) lysis in distilled water with freeze-thawing and boiling; (B) two-step lysis involving potassium hydroxide and dithiothreitol; and (C) the spanning protocol, using N-lauroylsarcosine. RESULTS: With method A, amplification efficiency was 66/120 (55%) and false-positive such as amplification failure or allele drop out was 42/120 (35%); with B, 96/120 (80%) and 21/120 (17.5%); and with C, 111/120 (92%) and 5/120 (4.2%), using single blastomeres and unaffected lymphocytes from male. Occurrence of false-negative such as contamination of another DNA with method A was 4/120 (3.3%); with B, 10/120 (8.3%); and with C, 2/120 (1.7%) from using single lymphocytes from affected males. CONCLUSION: The spanning protocol was most efficient for extracting DNA from a single cell and should be particularly useful for preimplantation genetic diagnosis.

Blastomeres↗

PubMed2026

First report of tomato spotted wilt virus (Orthotospovirus tomatomaculae) and phytoplasma in China aster and development of duplex PCR, LAMP, and qPCR assays for rapid detection.

UNLABELLED: China aster (Callistephus chinensis) is an economically important ornamental crop widely cultivated for cut flowers and landscaping. During field surveys conducted in three districts of Karnataka, India, China aster plants exhibiting chlorotic and necrotic ring spots, leaf deformation, and witches' broom symptoms were collected and analyzed to determine the causal agents. Mechanical inoculation of symptomatic leaf sap onto cowpea (Vigna unguiculata cv. C-152) produced characteristic chlorotic and necrotic ring spots on newly emerging leaves indicating the presence of an infectious viral agent. Serological assay by DAC-ELISA followed by RT-PCR confirmed the presence of tomato spotted wilt virus (TSWV, Orthotospovirus tomatomaculae) in symptomatic plants. Similarly the plants exhibiting witches' broom symptoms tested positive for phytoplasma infection using universal and Nested primers PCR assays targeting the 16S rRNA gene. Sequence analysis of TSWV CP gene revealed more than 97% nucleotide identity with TSWV isolates reported from India and other countries. Based on these results, one representative isolate was selected for complete genome sequencing. The complete sequences of the L, M, S RNA segements were amplified cloned, and sequenced showing more than 97% nucleotide identity with global TSWV isolates available in database. Sequence analysis of 16S rRNA gene of the phytoplasma associated with witches' broom symptoms was identified as 'Candidatus Phytoplasma australasiaticum' belonging to the 16SrII-D subgroup, sharing 99.2% nucleotide identity with previously reported isolates. Phylogenetic analysis further supported the placement of both the TSWV and phytoplasma isolates within their respective taxonomic groups. To facilitate rapid and sensitive diagnosis, quantitative PCR (qPCR) and RT-LAMP assays were developed for TSWV detection. In addition a duplex PCR assay was optimized for simultaneous detection of TSWV and phytoplasma from infected China aster plants in a single reaction. This study represents the first reports of the complete genome characterization of TSWV and phytoplasma infection in China aster in India along with the development of sensitive qPCR, RT-LAMP, and duplex PCR assays for rapid detection of these pathogens providing valuable tools for disease diagnosis, epidemiological studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s13205-026-05038-w.

China aster↗

PubMed1993

Detection and typing of human herpesviruses by multiplex polymerase chain reaction.

A new approach to simultaneous detection and typing of related agents by the multiplex polymerase chain reaction (PCR) is described. The reaction was been applied to human herpesviruses by nested amplification of fragments of the DNA polymerase genes. During the first amplification, primers were used as two equimolar mixtures of non-degenerate oligonucleotides, aligning the 3'-ends with selected consensus regions, and their 5'-ends with the non-related sequences of each herpesvirus to be amplified. The specific fragments obtained were the substrate for a second, multiplex reaction for which primers were designed to produce different-size fragments for each related virus. The results showed high specificity for the detection and typing of the human herpesviruses with known sequences and no amplification of human DNA, in spite of the presence of the same consensus regions within human DNA polymerase alpha. It is concluded that this new approach would be useful for the differential diagnosis of herpesviruses, as well as for other groups of agents with conserved regions in their genomes and causing similar syndromes.

Amino Acid Sequence↗

PubMed1993

Detection of bovine viral diarrhea virus RNA in formalin-fixed, paraffin-embedded brain tissue by nested polymerase chain reaction.

Isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissues is delicate due to its fragility and ubiquitous ribonucleases. For retrospective studies, however, a convenient procedure for the detection of RNA in archived material is of great value. Bovine viral diarrhea (BVD) virus is a member of the pestivirus genus in the family Flaviviridae. Different protocols for the isolation of BVD virus RNA from fresh and autolytic as well as from routinely formalin-fixed and paraffin-embedded brain tissue of BVDV-infected calves were compared. The polymerase chain reaction (PCR) after reverse transcription (RT-PCR) was carried out subsequently for the detection of viral RNA. Using proteinase K digestion of deparaffinized tissue sections without additional ribonuclease inhibitors and subsequent nested PCR, a 803 bp fragment of the gene coding for the nonstructural protein p125 of BVD virus could be consistently detected. In addition, BVD virus RNA was detected by RT-PCR from non-fixed brain tissue after 10 days of autolysis.

Animals↗

PubMed2005

Identification of the infectious source of an unusual outbreak of histoplasmosis, in a hotel in Acapulco, state of Guerrero, Mexico.

Three isolates of Histoplasma capsulatum were identified from mice lung, liver, and spleen inoculated with soil samples of the X hotel's ornamental potted plants that had been fertilized with organic material known as compost. The presence of H. capsulatum in the original compost was detected using the dot-enzyme-linked immunosorbent assay. Nested-PCR, using a specific protein Hcp100 coding gene sequence, confirmed the fungal identification associated with an unusual histoplasmosis outbreak in Acapulco. Although, diversity between the H. capsulatum isolate from the hotel and some clinical isolates from Guerrero (positive controls) was observed using random amplification of polymorphic DNA based-PCR, sequence analyses of H-anti and ole fragment genes revealed a high homology (92-99%) between them.

Animals↗

PubMed2004

Phylogenetic relationships among Pneumocystis from Asian macaques inferred from mitochondrial rRNA sequences.

The presence of Pneumocystis organisms was detected by nested-PCR at mitochondrial large subunit (mtLSU) rRNA gene in 23 respiratory samples from Asian macaques representing two species: Macaca mulatta and M. fascicularis. A very high level of sequence heterogeneity was detected with 18 original sequence types. Two genetic groups of Pneumocystis could be distinguished from the samples. Within each group, the extent of genetic divergence was low (2.5+/-1.4% in group 1 and 2.3+/-1.7% in group 2). Genetic divergences were systematically higher when macaque-derived sequence types were compared with Pneumocystis mtLSU sequences from other primate species (from 5.3+/-2.7% to 19.3+/-3.0%). The two macaque-derived groups may be considered as distinct Pneumocystis species. Surprisingly, these Pneumocystis species were recovered from both M. mulatta and M. fascicularis suggesting that host-species restriction may not systematically occur in the genus Pneumocystis. Alternatively, these observations question about the species concept in macaques.

Animals↗

PubMed1999

First-trimester fetal sex prediction by deoxyribonucleic acid analysis of maternal peripheral blood.

OBJECTIVES: We investigated whether the number of weeks of gestation influences the accuracy of first-trimester fetal sex prediction by analysis of deoxyribonucleic acid extracted from whole maternal blood. A comparison was also made to determine whether a difference exists between this approach and the deoxyribonucleic acid analysis of transcervical cells performed on the same group of subjects. STUDY DESIGN: Deoxyribonucleic acid was isolated from 50 maternal blood samples taken between gestational weeks 7 and 11. The sex of the fetus was assessed by nested polymerase chain reaction specific for the amelogenin gene. A receiver-operating characteristic curve analysis was used to correlate the accuracy of fetal gender prediction with the gestational age and also to compare the goodness of the 2 methods under investigation. RESULTS: Analysis of the receiver-operating characteristic curve provided a cutoff value of 9 weeks 4 days of gestation for both tests, indicating that a higher degree of accuracy in the sex assignment was obtained in those samples taken before or at this time. However, this difference was statistically significant only for analysis of deoxyribonucleic acid from maternal blood. The comparison between tests of deoxyribonucleic acid from maternal blood and from transcervical cells showed that the first approach is better, although a statistically significant difference was not found. CONCLUSION: Analysis of maternal blood deoxyribonucleic acid is a better approach than analysis of trans-cervical cell deoxyribonucleic acid in fetal sex prediction. The highest degree of accuracy is obtained when blood is drawn before 10 weeks of gestation. This can be important when sampling of chorionic villi should be avoided because of the risk of an X-linked disease when the fetal sex is female.

Amelogenin↗

PubMedDate not supplied

First reported case of Mycobacterium ulcerans infection in a patient from China.

Buruli ulcers have not been previously described in China, and only once at higher latitudes on the northern hemisphere. A patient who travelled in the Shan Dong Province in the People's Republic of China developed an ulcer which was proven to be a Buruli ulcer. The clinical picture and histopathological findings from biopsy specimens are characteristic for a Buruli ulcer, and also the growth in culture (Coletsos medium) at a restricted temperature of 30 degrees C. A multiplex polymerase chain reaction (PCR) based on the amplification of the gene encoding for 16S ribosomal RNA and a nested PCR based on the Mycobacterium ulcerans specific repeated sequence 2404 were performed. These PCR investigations identified the bacteria as M. ulcerans, subspecies shinshuense. The patient was initially treated with clarithromycin and rifampicin, which was changed to ciprofloxacin and rifabutin when rifampicin resistance of the first isolate was established. There were no signs of reactivation of the disease 6 months after the end of treatment. M. ulcerans infection occurs above 30 degrees latitude on the northern hemisphere in China and is caused by M. ulcerans, subspecies shinshuense. This case appears to be cured by chemotherapy alone, in contrast to the general experience that surgical treatment is indicated. The granulomatous reaction with only fragments of acid-fast bacteria in the biopsy at the end of treatment many indicate the development of an adequate cell-mediated immune response leading to resistance to the infection.

Adult↗

PubMed2002

Detection of Neorickettsia (Ehrlichia) risticii in tissues of mice experimentally infected with cercariae of trematodes by in situ hybridization.

Neorickettsia (Ehrlichia) risticii was demonstrated to occur in cercariae developing in Juga yrekaensis snails by experimental transmission, genetic detection and histopathology. Cercariae were isolated from the digestive glands of snails collected in a fresh stream water area of Siskiyou County, CA, and inoculated into CF1 mice. Mice developed clinical signs, splenomegaly and histopathologic abnormalities. The agent was maintained by serial passages of whole blood in CF1 mice. A 527-bp product of the 16S rRNA gene of N. risticii was serially detected by nested PCR in blood, feces, salivary gland, suprarenal gland, spleen, intestine and bone marrow of inoculated mice. N. risticii DNA was detected by in situ hybridization with DIG-labeled probe in PCR-positive salivary gland, intestine and spleen tissue sections of experimental mice on day 30 after inoculation. Infection in mice was established when cercariae were inoculated by either IP or SC routes but not established following intraoral route. N. risticii was detected by PCR in spleen, intestine and bone marrow even after 73 days post-inoculation whereas blood from the same animals became negative at 58 days. N. risticii was observed by in situ hybridization in salivary gland, spleen and intestine of mice infected by IP or SC inoculation. This ISH protocol should aid investigations on the host range of the Neorickettsiosis and pathogenesis of neorickettiosis in vector, animal or human.

Animals↗