Analysis of antibiotic-induced drug resistance of Salmonella enteritidis and its biofilm formation mechanism.

This research was to explore antibiotic-induced drug resistance of Salmonella enteritidis and its biofilm formation mechanism. Kirby-Bauer (K-B) disk method recommended by Clinical and Laboratory Standards Institute (CLSI) was used to test drug sensitivity of Salmonella enteritidis to 16 kinds of antibiotics including ß-lactams, aminoglycosides, quinolones, sulfonamides, chloramphenicols, and tetracyclines. Polymerase chain reaction (PCR) was performed to detect carrying of drug resistance genes of 29 kinds of antibiotics including ß-lactams, aminoglycosides, quinolones, sulfonamides, chloramphenicols, and tetracyclines of Salmonella enteritidis. The expressions of esp, ebpA, ge1E, and fsrB genes in biofilm group and plankton group were detected when Salmonella was induced, and difference of gene expression was detected by FQ-PCR. The drug resistance rates of Salmonella enteritidis to nalidixic acid, ampicillin, streptomyces, and cefoperazone were high, which were 94.5%, 75%, 67%, and 52%, respectively. 94 strains of Salmonella enteritidis formed 22 kinds of drug resistance spectrum, the strains were generally resistant to 4-5 antibiotics, and some strains formed fixed drug resistance spectrum as follows: AMP-CFP-STR-NA-TE (22.6,21.7%), AMP-STR-NA-TE (17,16%), and AMP-CFP-STR-NA (11.1,10.6%). During biofilm formation, fsr can increase expression of ge1E and decrease expression of esp and ebpA. Consequently, Salmonella enteritidis was generally resistant to nalidixic acid, ampicillin, and streptomycin, and the multidrug resistance was severe. The drug resistance genes sul2, sul3, blaTEM-1-like, tet(A), and tet(G) were highly carried in Salmonella enteritidis. Esp, ebpA, ge1E, and fsrB genes were closely related to biofilm formation of Salmonella enteritidis.

Detection of KIT Genotype in Pigs by TaqMan MGB Real-Time Quantitative Polymerase Chain Reaction.

The dominant white phenotype in domestic pigs is caused by two mutations in the KIT gene: a 450 kb duplication containing the entire KIT gene together with flanking sequences and one splice mutation with a G:A substitution in intron 17. The purpose of this study was to establish a simple, rapid method to determine KIT genotype in pigs. First, to detect KIT copy number variation (CNV), primers for exon 2 of the KIT gene, along with a TaqMan minor groove binder (MGB) probe, were designed. The single-copy gene, estrogen receptor (ESR), was used as an internal control. A real-time fluorescence-based quantitative PCR (FQ-PCR) protocol was developed to accurately detect KIT CNVs. Second, to detect the splice mutation ratio of the G:A substitution in intron 17, a 175 bp region, including the target mutation, was amplified from genomic DNA. Based on the sequence of the resulting amplified fragment, an MGB probe set was designed to detect the ratio of splice mutation to normal using FQ-PCR. A series of parallel amplification curves with the same internal distances were obtained using gradually diluted DNA as templates. The CT values among dilutions were significantly different (p < 0.001) and the coefficients of variation from each dilution were low (from 0.13% to 0.26%). The amplification efficiencies for KIT and ESR were approximately equal, indicating ESR was an appropriate control gene. Furthermore, use of the MGB probe set resulted in detection of the target mutation at a high resolution and stability; standard curves illustrated that the amplification efficiencies of KIT1 (G) and KIT2 (A) were approximately equal (98.8% and 97.2%). In conclusion, a simple, rapid method, with high specificity and stability, for the detection of the KIT genotype in pigs was established using TaqMan MGB probe real-time quantitative PCR.

Correlations between Epstein-Barr virus and acute leukemia.

Infection with Epstein-Barr virus (EBV) may be correlated to the onset of acute leukemia (AL). Studies were performed to investigate the relationship between EBV infection and immunophenotyping of acute lymphoblastic leukemia (ALL) and chromosome aberrations. Additionally, the effects of EBV on clinical prognosis were described. Fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect EBV-DNA copy numbers from bone marrow in 110 cases of patients with ALL, 75 cases of patients with acute myeloid leukemia (AML), and 37 cases of hematologically healthy control subjects. EBV-DNA copies were found in 64 of 185 (34.6%) patients with AL and 2 of 37 healthy control subjects (P < 0.001). The EBV infection rate of ALL patients, AML patients, and healthy controls was 40.9% (45/110), 25.3% (19/75), and 5.4% (2/37), respectively. The positive rate of EBV in the ALL and AML groups was higher than in healthy subjects (P < 0.05). EBV-DNA copies were found in 1 of 12 (8.3%) T-ALLs and 44 of 98 (44.9%) B-ALLs, the positive rate of EBV in B-ALLs increased significantly compared to the rate of T-ALLs (P < 0.05). Chromosome karyotype analysis showed that EBV infection rates in B-ALL, T-ALL, and AML patients with chromosome abnormalities were 46.3% (25/54), 33.3% (1/3), and 30% (9/30), respectively. There was no statistical significance between chromosome abnormality and EBV infection (P > 0.05). In addition, EBV+ -ALLs had higher relapse and mortality rates than that of EBV- -ALLs. Together, we conclude that EBV infection may be correlated with the incidence of AL, and the infection rate of EBV in B-ALL was higher than that of T-ALL. EBV-positive patients showed an unfavorable prognosis.

Expression of Epstein-Barr Virus DNA in Peripheral Blood Lymphocytes and Plasma in Patients with EBV-Associated Diseases / 现代检验医学杂志

Objective To explore the expression of Epstein-Barr virus DNA(EBV DNA)in the peripheral blood lymphocytes and plasma of patients with EBV-associated diseases.Methods The whole blood samples were collected from 112 patients with suspected EB virus infection diseases,including 14 cases of nasopharyngeal carcinoma(NPC),16 cases of infectious mononucleosis(IM),22 cases of lymphoma,23 cases of autoimmune disease,26 cases of upper respiratory tract infection, and 11 cases of abnormal liver function.The levels of EBV DNA in lymphocytes and plasma of the same sample were detec-ted by fluorescence quantitative PCR(FQ-PCR).Results The EBV DNA positive rates in lymphocytes and plasma of all 112 patients were 83.0%(93/112)and 27.7%(31/112)respectively,with statistically significant difference(χ2=60.02,P<0.01).The positive rate and the load of EB virus DNA in lymphocytes and plasma of 14 patients with nasopharyngeal car-cinoma(NPC)had no statistical difference(χ2=2.25,t=-1.04,all P>0.05).However,patients with lymphoma,infec-tious mononucleosis,upper respiratory tract infection,autoimmune disease or abnormal liver function,the positive rates and the concentration of EBV DNA in the plasma were dramatically lower than those in the peripheral blood lymphocytes,and the difference was statistically significant(χ2=4.17~15.06,all P<0.05;t=3.94~10.45,all P<0.01).Conclusion The detection of EB DNA in peripheral blood lymphocytes of non NPC patients by FQ-PCR might be better than that in plasma. There was no statistical difference between the detection of EBV DNA in lymphocytes and plasma of patients with nasopha-ryngeal carcinoma.Appropriate specimen type could be selected according to clinical consideration.

Detection of decorin expression in keloid with fluorescent quantitative-PCR / 中华医学美学美容杂志

Objective To detect the expression and content of decorin in fibroblasts of keloid to deeply reveal the mechenism and the role of decorin plays in scar formation.Methods Fibroblasts of keloid,normal scar and normal skin were cultured in vitro,and the morphology,activity,apoptosis of fibroblast were observed under light microscope and electron microscope; the mRNAs of decorin and TGF-β1 were detected and analyzed with real-time fluorescent quantitative-PCR (FQ-PCR).Results Fibroblasts of keloid showed irregular morphology,larger size and disorder arrangement.There were a large number of mitochondria,swelling rough endoplasmic reticulum,and euchromatin-rich in nucleus of fibroblasts,suggesting the protein synthesis of keloid fibroblast was very active.Compared with normal skin,the expression of decorin was significantly lower in keloid fibroblast; On the contrary,the expression of TGF-β1 was significantly higher in keloid fibroblast than in normal scar and normal skin.Conclusions Compared with normal skin,the expression of decorin in keloid fibroblast is significantly lower.Lower content of decorin in early stage of wound healing may induce weakly suppression of proliferation and synthesis of fibroblast,and up-regulate the activity of TGF-β1,which promotes the proliferation,migration and excessive collagen synthesis of the fibroblast of keloid.Thus,decorinis an suppressor factor of keloid formation.

Expression and clinical pathological significance of BCSG1in invasive breast cancer / 中华内分泌外科杂志

Objective To investigate the expression of breast cancer specific gene 1( BCSG1) in breast cancer tissues and its clinical significance.Methods The expression of BCSG1in 51cases of breast cancer tissues and 35 cases of adjacent tissues was determined by fluorescence quantitative polymerase chain reaction (FQ-PCR) and immunohistochenistry.Results FQ-PCR showed the expression level of BCSG1mRNA in breast cancer and adjacent tissues was 1.072 ± 0.178 and 0.324± 0.135 respectively.Immunohistochemistry showed the expression rate of BCSG1protein in breast cancer and adjacent tissues was 74.5％ (38/51) and 14.3％ (5/35).The expression of BCSG1mRNA and protein was dramatically increased in breast cancer tissues compared with that in normal background breast tissues (P ＜ 0.05).BCSG1mRNA and protein expression was significantly different in patients with lymph node metastasis and higher Nottingham prognostic index (P ＜ 0.05 ),and there was no significant difference with the pathological type of breast cancer,or status of ER,PR,and c-erbB2 ( P ＞ 0.05 ).Conclusions BCSG1is highly expressed in breast cancer tissues,which is closely related to the development and clinical prognosis of breast cancer.BCSG1is very likely to play an important role in the pathogenesis of breast cancer.

Fluorescence quantitative PCR in diagnosing and treating infantile cytomegalovirus infection / 国际儿科学杂志

Fluorescence quantitative PCR (FQ-PCR) is a more sensitive and specific method to diagnose and monitor cytomegalovirus(CMV) infection, and it is closely correlated with pp65 antigenemia levels. Measuring the CMV-DNA levels in infant urine and blood can be helpful to regulate the treatment and monitor the effects of the antivirus therapy.

Development of an HBV genotyping method by nucleic acid strip / 中华微生物学和免疫学杂志

Objective To develop an rapid, visuable method to detect the genotypes of HBV.Methods According to the published full-length sequence with known genotypes in GenBank, the specific primer and biotin-lablled probe were designed. We established a nucleic acid strip method for genotyping hepatitis B virus(HBV) isolates among 150 HBV infected patients and 20 healthy controls, and compared the results with those obtained by real-time PCR. Results There was 34.00% genotype B , 61.33% genotype C, 4.00% genotype B/C in 150 samples, respectively, while the remaining 0.67% unknown. The results for this assay were high comparable to the method of real-time PCR. Conclusion With the similar sensitivity and specifity when compared with real-time PCR, this rapid method is suitable to the clinical setting.

Antiviral Activity of Recombinant Cyanovirin-N against HSV-1 / 中国病毒学·英文版

In this study,a standard strain of HSV-1(strain SM44)was used to investigate the antiviral activity of the recombinant Cyanovirin-N(CV-N)against Herpes simplex virus type 1(HSV-1)in vitro and in vivo.Cytopathic effect(CPE)and MTT assays were used to evaluate the effect of CV-N on HSV-1 in Vero cells.The number of copies of HSV-DNA was detected by real-time fluorescence quantitative PCR(FQ-PCR).The results showed that CV-N had a low cytotoxicity on Vero cells with a CC50 of 359.03±0.56 μg/mL,and that it could not directly inactivate HSV-1 infectivity.CV-N not only reduced the CPE of HSV-1 when added before or after viral infection,with a 50% inhibitory concentration(IC50)with 2.26 and 30.16μg/mL respectively,but it also decreased the copies of HSV-1 DNA in infected host cells.The encephalitis model for HSV-1 infection was conducted in Kunming mice,and treated with three dosages of CV-N(0.5,5 & 10 mg/kg)which was administered intraperitoneally at 2h,3d,5d,7d post infection.The duration for the appearance of symptoms of encephalitis and the survival days were recorded and brain tissue samples were obtained for pathological examination(HEstaining).Compared with the untreated control group,in the 5mg/kg CV-N and 10mg/kg CV-N treated groups,the mice suffered light symptoms and the number of survival days were more than 9d and 14d respectively.HE staining also showed that in 5mg/kg CV-N and 10mg/kg CV-N treated groups,the brain cells did not show visible changes,except for a slight inflammation.Our results demonstrated that CV-N has pronounced antiviral activity against HSV-1 both in vitro and in vivo,and it would be a promising new candidate for anti-HSV therapeutics.

Real-time fluorescent quantitative PCR assay for detection of human Bocavirus / 临床儿科杂志

Objective To establish the method for detecting human Bocavirus (HBoV) by a real-time fluorescent quantitative PCR (FQ-PCR) and to assay HBoV from nasopharyngeal samples of children with acute respiratory tract infections.Methods The specific primers and Taqman probes of targeted HBoV NP1 gene were designed.The aimed fragment of NP1 gene was amplified with PCR and ligated into a pCEM-T Easy vector to set up the standards.The method for detection of NP1 gene using FQ-PCR was established.The sensitivity and specificity of the method had been tested.Then 195 clinical specimens were subsequently tested.Results The specificity of this method was excellent.The linear amplification of the assay ranges from 10 copies/μl to 10~8 copies/μl.Twelve specimens were tested positive for HBoV in 195 clinical specimens (6.2%).Conclusions The method for detection of HBoV NP1 geneby real-time fluorescent quantitative PCR was established successfully.It can offer fast and reliable diagnosis for HBoV infection.

Analysis of Chlamydia trachomatis Infection among 1509 Female Patients / 中华医院感染学杂志

OBJECTIVE To investigate the infection conditions of Chlamydia trachomatis in the women who were suffering from vaginitis,cervicitis or pelvic inflammatory disease.METHODS Collecting cervical secretion and using fluorescent quantitative polymerase chain reaction(FQ-PCR) technology to examine the infection conditions of C.trachomatis in 1001 vaginitis,302 cervicitis and 206 pelvic inflammatory disease women.RESULTS In 1001 vaginitis patients,113 samples were C.trachomatis positive(11.29%).Among 302 cervicitis patients,45 samples were C.trachomatis positive(14.09%).In 206 pelvic inflammatory disease patients,29 samples were C.trachomatis positive(14.08%).CONCLUSIONS The infection rate of C.trachomatis in vaginitis,cervicitis and pelvic inflammatory disease women is relatively higher.

By Fluorescence Quantitative Polymerase Chain Reaction CMV Viral Hepatitis in the Application / 医学研究杂志

Objective To investigate the fluorescence quantitative polymerase chain reaction(FQ-PCR)in the giant cell viral hepatitis in value.Methods 50 cases of serum IgM HCMV-positive patients with hepatitis and 25 cases of healthy people to do the control group respectively serum FQ-PCR detection of its content HCMVDNA its detection rate compared with the liver function and the relationship.Results Of 50 cases of serum IgM HCMV-positive patients with hepatitis HCMVDNA>103Copies/ml those 35 cases,HCMVDNA positive to the negative group than HCMVDNA serum total bilirubin(TBiL)alanine aminotransferase(ALT)aspartate aminotransferase(AST)to the high statistics P <0.05 the difference was significant.25 cases of healthy control group and HCMVDNA HCMV-IgM were negative.Conclusions FQ-PCR in cytomegalovirus infection with hepatitis reliable early diagnosis of the characteristics through HCMVDNA content of the clinical evaluation of drug efficacy has certain clinical value.

An exploration of relationship between the infection of Ureaplasma Urealyticum and Chlamydia Trachomatis and male sterility / 中国艾滋病性病

Objective To discuss the relationship between the infection of Ureaplasma Urealyticum(Uu) and Chlamydia trachomatis(Ct) and male sterility.Methods To detect 476 specimens of sperm by FQ-PCR,326 specimens were collected from sterile men(sterility group) and 150 specimens from normal men(control group).Results The total detection rate of Uu and Ct was 49.69% in sterility group,being significantly higher than 16.67% in control group(?~2=46.98,P

Detection of RFC,DHFR and GST-? expression in human osteosarcoma cell line with MTX-resistence by FQ-PCR / 中国矫形外科杂志

[Objective]To study the difference in expression of RFC, GST-?, DHFRmRNA between human osteosarcoma U2-OS cell line and the MTX-resisitant variants U2-OS/R1-R3, and to investigate the significance in MTX resistance for human osteosarcoma. [Methods]Three resistant MTX human osteosarcoma cell lines were established by pulse exposure parental cell line(U2-OS) in gradually increased dose of MTX . The expression of RFC,GST-?,DHFRmRNA were assayed by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR).[Results]Three MTX-resistant variants(U2-OS/R1-R3) were successfully established , the results of the FQ-PCR revealed that the MTX resistance was associated with the decreased expression of the RFC mRNA and increased expression of DHFR mRNA and GST-? mRNA.[Conclusion]The author investigated the MTX resistant mechanism of human osteosarcoma cell line at a gene level. The decreased expression of RFC mRNA and the increased expression of DHFR mRNA and GST-? mRNA participate in the MTX resistance in human osteosarcoma cell lines U-2 OS. This provides the evidence for exploring the MTX resistance mechanism in clinical osteosarcoma patients ,and helps to screen the patients who are insensitive to MTX chemotherapy.

Detection of Kallkreins 4 mRNA in human breast cancer tissue using a quantitative real-time PCR method / 临床检验杂志

Objective To develop a real-time fluorescence quantitative PCR(FQ-RCR)method for detection of Kallkreins(KLK)4 gene expression in human breast cancer,and to investigate KLK4 expression levels in breast cancer and normal tissues.Methods KLK4 expression levels of 25 normal breast tissues and 39 cancer tissues were detected by the developed real-time quantitative PCR method.The statistical analysis for the relationship between KLK4 expression and several pathological parameters was performed by t test.Results The levels of KLK4 mRNA in normal breast and breast cancer tissue were 0.0120?0.0044 and 0.0272?0.0067 respectively(P0.05).Conclusions The level of KLK4 mRNA in breast cancer tissue was higher significantly than that in normal breast tissue.The results indicated that KLK4 gene expression may have relevance with breast cancer development but have no significant relevance with ER,PR,CerbB-2 and tumor metastasis.

Serum IL-18 Level in HBV-infected Patients and Its Clinical Significance / 中华医院感染学杂志

OBJECTIVE To investigate the relationship between HBV DNA and serum IL-18 level in patients with hepatitis B virus. METHODS They were divided by MEIA method into 3 groups. group A:HBsAg+, HBeAg+ and HBcAb +(A); group B:HBsAg+, HBeAb+ and HBcAb+(B); and group C:HBsAb+(as normal controls ). Serum IL-18 level was determined by ELISA method in hepatitis patients and normal controls and HBV DNA was determined continuously . RESULTS Concentration of serum IL-18 in patients with different type of viral hepatitis was significantly higher than those in healthy volunteers(P

Effect of Astragalus injection on the expression of p53 mRNA in human nasopharyngeal carcinoma cell line CNE2 xenograft in nude mice / 中国药理学通报

Aim To study the effect of Astragalus injection on tumor growth inhibition of human nasopharyngeal carcinoma cell strain CNE2 xenograft in BALB/c nude mice,and explore the possible mechanisms.Methods CNE2 cells were injected subcutaneously into nude mice to establish model of transplanted tumor.Twenty one model nude mice were divided randomly into three groups treated with intraperitoneal injection,viz the model group given normal saline 10 ?l?(g?d)~-1,the positive control group given Cisplatin 30 mg?m~-2 every fourteen days and given normal saline sodium 10 ?l?(g?d)~-1 in other days,and the treatment group given Astragalus injection 10.40 mg?(g?d)~-1.The above administrations for groups lasted 4 weeks.The inhibitory effect of Astragalus injection on the growth of tumors in nude mice was observed,and the inhibitory rate and the relative tumor proliferation rate were calculated.The expression levels of p53 mRNA in tumor tissues were determined by FQ-PCR.Results After four weeks′treatment,the volumes of tumors in nude mice from the model group,the Cisplatin group,the Astragalus injection group were(1.51?0.25)cm~3,(0.91?0.35)cm~3 and(1.04?0.51)cm~3 respectively.Compared with the model group,the tumor volumes in the Cisplatin group and in the Astragalus injection group were smaller(P0.05).Conclusions The results suggest that Astragalus injection shows inhibitory effect on the growth of human nasopharyngeal carcinoma cell strain CNE2 xenograft in BALB/c nude mice,while it can not be claimed that Astragalus injection may affect the expression level of p53 mRNA in transplanted tumor tissue.

The Study on the Levels of HBV-DNA in Culture Supernatants of Peripheral Blood Mononuclear Cells From HBeAg Positive Carriers / 中国医师杂志

5000 copy/ml were both significantly higher than those in the controls.The levels of HBV-DNA positive in cultured supernatants of PBMC from suffering grave hepatitis B were higher than those in the sera by qualitative PCR of the same period.They were similar between the positive rate of quantitative PCR of HBV-DNA in sera and FQ-PCR in cultured supernatants of PBMC from hepatitis B types.They were not obviously related with the levels of ALT and TBiL.Conclusions The IL-12 and ? IFN secretion increased when the HBV-DNA of PBMC from carriers of HBeAg were at high-reproducing state.The HBV-DNA positive rate in cultured supernatants of PBMC of grave hepatitis detected by FQ-PCR was higher than that in sera by qualitative PCR.

Establishment of the technique for the real-time fluorescence quantitative reverse transcription polymerase chain reaction by DNA melting curve analysis for detecting the CDR3 skewing of TCR alpha gene repertoire in the human peripheral blood / 中国免疫学杂志

Objective:To establish the technique for real-time fluorescence quantitative reverse transcription polymerase chain reaction(FQ-PCR)by DNA melting curve analysis for detecting the CDR3 shewing of TCR alpha gene repertoire in human peripheral blood.Methods:Total RNA of peripheral blood mononuclear cell(PBMC)from 4 healthy donors and 2 patients with lymphomatous leukemia were transcripted reversely into cDNA.The cDNA of 32 TRAV gene family CDR3 was amplified by FQ-PCR.Analysis of the monoclonal/oligoclonal/polyclonal CDR3 spectratyping with DNA melting curve.Results:The FQ-PCR products of 32 TRAV family CDR3 were showedas a blur land at the predicted of products size in healthy donors and parts of TRAV family CDR3 products disappeared in patients on 1.5% agarose gel by Gold-View staining.The 32 TRAV family CDR3 were showed with different frequencies by relative fluorescence quantitative in healthy donors and the patients.The CDR3 spetratyping for 32 TRAV families was showed as polyclonal peak(Gaussian distribution)in healthy donors but showed as different monoclonal/oligoclonal/polyclonal peak in the patients with lymphomatous leukemia with DNA melting curve analysis(we called "melting curve spectratyping of CDR3")Conclusion:The study suggests that the technique of "FQ-PCR with DNA melting curve analysis be convenience and celerity for detecting the CDR3 skewing of TCR alpha gene repertoire in human peripheral blood.

Study on PBR expression in telencephalon of schistosomiasis mice with liver fibrosis / 中国血吸虫病防治杂志

Objective To explore the change of peripheral benzodiazepine receptors (PBR) expression in the telencephalon of mice with schistosomiasis. Methods The changes of PBR mRNA in the telencephalon of schistosomiasis mice were observed by the real-time FQ-PCR method. The results were judged with circle number of times (C_T) when the level of amplification exponent achieved detecting liminal value. Results Comparing the experiment group (n=10) with the healthy control (n=10) at the 8th、12th、20th week, the changes of C_T were significant(P