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Results for “Nested Genes”

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PubMed2006

Helicobacter pylori and other Helicobacter species DNA in human bile samples from patients with various hepato-biliary diseases.

AIM: To investigate the presence of Helicobacter species by nested PCR of 16S rRNA genes followed by the presence of Helicobacter pylori (H pylori) 16S rRNA, ureA, cagA genes in bile obtained at endoscopic retrograde cholangio-pancreatography (ERCP) from 60 Indian subjects. METHODS: Sixty bile samples were obtained from patients diagnosed with various hepato-biliary diseases and control subjects at ERCP. PCR analysis was carried out using primers for Helicobacter genus 16S rRNA gene and H pylori (16S rRNA, ureA and cagA) genes. Gastric H pylori status was also assessed from biopsies obtained at endoscopy from patients with various hepato-biliary diseases and controls. The control group mainly consisted of subjects with gastric disorders. Sequencing analysis was performed to confirm that PCR products with 16S rRNA and cagA primers were derived from H pylori. RESULTS No Helicobacters were grown in culture from the bile samples. Helicobacter DNA was detected in bile of 96.7% and 6.6% of groups I and II respectively. Ten from group I were positive for 16S rRNA and ureA and 9 were positive for cagA gene. In contrast of the 2 from the control, 1 amplified with 16S rRNA, ureA and cagA primers used. The sequences of the 16S rRNA genes and cagA were 99% similar to Helicobacter pylori. CONCLUSION: Helicobacters are associated with the pathogenesis of various hepato-biliary disorders.

Adult↗

PubMed2002

Modalities of testing Helicobacter pylori in patients with nonmalignant bile duct diseases.

AIM: This paper describes the procedure of detection of Helicobacter pylori (H. pylori) in bile specimens in patients suffering from benign diseases of biliary ducts (lithiasis with/without nonspecific cholangitis). METHODS: The group of 72 patients entering the study consisted of 32 male and 40 female (45% and 55%, respectively). Bile was obtained during ERCP in 68 patients, and during cholecystectomy in 4 patients. A fast urease test (FUT) to determine the existence of H. pylori in gastric mucosa was carried out for all the patients during the endoscopic examination. The existence of genetic material of H. pylori was determined by detection of ureA gene by the method of nested PCR. The results of this reaction were shown by electrophoresis on 10g.L(-1) agarose gel in a band of 256bp. RESULTS: The majority of the patients included in our study had biliary lithiasis without signs of cholangitis (48 patients, 67%), whereas other patients were complicated by cholangitis (17 patients, 24%). Seven patients (9%) had normal ERCP, forming thus the control group. In the group of patients with lithiasis 26 patients (54.2%) had positive PCR of H. pylori in bile and among the patients with associated cholangitis positive PCR was detected in 9 patients (52.9%). Among the seven patients with normal ERCP only one (14%) had positive PCR of H. pylori. A high percentage of H. pylori infection of gastric mucosa was observed (57 patients, 79%). It was also observed that its slightly higher positivity was in the patients with distinct bile pathology: 81% FUT positive patients in the group with choledocholithiasis alone and 76% in the group with choledocholithiasis associated with cholangitis. Seventy-one percent of the patients with regular findings had positive FUT. CONCLUSION: The prevalence of H. pylori infection both in bile and in gastric mucosa in patients with benign diseases of biliary ducts does not show a statistically significant difference in relation to the prevalence of the same with the patients with normal ERCP. The existence of H. pylori infection possibly does not play a role in pathogenesis of benign biliary diseases.

Adolescent↗

PubMed2005

[Detection of human papilloma virus in cytologic samples or biopsies of the cervix].

BACKGROUND: The DNA quality for the detection and typification of Human Papilloma Virus (HPV) varies according to the type of sample in which it is studied. This may affect the sensitivity and specificity of the method employed. AIM: To study the yield and specificity of HPV detection and typification in uterine cervical samples obtained by cervical brushing fresh frozen and formalin fixed tissue. MATERIAL AND METHODS: Cytological, fresh frozen and fixed tissue samples from 44 patients (nine with low grade lesions and 35 with high grade lesions) were studied. Nested polymerase chain reaction for genes E6/E7 was used to typify HPV groups as low risk or high risk. RESULTS: Of all the cytological samples obtained by brushing 84% of fixed samples and 43% of fresh frozen samples were positive for HPV. The yields were significantly different when comparing brushing with fixed tissue or fresh frozen tissue and fixed tissue with fresh frozen tissue (p <0.05). The frequency of high risk HPV fluctuated from 41% in fresh frozen tissue to 98% in cytological samples. Low risk HPV was detected in 16% of fresh frozen tissue and 68% of cytological samples. A mixed infection was detected in 66%, 41% and 14% of cytological, fresh frozen and fixed tissue samples respectively. CONCLUSIONS: Cytological samples obtained by brushing had the highest yield for the detection of cervical infection with HPV.

Biopsy↗

PubMed1998

[Molecular epidemiology of TT virus infection in some parts of China].

OBJECTIVE: To study the molecular epidemiology of new hepatitis TT virus (TTV) infection, its distribution in population and its role in pathogenesis of hepatitis in some parts of our country. METHODS: TTV DNA in serum samples was detected by nested-polymerase chain reaction, partial gene of various geographic strains of TTV was cloned and sequenced, and their genetic variation was analyzed. RESULTS: Forty-four of 112 cases with non-A to G hepatitis from Shenzhen, Guangdong province, Nanjing, Jiangsu province, Beijing and Shengyang, Liaoning province were positive for TTV DNA with a positive rate of 42.9%, but only three of 102 cases of hepatitis A-G was positive, with a positive rate of 2.9% (chi 2 = 42.8, P < 0.01). Positive rate of TTV DNA was significantly higher in blood donors with abnormal ALT but without infection markers of hepatitis A-G (34.6%) than in those with normal ALT (16.8%) (chi 2 = 4.5, P < 0.05). Sequencing analysis showed that more than 98% of their nucleotides were analogous between strains of TTVCHN002 from Shenzhen and Nanjing, and TTVSHB015 from Beijing, and more than 97% analogous between the above-mentioned strains and Japanese ones. CONCLUSION: There existed TTV infection in both north and south China. TTV infection correlated closely with abnormal ALT, which might be an important pathogen for non-A, non-B, hepatitis G. There were somebodies infected with TTV in the normal healthy population, similar to that of "chronic carrier status" in hepatitis B.

Base Sequence↗

PubMed1999

[Relationship between methylation status of gamma-glutamyl transpeptidase (GGT) genes and abnormal expression of its enzyme proteins in tissues of human hepatomas].

OBJECTIVE: To explore the mechanism of expression and alteration of gamma-glutamyl transpeptidase (GGT) genes during the development of human hepatomas. METHODS: The total GGT protein and total RNA were purified in human hepatomas, adjacent paracancerous and distal cancerous tissues. The specific activities of total GGT, membrane-combine GGT and soluble GGT were investigated, the GGT gene of 5'-NC region was amplified by using a nest RT-PCR assay, and methylation status of GGT gene M3 site were analyzed in the present study. RESULTS: An increasing tendency (P < 0.05) of total RNA concentrations was found from cancer in distal cancerous tissues; the specific activities (U/g) of total GGT, membrane-combine GGT and soluble GGT were significantly higher (P < 0.05) in hepatomas than those in adjacent paracancerous or and that distal cancerous tissues; and the frequencies of amplified fragment and hypomethylated M3 site of GGT 5'-NC region genes were 100% and 75% in hepatomas, 85% and 55% in adjacent cancerous, and 75% and 50% in distal cancerous tissues, respectively. CONCLUSIONS: The present data suggest that the abnormal expression of GGT proteins in hepatomas was related to hypomethylation status of GGT genes, and that the fragment analysis of the GGT genes might be a sensitive assay to monitor the hepatic cell canceration.

Carcinoma, Hepatocellular↗

PubMed2000

PCR-based evidence of bacterial involvement in eyes with suspected intraocular infection.

PURPOSE: To assess the usefulness of polymerase chain reaction (PCR) in detection of bacteria in ocular samples. METHODS: Thirty-seven samples (aqueous and vitreous) were collected from 25 eyes showing typical symptoms and clinical signs of bacterial endophthalmitis. Ocular samples were also collected from 38 eyes that underwent routine surgery and from 15 eyes with intraocular inflammation due to nonbacterial causes. Panbacterial PCR was performed with a nested pair of 16S rRNA gene primers. Subsequent bacterial identification was completed for 18 paired samples (nine eyes) using restriction fragment length polymorphism (RFLP) and DNA sequencing. RESULTS: A 100% concordance was obtained between PCR and culture-positive samples. A PCR product was amplified from all 37 intraocular samples from eyes with suspected infection, whereas only 15 of 22 vitreous samples and 5 of 15 aqueous samples were culture positive. Culture-negative PCR-positive samples contained a preponderance of gram-negative bacterial sequences. Cloning and DNA analysis revealed 30 DNA sequences and included eight bacterial 16S rDNA, which currently remain unidentifiable. The presence of bacterial DNA was associated with an inflammatory response suggestive of infection and not colonization. All 15 samples from inflamed eyes with diverse uveitis diagnoses were PCR negative. The false-positive rate, due to contamination during sampling, was 5%. CONCLUSIONS: Bacterial DNA was detected in all patients with typical clinical signs of endophthalmitis. Gram-negative organisms seem to play a much more important role in the pathogenesis of this disease than previously thought. PCR-based techniques have great value in the confirmation of the diagnosis of bacterial endophthalmitis especially in culture-negative eyes.

Adult↗

PubMed2001

[Genetic subtyping of HIV-1 in Liaoning province of China].

OBJECTIVE: To study the prevalence of HIV-1 in Liaoning province of China. METHODS: Nuclear acids were extracted from blood samples of 16 HIV-1 infected individuals collected locally in Liaoning province of China from Jun. 1997 to Dec. 2000. The 0.7 kb or 1.2 kb segments of HIV-1 env gene were amplified using nested-PCR and the HIV-1 genetic subtypes were then assayed by heteroduplex mobility assay. RESULTS: Fifteen of 16 samples were positive by PCR amplification of HIV-1 env region and samples were found to be genetic subtype A,B',C,E. The proportion due to sexual transmission in all HIV infection was 31.25% (5/15), among which subtype B' (3/5) was the majority. A man who returned from Africa together with his spouse both had type A (2/5) infection. Intravenous drug users (IDUs) took up 31.25% (5/15) of all the HIV infections. Subtype C (2/4) and E were predominant among intravenous drug users. However, there was one IDU with subtype B or E. Nearly all blood recipients and blood donors were B' (4/5) except one with C. CONCLUSION: There have been several subtypes of HIV-1 existed in Liaoning province, demonstrating the complexity of HIV epidemology in Liaoning province and the difficulty conducting prevention and treatment.

Acquired Immunodeficiency Syndrome↗

PubMed2003

[Sex determination of human preimplantation embryo using nested polymerase chain reaction].

OBJECTIVE: Using nested polymerase chain reaction (PCR) to perform preimplantation gender diagnosis. METHODS: One (or two) lymphocyte and blastomere (n=50/group) were collected and prepared under the following conditions: (1) water only (H(2)O); (2) freeze-thaw liquid nitrogen, then boiling; (3) potassium hydroxide/dithiotheriol, heated to 65 degree centigrade, followed by acid neutralization (KOH). Cells were analyzed by PCR using nested primers amplification with amelogenin gene. RESULTS: The amplification rate and allele dropout (ADO) rate for male lymphocytes by the three methods were 83%, 94%, 95% and 24%, 12%, 4%, respectively. Using two cells per reaction did not increase the amplification rate for the KOH method. CONCLUSION: The KOH method for DNA preparation is superior to the other methods evaluated. Dual blastomere biopsy and independent blastomere analysis may improve preimplantation diagnostic reliability.

Amelogenin↗

PubMed1999

[A mutation specific polymerase chain reaction for detecting hepatitis B virus genome with A-to-C mutation at nt585].

OBJECTIVE: To investigate the prevalence of hepatitis B virus (HBV) nt585 A-to-C mutants in China. METHODS: A mutation specific polymerase chain reaction (msPCR) was established for amplifying HBV DNAs with A-to-C mutation at nt585. Two sets of primer pairs with same sequences except for one base at 3' terminus were designed and synthesized according to 48 of the known HBV genome sequences and the popular HBV subtypes, adr and adw, in China. RESULTS: 57 of HBV S gene fragments amplified by a nested PCR from different and child adult hepatitis B patients were characterized by using the msPCR. It was shown that HBV mutants with A-to-C mutation at nt585 could be confirmed. CONCLUSION: The msPCR is a specific and sensitive method for detecting the HBV nt585 A-to-C mutants.

Adult↗

PubMed2004

The role of the polymerase chain reaction in the diagnosis of bacterial meningitis in Vietnam.

Bacterial meningitis remains an important cause of morbidity and mortality in Vietnam. Diagnosis is hampered by the ready availability of antibiotics in the community, leading to late presentation, masked clinical signs, and poor organism detection during the microscopical examination and culture of cerebrospinal fluid (CSF). In order to improve organism detection at the Hospital for Tropical Diseases in Ho Chi Minh City, a diagnostic PCR-based protocol was developed. This protocol was followed in the investigation of CSF samples from 36 patients with clinical signs of bacterial meningitis. Each sample was first tested in a semi-nested PCR using primers for the 16sRNA gene common to all bacteria. The products of this reaction were then amplified using a 16sru8 primer and primers specific for Neisseria meningitidis, Haemophilus influenzae or Streptococcus spp. The samples found positive for Streptococcus were further investigated in a nested PCR using primers specific for the pneumolysin gene of S. pneumoniae. The sensitivity of detection was increased from 36% with culture to 44% with PCR. Although the sample size was small, the results indicate that PCR would be a feasible and useful adjunct in the diagnosis of bacterial meningitis, particularly in areas where community antibiotic use is common.

Antigens, Bacterial↗

PubMed2000

Monkeys of the rainforest in French Guiana are natural reservoirs for P. brasilianum/P. malariae malaria.

Monkey blood samples were collected from 214 monkeys relocated as part of the wildlife rescue organized in French Guiana during the filling of the Petit Saut Dam on the Sinnamary River. These samples were tested for malaria parasites by microscopy of thick blood filsm and by nested PCR for small subunit rRNA genes (SSUrRNA). Parasitic blood forms similar to Plasmodium brasilianum were detected in 4 monkey species: Alouatta seniculus macconnelli, Saguinus midas midas, Pithecia pithecia and Ateles paniscus paniscus, with the highest prevalence in Alouatta monkeys. PCR was more sensitive than the conventional method for detecting low-grade parasitaemia in positive monkeys. The examination of blood films indicated that 5.6% of the animals carried parasites whereas the nested PCR for ribosomal DNA indicated a prevalence of 11.3%. The P. brasilianum SSUrRNA gene sequence was analysed and aligned with those from P. malariae, P. falciparum and P. vivax. This suggested that P. brasilianum and P. malariae are very closely related. Similar results were obtained from analysis of the sequences in P. malariae and P. brasilianum isolates of a polymorphic gene fragment analogous to the merozoite surface protein-1 (MSP-1) gene of P. falciparum. The P. brasilianum/P. malariae sequences were more similar to those of P. vivax than to those of P. falciparum, at least in the gene region examined. The high degree of DNA homology in the sequences of the SSUrRNA and msp1-like genes is consistent with other characterizations demonstrating a taxonomic relationship between P. brasilianum and P. malariae species. Our results provide further evidence that P. brasilianum and P. malariae are virtually identical and should probably be considered to be a single malaria species. This raises the question as to whether monkeys living in the rainforest are natural reservoirs for both simian and human malaria. These results have implications for the interpretation of the current epidemiological situation in French Guiana.

Amino Acid Sequence↗

PubMed2005

Detection of Pneumocystis jirovecii dihydropteroate synthase polymorphisms in patients with Pneumocystis pneumonia.

In the present study, in order to improve the detection of Pneumocystis jirovecii dihydropteroate synthase (DHPS) mutations in pulmonary specimens of HIV-infected patients with P. jirovecii pneumonia, we evaluated a microfiltration procedure for the removal of human cell contamination and a nested-PCR method, for amplification in specimens with low parasite load. In the studied population, PCR amplification of the DHPS gene was more successful in unfiltered than in filtered specimens, with both touchdown-PCR and nested-PCR procedures (p<0.05 and p<0.001, respectively), but the amount of host DNA in the samples analysed seems to be inversely related with the successful PCR parasite detection. Amplification of P. jirovecii DHPS gene with nested-PCR was achieved in 77.5% of the specimens studied, demonstrating that this is a useful method for the identification of mutations in pulmonary specimens, including samples with low parasite loads, and will facilitate the evaluation of the relationship between the P. jirovecii DHPS polymorphisms and clinical resistance to sulfa drugs.

Bronchoalveolar Lavage Fluid↗

PubMed2006

Genetic organization and embryonic expression of the ParaHox genes in the sea urchin S. purpuratus: insights into the relationship between clustering and colinearity.

The ANTP family of homeodomain transcription factors consists of three major groups, the NKL, the extended Hox, and the Hox/ParaHox family. Hox genes and ParaHox genes are often linked in the genome forming two clusters of genes, the Hox cluster and the ParaHox cluster, and are expressed along the major body axis in a nested fashion, following the relative positions of the genes within these clusters, a property called colinearity. While the presences of a Hox cluster and a ParaHox cluster appear to be primitive for bilaterians, few taxa have actually been examined for spatial and temporal colinearity, and, aside from chordates, even fewer still manifest it. Here we show that the ParaHox genes of the sea urchin Strongylocentrotus purpuratus show both spatial and temporal colinearity, but with peculiarities. Specifically, two of the three ParaHox genes-discovered through the S. purpuratus genome project-Sp-lox and Sp-Cdx, are expressed in the developing gut with nested domains in a spatially colinear manner. However, transcripts of Sp-Gsx, although anterior of Sp-lox, are detected in the ectoderm and not in the gut. Strikingly, the expression of the three ParaHox genes would follow temporal colinearity if they were clustered in the same order as in chordates, but each ParaHox gene is actually found on a different genomic scaffold (>300 kb each), which suggests that they are not linked into a single coherent cluster. Therefore, ParaHox genes are dispersed in the genome and are used during embryogenesis in a temporally and spatially coherent manner, whereas the Hox genes, now fully sequenced and annotated, are still linked and are employed as a complex only during the emergence of the adult body plan in the larva.

Amino Acid Sequence↗

PubMed1991

Identification of deletion mutations and three new genes at the familial polyposis locus.

Small (100-260 kb), nested deletions were characterized in DNA from two unrelated patients with familial adenomatous polyposis coli (APC). Three candidate genes located within the deleted region were ascertained and a previous candidate gene, MCC, was shown to be located outside the deleted region. One of the new genes contained sequence identical to SRP19, the gene coding for the 19 kd component of the ribosomal signal recognition particle. The second, provisionally designated DP1 (deleted in polyposis 1), was found to be transcribed in the same orientation as MCC. Two other cDNAs, DP2 and DP3, were found to overlap, forming a single gene, DP2.5, that is transcribed in the same orientation as SRP19.

Adenomatous Polyposis Coli↗

PubMed2006

Panmixia: an example from Dawson's burrowing bee (Amegilla dawsoni) (Hymenoptera: Anthophorini).

Dawson's burrowing bee is a large, fast-flying solitary nesting bee endemic to the arid zone of Western Australia. In this study the population structure of the species was examined with molecular markers. Using eight microsatellite loci, we genotyped 531 adult female bees collected from 13 populations of Dawson's burrowing bee, Amegilla dawsoni, across the species range. The mean number of alleles per locus ranged from 4 to 38 and expected heterozygosity was uniformly high with a mean of 0.602. Pairwise comparisons of F(ST) among all 13 populations ranged from 0.0071 to 0.0122 with only one significant estimate and an overall F(ST) of 0.001. The entire sample collection was in Hardy-Weinberg equilibrium and there was no evidence of inbreeding with a mean F(IS) of 0.010. The mating and nesting behaviour of this bee suggests that gene flow would be limited by monandry and the fact that almost 90% of females mate immediately on emergence. Nevertheless there is obviously sufficient gene flow to maintain panmixia, and we suggest that this results from infrequent and unreliable rainfall in the species range, which causes the bees to congregate at limited food resources, allowing a small number of unmated females from one emergence site to come into contact with males from another population. In addition, when drought eliminates food resources near an emergence site, the whole population may move elsewhere, increasing gene flow across the species range.

Alleles↗

PubMed2000

A formal assessment of gene flow and selection in the fire ant Solenopsis invicta.

Recent studies of the introduced fire ant Solenopsis invicta suggest that introduced polygyne (with multiple queens per nest) populations are strongly influenced by male-mediated gene flow from neighboring monogyne (single queen per nest) populations and selection acting on a single locus, general protein-9 (Gp-9). This investigation formally tests this hypothesis and determines if these processes can account for the genotypic structure of polygyne S. invicta. To increase the statistical power of this test, we considered the genotypes of polygyne queens and workers at both Gp-9 and the closely linked, selectively neutral locus Pgm-3. We then constructed and analyzed a novel mathematical model to delimit the effects of monogyne male gene flow and selection on the joint genotypes at the Pgm-3/Gp-9 superlocus. Using this framework, a hierarchical maximum-likelihood method was developed to estimate the best-fitting gene flow and selection parameters based on the fit of our model to data from both the current study and an earlier one of the same population. In each case, selection on polygyne queens and workers alone, with no monogyne male gene flow, provides the most parsimonious explanation for the observed genotype frequencies. The apparent discrepancy between this result and the empirical evidence for monogype male gene flow indicates that undocumented factors, such as other forms of selection in polygyne males or workers, are operating in introduced polygyne S. invicta.

Animals↗

PubMed2001

PCR-based detection, restriction endonuclease analysis, and transcription of tonB in Haemophilus influenzae and Haemophilus parainfluenzae isolates obtained from children undergoing tonsillectomy and adenoidectomy.

We developed and evaluated a PCR-based-restriction endonuclease analysis method to detect and analyze the tonB gene of Haemophilus influenzae and Haemophilus parainfluenzae from pediatric patients undergoing tonsillectomy and adenoidectomy. Multiple sites from the same patient, including the surface of adenoids and tonsils, as well as the core of tonsils, were cultured on chocolate agar and identified using standard procedures and the API NH Kit. A total of 55 H. influenzae isolates were recovered from different sites of 20 patients, and 32 H. parainfluenzae isolates were recovered from various sites of 12 patients. DNA was extracted from American Type Culture Collection strains and test isolates by the PureGene kit. Two primers, G1 (21-mer) and G2 (23-mer), were designed by us to amplify by PCR the tonB gene that consists of an 813-bp fragment. A nested PCR using primers T1 (23-mer) and T2 (24-mer) that flank an internal sequence to the gene of the order of 257 bp and restriction endonuclease digestion using XhoI and BglII were done to detect whether heterogeneity within the gene exists between the two species. Reverse transcription-PCR (RT-PCR) was finally done to detect transcription of the gene in both species. Our data have shown that the tonB gene was detected in both species. It is known to encode a virulent protein, TonB, in H. influenzae; however, demonstration of its presence in H. parainfluenzae is novel. Nested-PCR and restriction endonuclease analysis have shown that the tonB gene is apparently structurally the same in both species, with possible differences that may exist in certain H. parainfluenzae isolates. RT-PCR done on selected numbers of H. influenzae and H. parainfluenzae have shown that the tonB gene was transcribed in both species. This shows that the TonB protein, if expressed, may play a different role in the virulence in H. parainfluenzae since it is not needed for heme or heme complexes uptake as with H. influenzae.

Adenoidectomy↗

PubMed2003

Prospects for tissue-specific analysis of gene expression in Xenopus embryos through laser-mediated microdissection of histological sections.

Analysis of spatiotemporal patterns of gene expression is an important prerequisite for understanding the molecular basis of embryogenesis. Tissue-specific resolution is desirable, but often not achieved owing to methodical limitations. We used a common model system for embryonic development--the South African clawed frog Xenopus laevis--to demonstrate that laser microdissection and laser-mediated catapulting of tissue samples from histologic sections are feasible even for yolk-rich, fragile embryonic tissue. A combination with RT-PCR provides the possibility of detecting tissue-specific gene expression with high resolution and fidelity. We show that specimens of various sizes and shapes can easily be procured by laser microdissection and pressure catapulting (LMPC). Subsequent RNA-isolation and nested RT-PCR for marker genes revealed that the combination of these methods allows for analysis of specific gene expression in micro-areas. We report on the efficiency and reliability of detection of marker genes in dissected tissue. We further discuss the question of whether such a combination can be applied to certain issues raised in developmental biology with regard to other techniques.

Animals↗