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In Zherong county, Fujian province, the black spot of Pseudostellaria heterophylla often breaks out in the rainy season from April to June every year. As one of the main leaf diseases of P. heterophylla, black spot seriously affects the yield and quality of the medicinal material. To identify and characterize the pathogens causing black spot, we isolated the pathogens, identified them as a species of Alternaria according to Koch's postulates, and then tested their pathogenicity and biological characteristics. The results showed that the pathogens causing P. heterophylla black spot were A. gaisen, as evidenced by the similar colony morphology, spore characteristics, sporulation phenotype, and the same clade with A. gaisen on the phylogenetic tree(the maximum likelihood support rate of 100% and the Bayesian posterior probability of 1.00) built based on the tandem sequences of ITS, tef1, gapdh, endoPG, Alta1, OPA10-2, and KOG1077. The optimum conditions for mycelial growth of the pathogen were 25 ℃, pH 5-8, and 24 h dark culture. The lethal conditions for mycelia and spores were both treatment at 50 ℃ for 10 min. We reported for the first time the A. gaisen-caused black spot of P. heterophylla. The results could provide a theoretical basis for the diagnosis and control of P. heterophylla leaf spot diseases.
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Bayes Theorem , Phylogeny , Caryophyllaceae , Alternaria , MyceliumABSTRACT
Objective To assess the significance of screening for inherited metabolic diseases in the treatment and diagnosis of infantile cholestatic hepatopathy,and to analyze the biochemical changed character‐istics of patients who were diagnosed with gene mutations. Methods From January 2016 to January 2017,69 children who were diagnosed as intrahepatic cholestasis in the Pediatric Gastroenterology Department of Shengjing Hospital Affiliated to China Medical University were enrolled. The medical history,physical exami‐nation,biochemical test and genetic metabolism screening results were recorded. Results Sixty‐seven cases of 69 children made tandem mass spectrometry(MS/MS),gas chromatography‐mass spectrometry(GC/MS) or genetic testing. Compared with the normal hereditary metabolic disease screening group, the abnormal group had higher levels of alkaline phosphatase,total bilirubin,direct bilirubin,and total bile acid,the differ‐ence was statistically significant (P<0. 05). The most common abnormal in MS/MS were elevation of free carnitines and arginine,citrulline,methionine,and the most common abnormal in GC/MS were elevation of 3‐hydroxyl propionic acid,4‐hydroxyl phenyllactic acid,4‐hydroxyl phenylacetic acid. In 6 children with posi‐tive genetic test results,the MS/MS and GC/MS of 4 neonatal intrahepatic cholestasis caused by citrin defi‐ciency showed aminoacidemia(citrullinemia,tyrosinemia) and elevations of urine organic acids. Five muta‐tions of SLC25A13 gene were found in the neonatal intrahepatic cholestasis caused by citrin deficiency pa‐tients,including IVS6+5G >A,851del4,IVS11 +1G >A,851 854de and 852 855del. The main clinical manifestations of progressive familial intrahepatic cholestasis type 2 ( PFIC2) were cholestatic jaundice and pruritus,γ‐glutamyl transpeptidase was normal,and with the c. 667C>T defection in the ABCB11 gene. The TALDO1 gene mutation type of one transaldolase deficiency was c. 716G>A and c. 854dupA heterozygous mutation. Conclusion MS/MS and GC/MS play a vital role in the early identification of cholestasis caused by genetic and metabolic disorders. Genetic testing can provide accurate diagnosis for rare genetic metabolic diseases.
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Objective To investigate rickettsiae infection from host animals and vector arthropods in some areas of Yunnan Province. Methods Rat clip and cage traps were used to capture mice. Chiggers from body surface of mice and ticks from body surface of farm cattle were collected. DNAs were extracted from mice spleens, chiggers and ticks. Rickettsiae groEL segment were amplified by nested-polymerase chain reaction (nPCR), sequenced to analyze the homology with other known sequences. Results A total of 410 samples were collected for rickettsiae groEL segment detection with nPCR and 19 samples (4.63%) showed positive for rickettsiae groEL segment . Among them, 2.68%(11/410)were positive for Orientia tsutsugamsushi (Ot) groEL segment, and 1.22%(5/410)were positive for spotted fever group rickettsia (SFGR) groEL segment, and 0.49%(2/410)were positive for rickettsia mooseri (Rm) groEL segment, and 0.24%(1/410)were positive for rickettsia endosymbiont(Re) groEL segment. When analyzed the homology with other known sequences, 11Ot strains with 93.6%-100% similarities among them in this study shared the highest similarity with other Ot strains from GenBank respectively, reached up to 96.1%-100%; The groEL segments of 5 SFGR strains with 92.1%-99.5% similarities among them in this study shared highest similarity with other SFGR strains from other GenBank respectively, reached up to 98.9%-100%; In this study groEL segments of 2 Rm strains all showed 100% similarity with Wilmington strain (GenBank No:AE017197); One groEL segment of Re showed 98.9% similarity with Re strain (GenBank No:EU435143). Conclusion There were kinds of rickettsiaes infection in host animals and vector arthropods in Yunnan Province, so the monitoring and prevention of the Rickettsiosis should be strengthened.
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Objective To analyze the genetic structure and recombination characteristics of a new-ly discovered HIV-1 unique recombinant strain in Yunnan Province. Methods During a test for drug-resist-ant HIV genotypes in Yunnan Province in 2016, a recombinant fragment was found in the pol region of a HIV-1 strain isolated from a patient. Two overlapping segments of the HIV-1 genome were amplified by RT-PCR, and then the products were sequenced. Recombination analysis was performed using RIP, jpHMM and SimPlot3. 5 software. A phylogenetic tree was constructed for homology analysis by Neighbor-joining method using MEGA6. 06 software. Results A nearly full-length HIV-1 gene sequence with 8590 bp in length was obtained. Breakpoint analysis indicated that the sequence consisted of CRF01_AE and fragments of B and C subtypes. CRF01_AE was used as the backbone with B and C subtype fragments inserted. The positions were 791 to 1171 for CRF01_AE, 1172 to 2652 for C subtype fragment, 2653 to 2977 for B subtype frag-ment, and 2978 to 9380 for CRF01_AE using HIV-1 HXB2 as the reference strain. Conclusions Some new strains formed by cross-recombination of CRF01_AE and B and C subtypes were discovered in Yunnan Province in recent years. It was found that the recombination pattern of the newly discovered strain was com-plex, suggesting that close attention should be paid to the changes in epidemic trends, which was of great im-portance to understand the current prevalence and epidemic trends of HIV-1.
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We investigated epidemiological characteristic of scrub typhus in Yongshan County,Yunnan Province,China. The serum samples were collected from the patients with fever for detecting the antibody against Orientia tsutsugamsushi (Ot) by colloidal gold immunoassay assay.Rat traps were used to capture rodents.The spleen tissues of the captured rodents were detected by nested-polymerase chain reaction for rickettsia groEL segment.The groEL segments were sequenced and analyzed the homology with the other known sequences.Thirty-four scrub typhus cases were found in Yongshan County,Yunnan Prov-ince from May 2015 to October 2015.Among them,21 cases were confirmed by laboratory tests and 13 cases were clinical di-agnosis diseases.Of these patients,32.35% of the cases occurred in June.The 32.35% were in the group of the 40-49 year-old,and 79.41% were farmers,94.12% exhibited eschar or skin ulcer(31.25% were observed in groin of these cases),and rash developed in 50%.In 39 spleen tissue samples of Rattus flavipectus,9 samples showed positive for groEL gene Ot,but gro-EL gene of Typhus group rickettsia and spotted fever group rickettsia were negative.Sequence analysis showed that YSP30 was closely related to some Saitama related strains of Ot,such as HSB1,FAR1 and UAP4,while the other 8 strains were closely related to some Karp related strains of Ot,such as UT213,UT221 and SH205.It was confirmed that the Yongshan County was the natural foci of scrub typhus by the serological and molecular biological detections.There are Karp and Saitama genotype related Ots in the natural foci.
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To understand pathogen spectrum of nontuberculosis Mycobacteria (NTM) and the dominant NTM in Gansu Province and provide the scientific basis for the effective prevention and treatment of NTM diseases,875 Mycobacteria isolates were collected from 2012 to 2014 in Lanzhou Pulmonary Hospital,NTM species were identified by means of PNB/TCH differentiate medium and 16S rRNA gene sequence analysis respectively.Forty-six isolats of NTM were identied from 875 PNB/TCH.Then with 16S rRNA gene sequence analysis,the NTM strains were identified to 3 strains of Nocadia and 43 strains of NTM,including M.intracellulare,M.kansasii,M.avium,M.senegalense,M.gordonae,M.szulgai,M.peregrinumand M.fortuitum.Among them,there were 31 strains of M.intracellulare,which accounted for 72.09％ of the total number of NTM strains.The dominant nontuberculosis Mycobacteria in Gansu Province were mainly M.intracellulare.The application of molecular biology can rapidly and accurately identify the species of nontuberculosis Mycobacteria,and can provide relevant evidence for clinical diagnosis and therapy.
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The ability to recognize and repair abnormal DNA structures is common to all forms of life. Physiological studies and genomic sequencing of a variety of bacterial species have identified an incredible diversity of DNA repair pathways. Despite the amount of available genes in public database, the usual method to place genomes in a taxonomic context is based mainly on the 16S rRNA or housekeeping genes. Thus, the relationships among genomes remain poorly understood. In this work, an approach of multiple gene sequence analysis based on genes of DNA repair pathway was used to compare bacterial genomes. Housekeeping and DNA repair genes were searched in 872 completely sequenced bacterial genomes. Seven DNA repair and housekeeping genes from distinct metabolic pathways were selected, aligned, edited and concatenated head-to-tail to form a super-gene. Results showed that the multiple gene sequence analysis using DNA repair genes had better resolution at class level than the housekeeping genes. As housekeeping genes, the DNA repair genes were advantageous to separate bacterial groups at low taxonomic levels and also sensitive to genes derived from horizontal transfer.
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Objective To clone the human mucin (MUC)5AC gene promoter and construct its luciferase reporter vector for human MUC5AC gene and analyze its transcriptional activity. Methods The 1 348 bp DNA sequence at the human MUC5AC gene 5 end was analyzed by the Vector NTI software.After the target sequence from human A549 cells genomic DNA was amplified by PCR method, and the product of PCR was sequenced.By promoter deletion analysis, 3 promoter segments with diferent lengths were amplified by PCR, then the products were identified by DNA sequencing, and 4 promotor segments were inserted into pGL3- enhancer vectors.Site-specific mutagenesis technique was used to establish mutants of specificity protein (SP)-l and nuclear factor-kappa B (NF-кB) site in MUC5AC gene promoter. The relative luciferase activities were detected in the transfected A549 cells. Results Sequence analysis indicated that there were many cis-acting elements in the regions of 1 348 bp DNA sequence at the human MUC5AC gene 5 end.The 4 reporter gene vectors with promoter segments with different lengths were constructed successfully.Dual-luciferase assay revealed the 372 bp fragment including activity with the minimal fragment. Neutrophil elastase (NE) could increase the expression of luciferase reporter gene plasmid containing mutated NF-кB version (P＜0.05 vs. contro1) of MUC5AC promoter in the transfected A549 cells. The induction by NE decreased markedly when the SP-l element in MUC5AC promoter were mutated. Conclusion This research may provide an important basis for the further study of human MUC5AC gene promoter activity and regulation of gene expression.There is an up-regulative element of gene transcription in the region of -324 to -64 bp in MUC5AC gene upstream. SP-l site of the promotor mediates NE-induced MUC5AC expression in human A549 cells.
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BACKGROUND: Acinetobacter spp. is increasingly implicated in hospital-acquired infections. We experienced a pseudooutbreak of Bordetella bronchiseptica bacteriuria identified with biochemical tests, that was later identified as Acinetobacter spp. by using 16S rRNA gene sequence analysis. MATERIALS AND METHODS: Five in-ward patients were found to have B. bronchiseptica bacteriuria without symptoms of urinary tract infection between September 23 and 26 of 2005. We conducted pulsed field gel electrophoresis (PFGE) of the bacteria and epidemiological investigation of this pseudooutbreak. In addition, 16S rRNA gene sequence analysis was performed for the verification of the strains. RESULTS: All 5 isolates were identified as B. bronchiseptica with similar antibiogram by VITEK system. There was no evidence of any symptom or sign of urinary tract infection. The source of this pseudooutbreak was not detected even after performing environmental culture and interviews with healthcare workers. We could not get the appropriate results from the first PFGE with XbaI restriction enzyme. B. bronchiseptica is an unusual organism in human so we conducted 16S rRNA gene sequence analysis for verification. The analysis of 16S rRNA gene sequence with 5 isolates demonstrated 99-100% similarity to a sequence of Acinetobacter spp. (AU1523). According to the results of 16S rRNA gene sequence analysis, we performed the second PFGE with SmaI restriction enzyme, which showed indistinguishable pattern among the all 5 isolates. CONCLUSION: This investigation suggests that the combined method of 16s rRNA gene sequence analysis and PFGE would be helpful for investigation of outbreak caused by unusual organisms
Subject(s)
Humans , Acinetobacter , Bacteria , Bacteriuria , Bordetella bronchiseptica , Delivery of Health Care , Electrophoresis, Gel, Pulsed-Field , Genes, rRNA , Microbial Sensitivity Tests , Sequence Analysis , Urinary Tract InfectionsABSTRACT
BACKGROUND: Acinetobacter spp. is increasingly implicated in hospital-acquired infections. We experienced a pseudooutbreak of Bordetella bronchiseptica bacteriuria identified with biochemical tests, that was later identified as Acinetobacter spp. by using 16S rRNA gene sequence analysis. MATERIALS AND METHODS: Five in-ward patients were found to have B. bronchiseptica bacteriuria without symptoms of urinary tract infection between September 23 and 26 of 2005. We conducted pulsed field gel electrophoresis (PFGE) of the bacteria and epidemiological investigation of this pseudooutbreak. In addition, 16S rRNA gene sequence analysis was performed for the verification of the strains. RESULTS: All 5 isolates were identified as B. bronchiseptica with similar antibiogram by VITEK system. There was no evidence of any symptom or sign of urinary tract infection. The source of this pseudooutbreak was not detected even after performing environmental culture and interviews with healthcare workers. We could not get the appropriate results from the first PFGE with XbaI restriction enzyme. B. bronchiseptica is an unusual organism in human so we conducted 16S rRNA gene sequence analysis for verification. The analysis of 16S rRNA gene sequence with 5 isolates demonstrated 99-100% similarity to a sequence of Acinetobacter spp. (AU1523). According to the results of 16S rRNA gene sequence analysis, we performed the second PFGE with SmaI restriction enzyme, which showed indistinguishable pattern among the all 5 isolates. CONCLUSION: This investigation suggests that the combined method of 16s rRNA gene sequence analysis and PFGE would be helpful for investigation of outbreak caused by unusual organisms
Subject(s)
Humans , Acinetobacter , Bacteria , Bacteriuria , Bordetella bronchiseptica , Delivery of Health Care , Electrophoresis, Gel, Pulsed-Field , Genes, rRNA , Microbial Sensitivity Tests , Sequence Analysis , Urinary Tract InfectionsABSTRACT
Cucumber mosaic virus was detected from infected Basella rubra L. with the indirect enzyme-linked immunosorbent assay. Total RNA was extracted from infected leaves and the cDNAs of coat protein gene of CMV-Ba were obtained by RT-PCR. The amplified cDNA fragments were then cloned into pMD 18-T vector and sequenced,the result showed that the CP gene was 657 nucleotides in length. This sequence was aligned with the obtained CP gene and some CMV strains or isolates of subgroup Ⅰ and subgroup Ⅱ in GenBank using DNA MAN software. The results showed that CMV-Ba shared 90.9%~93.8% and 76.1%~76.9% identity with the known CP genes of subgroup Ⅰ and Ⅱ respectively in nucleotide level,on the other hand,amino acids deduced from CMV-Ba CP gene shared 92.7%~97.7% and 72.4%~78.1% identity with the known CP protein of subgroup Ⅰ and Ⅱ,respectively. This suggested that CMV-Ba CP gene belongs to CMV subgroupⅠ.
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Eight Borrelia burgdorferi strains, which had been isolated from Ixodes nipponensis and Apodemus agrarius captured in the Chungju area of Korea, were characterized by ospC gene sequence analysis. Nucleotide sequence similarity among the Chungju strains ranged from 83.6 to 100%. Deduced amino acid sequence similarity of the Chungju strains ranged from 75.4 to 100%. In a molecular phylogenetic analysis, the Chungju strains were separated from B. burgdorferi and B. garinii reference strains, and formed a cluster with B. afzelii reference strains. Three (KK2, KM4, and KK5), two (CJ2 and CJ21), and one (CJ3) Chungju strains formed clusters with OspC serotype 5, OspC serotype 8, and OspC serotype 7 reference strains, respectively. However, two Chungju strains (KK1 and KM10) formed a distinctive cluster that was separated from other strains of B. afzelii reference strains. These results suggest that Chungju strains are very heterogeneous in clonality.
Subject(s)
Animals , Amino Acid Sequence , Base Sequence , Borrelia burgdorferi Group , Borrelia burgdorferi , Borrelia , Genetic Heterogeneity , Ixodes , Korea , Murinae , Sequence AnalysisABSTRACT
A 1 3kb DNA fragment was amplified in DNA samples extracted from periwinkle showed yellows The amplified fragment was ligated into pGEM T easy vector and then transformed into JM109 strain of E coli Cloned DNA fragments were verified by PCR, restriction endonuclease ( Eco RI) digestion and sequence analysis The result revealed that the ribosomal protein gene of PY strain consists of 1244 base pairs, which contains the entire rp l22 gene encoding 129 amino acids and rps3 gene encoding 252 amino acids These two genes are overlap The character of ribosomal protein gene of PY strain is very resemble with that of other phytoplasmas
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Objective:To acquire Chinese IL 18 mature peptide cDNA from adult PBMC,adult bone marrow cDNA libray,adult tonsil cDNA library,Embryonic cerebrum and cerebellum aged 3 months,Embryonic cerebrum and cerebellum aged 8 months.Methods:By RT PCR to aquire Chinese IL 18 mature peptide cDNA.Results:The six sequences of our seven species have basepair variation.Conclusion:The cDNA of Chinese IL 18 has polymorphism.