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Objective:To establish a real-time quantitative PCR assay to detect the copy number of IS711 transposase gene (orfA) in Brucella genome, and the assay is applied to identify the species and biovars of Brucella. Methods:To establish an orfA gene copy number detection system based on Taqman real-time quantitative PCR technique. Primers and probes of bcsp31 and orfA genes were designed, the contents of bcsp31 gene and orfA gene in the same strain with the same DNA concentration were simultaneously detected by real-time quantitative PCR assay, and cycle number (CT value) of the two genes were obtained. According to the differences of CT values of bcsp31 gene and orfA gene, the copy number of orfA gene in Brucella genome was calculated. At the same time, the DNA of Brucella 16M strain was double decreasing dilution to verify the stability of the detection system. Results:A real-time quantitative PCR assay was used to detect bcsp31 gene and orfA gene simultaneously, when the DNA concentration difference of 16M strain was 2 times, the mean difference of CT values measured was 1.00, 95% confidence interval was 0.95-1.05, standard deviation was 0.17, and coefficient of variation was 0.17. The orfA gene copy number of 30 Brucella strains was detected by this detection system. It was found that there were 6, 9, and 7 copy numbers in the biovars 1-3 of Brucella melitensis, respectively. The strain of Brucella suis biovar 2 had 10 copy numbers, which were different from those of the other 4 strains of biovars 1, 3-5. There were 37 copy numbers in Brucella ovis strain. The copy numbers were stable at 5-6 copies in 8 biovars (1-7, 9) of Brucella abortus strains. Conclusions:A real-time quantitative PCR assay for detection of orfA gene copy number in Brucella DNA has been established. This method could identify some Brucella species and biovars strains.
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Objective:To establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. Methods:Based on the differences in the entire genome sequence between Brucella S2 vaccine strain and other reference strains of Brucella, primers and probes were designed to establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. The DNA of 22 reference strains of Brucella and 8 non- Brucella control strains were obtained from the National Institute for Infectious Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. At the same time, environmental samples were obtained from the brucellosis vaccine manufacturers, and bacterial DNA from environmental samples was extracted using a blood/tissue genomic DNA extraction kit. The obtained DNA was pre-amplified by conventional PCR, and then subjected to quantitative real-time PCR secondary amplification (nested fluorescence quantitative PCR) using the amplified PCR product as a template. The specific fluorescence curve and corresponding number of cycles (Ct value) were observed, and the sensitivity was tested. Results:The quantitative real-time PCR detection system established did not detect specific fluorescence curves (without Ct values) for 21 reference strains of Brucella and 8 non- Brucella control strains, except for S2 vaccine strains. The established detection system had a minimum detection limit of 4.34 fg (genomic DNA) for detecting the DNA of Brucella S2 vaccine strain; DNA of Brucella S2 vaccine strain was detected in 3 of the 14 environmental samples collected. Conclusion:The quantitative real-time PCR detection system established can detect Brucella S2 vaccine strain in samples, with good sensitivity and specificity.
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Objective:A nested-PCR assay is developed to detect and identify the genomic DNA of Brucella vaccine A19 strain. Methods:The whole genomic sequences of Brucella vaccine A19 strain and other Brucella spp. strains were compared and analyzed. The primers were designed by nucleotide difference sites. The nested-PCR assay was established to detect and identify Brucella vaccine A19 strain. The genomic DNA of Brucella vaccine A19 strain was extracted and diluted. The diluted template DNA was tested for sensitivity of using nested-PCR assay. And the specificity of nested-PCR assay was tested for the genomic DNA of other Brucella spp. strains and non- Brucella spp. strains. Results:The minimum detection limit of the nested-PCR assay was 3.43 fg. The nested-PCR assay established for amplification of Brucella vaccine A19 strain showed 246 bp electrophoresis bands, while other Brucella spp. strains showed 314 bp electrophoresis bands, and non- Brucella spp. strains did not produce electrophoresis bands. Conclusions:The nested-PCR assay established has the characteristics of high sensitivity and specificity. It can be detected when there is one copy of Brucella vaccine A19 strain genomic DNA in the reaction system. This method is particularly suitable for the detection and identification of trace genomic DNA of Brucella vaccine A19 strain in sample.
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Objective@#To study the molecular-epidemiological characteristics of Brucella species isolated from different countries, using the multiple locus tandem-repeat (MLVA) analysis.@*Methods@#Eleven variable-number tandem-repeat (VNTR) loci were selected. VNTR strains of Brucella isolated from 48 different countries in 1953-2013, were analyzed by using the BioNumerics software. Unweighted Paired Arithmetic Average method was used to cluster and draw phylogenetic tree as well as the minimum spannin.@*Results@#The evolutionary relationship of Brucella phylogenetic tree was consistent with the classical biological typing method. However, the Brucella suis biovar 5 strains were different from the other Brucella suis biovars 1, 2, 3 and 4. Brucella ceti strains were divided into two parts and different from each other. Worldwide epidemics of brucellosis were emerged from 2005 to 2008 under the MLVA11 Orsay analysis. China has been a brucellosis-prone regions, with Brucella melitensis as the main epidemic Brucella species, followed by Brucella abortus. Brucella suis was mainly identified in the southern provinces, but Brucella canis was mainly found in dogs. No human cases were found.@*Conclusion@#Molecular-epidemiological characteristics of the Brucella strains were related to factors as time, region and hosts of isolation, which are important to setting up prevention and control programs on brucellosis.
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Objective To screen the most suitable medium for Brucella drug susceptibility test, and observe the resistance of human derived Brucella to different antibiotics. Methods Totally 180 strains of Brucella isolated from 25 provinces (municipalities, autonomous regions) in recent years were taken as observation objects. Mueller-Hinton ( MH ) agar , MH blood agar and Brinell agar were used to carried out the drug susceptibility test in vitro, and to compare the results of drug susceptibility test of different medium; the most suitable Brucella drug susceptibility test medium was used to detect the resistance of human derived Brucella to Doxycycline, Rifampicin, Streptomycin, Levofloxacin, Moxifloxacin, Ceftriaxone sodium, Co-trimoxazole and Amoxicillin/Clavulanic acid by K-B drug sensitive paper, and to observe the formation of antibacterial ring around the drug sensitive paper. Results The growth of Brucella on the MH agar and MH blood agar were slower than that on the Brinell agar, and the antibacterial rings were not obvious. All the 180 strains of Brucella were sensitive to seven antibiotics such as Doxycycline, Rifampicin, Streptomycin, Levofloxacin, Moxifloxacin, Ceftriaxone sodium, and Amoxicillin/Clavulanic acid; and 70 strains of Brucella were resistant to Co-trimoxazole, accounting for 39％ (70/180); Brucella strains resistant to Co-trimoxazole were found in 21 provinces. Conclusions Brinell agar is the most suitable medium for Brucella susceptibility test. The human derived Brucella is resistant to Co-trimoxazole; the resistant strains are distributed in 21 provinces ( municipalities , autonomous regions ) . It is recommended that relevant departm ents of the province ( municipalities , autonomous regions ) carry out epidemiological investigations on the resistance of Brucella, and strengthen the monitoring of drug resistance in clinical drugs of brucellosis patients.
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Objective To analyze the etiological characteristics of human Brucella strains isolated, and to improve the precision in control and prevention of brucellosis. Methods In 2016, blood samples were collected from patients in Jingyuan County Gansu Province, and tested via the Rose-Bengal Plate Agglutination Test (RBPT) and the tube agglutination test methods,and serological positive blood samples were inoculated to bidirectional blood culture bottle for culturing, and further identified by traditional biological classification method and the Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis species-specific PCR (AMOS-PCR). Multiple-locus variable number tandem repeat sequence analysis (MLVA) -16 was used to detect molecular typing and do cluster analysis. Results The isolated strain was identified by the traditional biological classification method, bacteria could grow in thionine and reddened dye, A and M factors agglutination tests were positive, Bk2phage treatment of bacterial strain cracking, but Tb, Wb phages were not cracked. AMOS-PCR amplification result showed a 731 bp band, which was a strain of Brucella melitensis. The results of MLVA-16 showed that there was a difference in the number of repeats on some Variaable Number of Tandem Repeat(VNTR)sites of the isolated strain. Clustering analysis showed that, the isolated strain was clusted into the same clade with the clustering of Brucella melitensis type 3 from GS-201605 in Gansu. And the clustering was similar compared with that of Zhejiang, Guangdong, Fujian and Yunnan. Conclusion Human brucellosis is a inputting transmission in Gansu Province, there is a genetic variation of genotype 3 sheep Brucella between Gansu Province and other domestic provinces.
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<p><b>OBJECTIVE</b>To evaluation the specificity and sensitivity of 5 kinds of serological detection methods about brucellosis.</p><p><b>METHODS</b>To investigate in the 4 autonomous banner (Cha You Hou Qi, Right-Wing Central Banner of Kerqin Region, Linxi County and Siziwangqi Banner) of Inner Mongolia autonomous region from January to December, 2013. Accepting criteria: professionals of breeding cattle and sheep, and slaughter,accompanied by Bloom's disease suspected symptoms such as fever, fatigue,arthralgia, ranging in age from 25 to 55 years old. To collect suspected patients venous blood 3-5 ml in the morning, a total of 236 samples were collected. To detect the Brucella antibody by using plate agglutination test (PAT), tiger red plate agglutination test (RBPT), standard test tube agglutination test (SAT), enzyme-linked immunosorbent assay (ELISA) and immune colloidal gold method (GICA), SAT was taken as a golden standard, analyzed the sensitivity and specificity of RBPT and SAT, ELISA and GICA.</p><p><b>RESULTS</b>SAT method of positive patients: 136 cases (57.6%). PAT method positive patients: 150 cases (63.6%). RBPT positive patients: 159 cases (67.4%), and 143 patients with ELISA method: positive (60.6%), 147 patients with positive GICA method (62.3%). The detection rate of Brucella antibody positive was different by different testing methods.There was no significant difference (χ(2)=0.52,P=0.264). To take the SAT method as the gold standard, PAT, RBPT, ELISA and GICA method of the sensitivity were 97.7% (133/136), 98.5% (134/136), 94.8% (129/136) and 94.1% (128/136), respectively. The specificity was lower,the rate were 70.0% (70/100), 75.0% (75/100), 86.0% (86/100) and 81.0% (81/100), respectively. The total coincidence rate were 86.0% (203/236), 88.5% (209/236), 91.1% (215/236) and 88.5% (209/236), respectively.</p><p><b>CONCLUSION</b>The specificity and sensitivity of ELISA and GICA method is higher in the diagnosis of disease. The two methods are rapid, GICA method can be used on-site testing, large sample test is suitable for using ELISA.</p>
Subject(s)
Adult , Animals , Cattle , Humans , Middle Aged , Agglutination Tests , Methods , Antibodies, Bacterial , Blood , Brucella , Brucellosis , Diagnosis , China , Enzyme-Linked Immunosorbent Assay , Sensitivity and Specificity , SheepABSTRACT
Objective To develop a one-step PCR assay for rapid discrimination of six Brucella species and some intraspecific biovars.Methods Using 6 pairs of primers in one-step PCR to differentiate six classical Brucella species and some biovar in ordinary PCR instrument.The tested strains including 27 reference strains of six Brucella species and 239 Brucella strains were estimated by the PCR assay and biological identification methods.Results The six Brucella species could be precisely differentiated by the onestep PCR assay from the tested strains.Five biovars and vaccine strain of B.suis species could be determined,and biovars 1,3,4 and biovars 2,5,6,7,9 of B.abortus species could be identified at the level of their biovar,moreover,biovars 1,2 and 3,and vaccine strain Rev 1 of B.melitensis species were also discriminated at the biovar and strain level.The accurate rates of the biological identification method and the PCR assay were 98.33％ and 100％ respectively.Conclusion One-step PCR assay was a rapid,specific,and low cost method for identification of Brucella species and discriminating biovars in ordinary PCR instrument.
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ObjectiveTo study the reasons and mechanism of cardiomyocyte apoptosis in chronic heart failure by using Losartan and to provide a theoretical basis for the treatment of chronic heart failure. Methods The models of chronic heart failure were produced by injecting Adriamycin and Losartan as intervention agents, the expression of apoptotic protein Bax, Bcl-2 and channel protein ERK1, JNK1 and P38MAPK were detected by immunohistochemistry and RT-PCR.Cardiomyocyte apoptosis and myocardial ultrastructure are detected by transmission electron microscopy and TUNEL staining.Results Compared with the model group of heart failure, after Losartan treatment, the ultra structure of myocardial cells were significantly improved, Apoptosis index was decreased significantly (P <0.01), The level of Bax and JNK1 decreased (Bax χ~2=6.6149, P=0.0078; JNK1 q=22.0156,P<0.01). However, the expressions of ERK1 and Bcl-2 were significantly increased (ERK1 q=15.3514,P<0.01;Bcl-2 χ~2=6.81,P=0.0074).Conclusion The effect of Losartan on chronic heart failure is related closely with the pathway of ERK1 and JNK1, and no p38 MAPK pathway.
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Objective To compare the three-dimensional eephalometric and three-dimensional CT measurements of adult zygomatic complex among a Sanjiang population.Methods 120 female natives of Sanjiang region from Chinese Han between 19 to 23 years of age (mean 21.4 years),were randomly selected from students of Jiamusi University.Three-dimensional cephalometric and CT measurements of the face and skull were taken,with reference to zygomatic size measurement by Prof.Qi Zuo-liang,for the comparative study, which included the upper,mid and lower facial width followed by the length,width and angle of malar process,and width of the facial bone.Statistical analysis was done with the obtained measurements.Results Three-dimensional CT analysis showed facial width ratio of 0.83 and 0.79 and skeletal facial profile width ratio of 0.81 and 0.77,respectively,when compared with three-dimensional cephalometric analysis that showed facial width ratio of 0.84 and 0.77.Both values showed no statistical significance (P>0.05).Conclusion Three-dimensional CT measurement as the same to three-dimensional cephalometric can be used in the diagnosis of prominent malar complex.
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objective To investigate the epidemiological and molecular typing features of the pathogenic Haemophilus influenzae(H.influenzae)by biotyping,serotyping and pulsed-field gel electrophoresis(PFGE).Methods A total of 273 invasive isolates of H influenzae were collected from the pediatric patients with pneumonia at Chengdu Children Hospital of Sichuan province from 1988 and 2004 to 2007.The idenbfication of H.influenzae strains were done according to the laboratory standard methodology described by Manual of Clinical Microbiology(American).All strains were biotyped according to Kilian's classification with the API[R]NH system.And serotyped by a slide agglutination assay with type a to f specific antlaerum as described by Pittman.PCR method for identification of H.influenzae were performed as described by Falla.One hundred of 273 strains were analyzed by PFGE as described by Saito with some modifications.The resuIts of PFGE were analyzed by Bionumerics soft(Version 4.0,Applied Maths BVBA,Belium).Restilts 78.2%of 273 cases occurred under 1 years old.Eight biotypes were found among the 273 H.influenzae isolates.17.6%(48/273)of all isolates belonged to biotype Ⅰ,43.6%(119/273)were biotype Ⅱ,22.7%(62/273)were biotype Ⅲ,7.3%(20/273)were biotype Ⅳ,5.9%(16/273)were biotype Ⅴ,0.4%(1/273)were biotype Ⅵ,1.8%(5/273)were biotype Ⅶ and 0.7%(2/273)were biotype Ⅷ.respeetively.99.6% of all 273 isolates were nontypeable.There was only one isolate was serotvpe f Ninty-six PFGE genotypes were obtained in this study.One hundred strains demonstrated a variety of genomic Datterns by PFGE.The most isolates of the flame PFGE genotype(type 35)was 3 isolates.Each of93 PFGE genotypes was represented by only a single isolate.The genotypes distribution didn't correlate with the time distribution of the strains were isolated.Conclusion Nontypeable H.influenzae primarily caused acute Dneumoma in children under 1 years old.They mostly belonged to biotype Ⅰ,Ⅱ and Ⅲ biotypes.The nontypeable H.influenzae strains appeared to more heterogeneous patterns by PFGE genotyping.Genotyping may helP understand the molecular characteristics of outbreak and endemicity according to the results of PFGE.PFGE genotyping proved to have a much stronger discriminatory power than either serotyping or biotyping.Our findings suggest that PFGE analysis is useful for the epidemiologieal study of H.influenzae infections.
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Objective To establish TaqMan real-time PCR method for detection and identification of Haemophilus influenzae and Streptococcus pneumonia. Methods Two sets of primers and FAM-labeled probes targeting different genes of Haemophilus influenzae and Streptococcus pneumoniae were designed and synthesized. The bexA gene was used for identification of Haemophilus influenzae and lytA for Streptococcus pneumoniae. The sensitivity and specificity of real-time PCR were assessed for different primers and probes. Cut-off values of cycle threshold (Ct) were determined. Two hundred and seventy-eight cerebrospinal fluid (CSF) specimens from suspected bacterial meningitis cases were detected by real-time PCR assay, latex agglutination test and bacteria culture simultaneously. Results Haemophilus influenzae isolates of serotype a to d could be detected and identified by bexA primers and probe. All Streptococcus pneumoniae isolates of different serotypes could be detected and identified by lytA primers and probe. The respective sensitivities for Haemophilus influenzae and Streptococcus pneumoniae were 10 and 90 genome DNA copies in each PCR reaction. Of the 278 CSF specimens, four were positive by Haemophilus influenzae and seven positive by Streptococcus pneumoniae when detected by real-time PCR. Of the four Haemophilus influenzae positive specimens, two were positive by culture and one positive hy latex. Of the seven Streptococcus pneumonia positive specimens, two were positive by culture and two positive by latex. Conclusions Real-time PCR could rapidly detect and identify Haemophilus influenzae of serotype a to d and Streptococcus pneumoniae of different serotypes with high sensitivity. TaqMan real-time PCR could be widely used for the diagnosis of invasive meningitis caused by Haemophilus influenzae and Streptococcus pneumoniae. It can improve the rate of positivity for diagnosis of suspicious bacterial meningitis cases.
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Haemophilus influenzae (Hi) is a pathogen exclusively found in humans. It causes a wide range of infections from the upper respiratory tract to serious invasive diseases. Such as pneumonia, septicemia and meningitis. Strains of Hi are usually classif ied into six serotypes a to f and nontypeable H. influenzae (NTHI) according to the antigenicities and compositions of their polysaccharide capsules. Hib was a common cause of serious infections in younger children. The polysaccharide-protein conjugate vaccines against Hib had almost eliminated H. influenzae as a cause of pediatric meningitis. However, NTHI remains an important pathogen, particularly in children and the elderly. Efforts to understand and control NTHI disease have been hampered by the diversity of these bacteria. This review introduced the study progress about pathogenic mechanism of NTHI. In order to provide the help for development of vaccine, clinic treatment and prevent the occurrence of diseases causing by NTHI.
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In order to control the quality of all parts of the process for culturing,preparing and using clinically the human embryo olfactory ensheathing cells (OECs),the conduct standard of culturing human OECs from olfactory bulb is formulated. Because the strict conduct process can reduce the human factor as much as possible and ensure the stability of the cell quality. Therefore,during the culturing process of human embryo OECs, as for the embryo sample of OECs from olfactory bulb,the standard of lab condition and cell culture must be set strictly. The key steps of conduct process must be regulated,the freezing and resuscitating process of OECs must be controlled,the test method for microbial contamination during cell culture must be proposed and the risk must be reduced,then the quality of the human OECs from olfactory bulb can be guaranteed during the clinical application.
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Objective To explore the double-labeling method of monitoring the GHRP regulatory function on [Ca2+]i and NO in cardiomyocytes of rats on real time under LSCM.Methods The reformed constant-flow Langendorff system and enzyme-dissociated was used to isolate cardiomyocytes.[Ca2+]i and NO in the cardiomyocytes of SD rats were double-labeled by their molecular probe Rhod-2/AM and DAF-FM/DA,respectively to monitor the regulatory function of GHRP on [Ca2+]i and NO on real time by LSCM.Results Ca2+ signal showed a red fluorescence and NO showed a green fluorescence while the overlapping of the two signals showed a yellow-green fluorescence by this system,and the similar effect presents in both double-labeled state and the single labeled one:GHRP induced a transient[Ca2+]i increase then followed by a plateau phase while there was not significant change in NO signal system after GHRP stimulation under the LSCM in the cardiomyocytes of rats.Conclusions After having established the double-labeling method we monitored the GHRP regulatory function on [Ca2+]i and NO on real time in cardiomyocytes of rats under LSCM causing the [Ca2+]i biphasic increase while no significant change in NO signal system.