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Objective To detect the distribution of H.pylori ureA, vacA s1 gene and cagA subtype(ABC, ABD, ABAB, AAD, et al) in patients with digestive diseases in Guangzhou and investigate the relationship with the pathological findings of gastric mucosa.Methods A total of 227 randomly selected gastric mucosa from patients with digestive diseases were enrolled in the research, including 46 without pathological changes, 130 with chronic gastritis, 29 with peptic ulcer, 15 with atrophic gastritis and 7 with gastric cancer.Real-time PCR assay were used to detect Helicobacter pylori ureA gene and vacA s1 gene.EPIYA motifs in the 3′ region of cagA were amplified by conventional PCR followed by subtype sequencing. The conserved gene ureA was used to detect H.pylori infection.Results Among the 227 patients with digestive diseases, 50.7% (115/227) patients were H.pylori positive, in which 91.3%(105/115) carried vacA s1 and 78.3% (90/115) carried cagA. Four types of cagA-EPIYA subtype were detected, including ABC 17.8%(16/90), ABD 78.9%(71/90), AAD 2.2%(2/90) and ABAB 1.1%(1/90).In the non-pathological change group, 32.6% (15/46) were H.pylori positive, in which 28.3% (13/46) carried vacA s1 and 26.1% (12/46) carried cagA;in chronic gastritis group, it was 48.5% (63/130), 43.8% (57/130) and 36.2% (47/130), respectively;in ulcer group, it was 72.4% (21/29), 65.5% (19/29) and 55.2% (16/29), respectively;in atrophic gastritis group, it was 66.7% (10/15), 66.7% (10/15) and 66.7% (10/15), respectively;in gastric cancer group, it was 85.7% (6/7), 85.7% (6/7) and 71.4% (5/7), respectively.The distribution of H.pylori among the 4 groups had statistical significance (χ2=16.72;P<0.01). H.pylori was more prevalent in ulcer, atrophic gastritis and cancer group than in inflammation group and non-pathological change group (χ2=16.02;P<0.01).In patients infected by H.pylori, there was no significant difference in the distribution of vacA s1 gene as high virulence factors among non-pathological change, inflammation, ulcer, atrophic gastritis and cancer group (χ2=2.00;P=0.74), as well as cagA (χ2=3.44;P=0.49) and EPIYA subtypes (χ2=3.66;P=0.45).Conclusions H.pylori infection is significantly associated with the pathological change of gastric mucosa for patients with digestive diseases in Guangzhou, while the relationship with the pathogenicity of H.pylori with high virulence genotype is not significant.More samples and diseases reclassification are needed to make an advanced analysis of the effect of H.pylori with high virulence in gastrointestinal diseases development.
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Objective To investigate the effect of low molecular weight substance extracted from chick embryo on mitochondrial DNA(mtDNA)deletion in senile mice induced bv D-galactose.Methods Senile mice induced by D-galactose were treated with low molecular weight substance extracted from chick embryo.The deleted fragment of mtDNA was examined by using polymerase chain reaction technique and agarose gel electrophoresis.A relative quantitation of band densities was performed by using densitometry scanning techniques.The deletion was identified by using direct sequencing analysis. Results The 4239 bD mtDNA deletion were present in liver,cerebral cortex and hippocampus tissues in all of the mice.and it was significantly higher in senile model mice than in control mice(all P＜0.01).Low molecular weight substance extracted from chick embryo reduced the mtDNA deletion in various tissues in senile model mice(all P＜0.05).Deletion was more abundant in liver than in cerebral cortex and hippoeampus. Conclusions The 4239 bp mtDNA deletion is a common deletion,and low molecular weight substance extracted from chick embryo could decrease the incidence of 4239 bD mtDNA deletion.
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The epidemiological effects of native and mutated YMDD motif in the HBV genome under the selective pressure of lamivudine were investigated. YMDD wild and mutation motif in HBV genome were detected by flow through reverse dot blots (FT-RDB) with KaiPuTM DNA HybriMax Rapid Hybridization Machine based on the principle of "Flow-through hybridization" and by the traditional Reverse Dot Blot assay. Sera from 1021 suspected lamivudine-resistant chronic HBV carders after more than 8 months of lamivudine therapy and the corresponding archived sera were collected and assayed. We found 35.94% were single type infections with 8.03% YMDD, 7.93% YIDD and 19.98% YVDD. It was also found that 64.06% were mixed infections including 1.96% YMDD and YIDD, 51.62% YMDD and YVDD, 1.96% YIDD and YVDD, 8.52% YMDD, YIDD and YVDD. The levels of infections containing YVDD motif reached 82.08%. The pretreatment infectious status were: YMDD single infection was 36.93%; YIDD single infection was 6.07%; YVDD single infection was 17.04%; YMDD and YIDD mixed infection was 0.97%; YMDD and YVDD mixed infection was33.99%; YIDD and YVDD mixed infection was 0.98%; YMDD, YIDD and YVDD mixed infection was 4.02%. Infections containing YVDD motif were only 56.03%. The 34.32% mutation rate of YMDD motif to YVDD was significantly higher than the 10.97% of YMDD to YIDD (U=10.98, P<0.05), as estimated by Mann-Whitney U-test for non-parametric data. HBV containing YVDD motif might have an evolutionary ascendancy and become the dominant type under the selective pressure of lamivudine.
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Objective To establish a convenient, fast, economic and hypersensitive low-density DNA array method to detect the genotypes of human papillomavirus (HPV) and evaluate its application in clinic services.Methods HPV in cervical swab samples from 355 suspected female patients collected in gynaecology and obstetrics clinic were genotyped by hybrid capture (HC) II method and low-density DNA array simultaneously. HPV in 730 clinic samples from the area of Pearl River delta were genotyped by low-density DNA array.Results Among 355 suspected samples positive HPV-DNA were detectable in 211 (59.4%) samples by DNA array and 222 (62.5%) samples by HCII method. The concordance rate between the two assays was 94.1%. In the HPV genotypes 15 high-risk type and 5 low-risk type were detected by low-density DNA array. The most common high-risk types were HPV-16, 52, 58 and 56. The peak age of HPV infection was 26-30 years. The distribution of genotypes was different from various degree of cervical changes.Conclusion Either single type or multiple type of HPV infection can be detected by low-density DNA array. The combination of HPV detection with cytology detection will provide instruction for cervical cancer screening.
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<p><b>OBJECTIVE</b>To establish a fluorescent polymerase chain reaction (F-PCR) method for detecting the coronavirus related to severe acute respiratory syndrome (SARS) and to evaluate its value for clinical application.</p><p><b>METHODS</b>The primers and the fluorescence-labeled probe were designed and synthesized according to the published sequence of the SARS-associated coronavirus genes. A F-PCR diagnosis kit for detecting the coronavirus was developed, and 115 clinical nasopharyngeal gargling liquid samples were tested.</p><p><b>RESULTS</b>The sequence of PCR amplified products completely matched the related sequence of the SARS-associated coronavirus genome. Forty-nine out of 67 samples from identified SARS patients and 8 of 18 samples from persons having close contact with SARS patients showed positive results. All 30 samples from healthy controls were negative.</p><p><b>CONCLUSION</b>The F-PCR method established may be a rapid, accurate and efficient way for screening and for the early diagnosis of SARS patients.</p>
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Humans , Fluorescence , Polymerase Chain Reaction , Methods , Severe acute respiratory syndrome-related coronavirus , Genetics , Severe Acute Respiratory Syndrome , VirologyABSTRACT
<p><b>OBJECTIVE</b>To establish a fluorogenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of bcr/abl mRNA fusion gene expression level in leukemia cells, and provide a useful tool for leukaemia diagnosis and minimal residual disease inspectation.</p><p><b>METHOD</b>The conventional RT-PCR was used to amplify bcr/abl gene from cultured K562 cells, the quantitative standard template was constructed with A-T clone method. The fluo-rogenic quantitative RT-PCR method by using Applied Biosystems 7700 Sequence Detector for detecting the expression of bcr/abl fusion gene was successfully. The sensitivity, stability and repetitiveness of this method was determined. The peripheral blood samples from 14 CML patients, one of whom before and one month after bone marrow transplantation (BMT) and 4 cases of ALL in the early stages were detected.</p><p><b>RESULTS</b>The sensitivity of FQ-RT-PCR for detecting bcr/abl fusion gene was about 10(-5) micro g RNA from K562 cell and 10 copies recombined plasmid. The repetition CT value (cycle threshold) and the coefficient variation (CV) among tubes and batches were 2.0% and 3.7%, respectively. The median bcr/abl fusion gene expression level of 14 CML patients was 5.15 x 10(4) copies/ micro g RNA. The products analyzed by electrophoresis showed that 11 cases were b2a2 and 3 cases b3a2. 1.2 x 10(5) copies/ micro g RNA in one CML patient before BMT was changed to 2.3 x 10(3) copies/ micro g RNA one month after BMT. B2a2 was observed in one of the four (25.0%) patients with ALL, and its level of expression was 8.2 x 10(5) copies/ micro g RNA.</p><p><b>CONCLUSION</b>The established FQ-RT-PCR method is sensitive, specific, reliable, accurate and good at repetitiveness. The results expressed in copies were easy for evaluation and comparation. Two different bcr/abl fusion gene form - b2a2, b3a2 can be detected by the method. It can be widely applied to diagnosis and detection of minimal residual disease for CML and some ALL patients.</p>
Subject(s)
Humans , Fusion Proteins, bcr-abl , Genetics , Genes, abl , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Genetics , RNA, Messenger , Genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Objective:Use a new F-PCR method to develop a hepatitis C virus(HCV) diagnostic kit, test the kit through clinical trial and compare it with immunological method. Methods: Fluorescence PCR(F-PCR) is a method which combines PCR and fluorescence probe hybridization together to measure DNA/RNA. Because in-tube monitoring of fluorescence signal can be done to stand for the quantitity of PCR product. Electrophoresis and UV detection are eliminated, so after-PCR cross-contamination which causes false positive can be avoided. Results:A clinical diagnostic kit for HCV with this method is developed. 512 clinical serum samples were tested with this kit, using HCV FLISA kit from Abbott Co.and HCV Fluorescence RT-PCR kit from Biotronics Co. (B-PCR) as control. The results shows :positive rate is 30.5%,sensitivity 97.3 % and specificity 98.1% . Conclusion: F-PCR is obviously superior to ELBA, and higher than B-PCR in sensitivity. The specificity of those three kits have no statistic difference. F-PCR can be used to monitor RNA of HCV in serum, and could be useful for clinical diagnose and therapy effects monitoring.
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【Objective】 To detect the copy numbers of brain-deriv ed neurotrophic factor (BDNF) gene in BDNF transfected PcDNA3.1(+)/BDNF/CHO cel ls with fluorescent quantitative polymerase chain reaction (FQ-PCR). 【Methods 】 BDNF DNA were amplified by GeneAmp 5700 Sequence Detection System with eq ual quantitative genomic DNA of PcDNA3.1(+)/BDNF/CHO, PcDNA3.1(+)/CHO and CHO cells as tamplates respectively. The process was repeated 30 times for every sam ples. The results were analyzed using q test. 【Results】 The copy numbers o f BDNF of PcDNA3.1(+)/BDNF/CHO cells and PcDNA 3.1(+)/CHO and CHO cells were 9 5 164±12, 31 622±10, 31 622±11 respectively. The copy numb ers of BDNF of PcDNA3.1(+)/BDNF/CHO cells were as three times as those of the P cDNA3.1(+)/CHO and CHO cells. The copy numbers of the two latters were the same . 【Conclusion】 The results clearly show that the PcDNA3.1(+)/BDNF/CHO cells h arbor two BDNF DNA copies.
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Objective To investigate the effects of low molecular weight polypeptide(LMP) extracted from chicken embryo on learning and memory in senile mice induced by D-galactose(Gal). Method The 16 d old chicken embryo was acid and alkali hydrolyzed,enzymolyzed and ultrafiltered to formulate LMP. 48 mice were divided randomly into four groups: control,aging model ,aging+low dose LMP,aging+high dose LMP. The aging model and two LMP groups were treated with Gal 80 mg/ (kg bw?d) by nape subcutaneous injection,while the control group with normal saline 8ml/(kg bw?d). Control and model groups were given i.g. normal saline 20 ml/(kg bw?d),and two LMP groups 10 ml or 20 ml/(kg bw?d) LMP respectively. Learning and memory of mice were tested with Morris water maze. Results The escape latency of model group was longer and the percentage of swimming distance in platform region higher than that of control group (P
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This study presented a systematic observation on the human peripheral nerves by Karnovsky histochemical method for acetylcholinesterase (AChE). Some major nerve trunks and their branches from twenty upper limbs, a part of the muscular and cutaneous nerves of the lower limbs, and the ventral and dorsal roots and the gray communicating rami of C_7 from six human cadavers were obtained within 3 hours postmorten for this study. In the sensory nerve fascicles, the myelinated axons failed to react and the unmyelinated fibres showed strong enzymatic activity. In the motor nerve fascicles, 58% of the myelinated axons showed enzymatic activity, while the unmyelinated fibres which showed strong enzymatic activity, were much less than those in the sensory nerve fascicles. 87% of the myelinated axons in the ventral roots from the C_7 showed morphologically the same enzymatic activity as those in the motor nerve fascicles. The myelinated axons failed to show an enzymatic activity and the unmyelinated fibres showed very slight enzymatic activity in the dorsal roots. A strong enzymatic activity was shown in the gray communicating rami. Thus it is clear that both myelinated and unmyelinated fibres show different enzymatic activities in the motor and sensory nerve fascicles, and that those myelinated fibres showing enzymatic activity originate from the ventral roots and the unmyelinated fibres showing strong enzymatic activity originate from the gray communicating rami.