基因扩增联合测序法对痰标本分离的60株真菌病原学鉴定

目的:应用PCR扩增真菌ITS2区基因,结合基因测序法对痰标本中分离的60株真菌进行病原学鉴别。方法:收集痰标本分离的真菌菌落60份及2种真菌标准菌株(白色念珠菌,烟曲霉菌)培养菌落,分别提取基因组DNA,半巢式PCR扩增ITS2区,扩增产物经测序后,结果与NCBI基因库中的核酸序列进行比对,确定致病菌的种属,并对其分布特征进行分析。结果 :2种真菌标准菌株的ITS2区基因序列比对结果与种属一致。痰标本中60株真菌测序鉴定结果为:曲霉菌属24株(其中烟曲霉菌17例、黑曲霉菌3例、黄曲霉菌2例、土曲霉菌1例、变色曲霉1例),青霉菌属3株(小刺青霉菌1株、娄地青霉菌1株、产黄青霉菌1株),念珠菌属29株(白色念珠菌20株、热带念珠菌3株、近平滑念珠菌2株、光滑念珠菌2株、鲁西坦念珠菌1株、季也蒙毕赤酵母菌1株),其它丝状菌还有暗孢节菱孢菌2株,淡紫拟青霉菌1株和串珠状赤霉菌1株。结论:基因测序法能快速、简便、准确地鉴别痰培养的真菌种属,为本地区呼吸道真菌性疾病病原菌流行病学调查及个性化治疗奠定基础。

Genome Sequencing,Probiotic Analysis,and Oxalate Degradation Modification of Limosilactobacillus reuteri Q35

Limosilactobacillus reuteri is a microbe intricately linked to humans and animal health.A thorough assessment of its safety and potential benefits is imperative prior to its application in human and animals.In this investigation,we performed a comprehensive analysis encompassing genome sequencing,genomic analysis,and phenotypic characterization of L.reuteri Q35,an exceptionally proficient producer of reuterin.The whole genome sequencing results showed that the complete genome sequence spans 2145158 bp with a GC content of 38.9%and encompasses 2121 genes.Initial identification of antibiotic-resistant genes,virulence factors,and toxin-coding genes in the genome substantiated the strain’s low-risk status.Subsequent tests for antibiotic resistance,acute oral toxicology,and hemolysis further confirmed its elevated safety level.The genome of L.reuteri Q35 was found to contain genes associated with adhesion and stress tolerance.Following exposure to artificial gastric juice and bile salt,the strain exhibited a higher survival rate and demonstrated a strong scavenging ability for hydroxyl free radicals in antioxidant capacity tests.These findings suggested that L.reuteri Q35 possesses unique probiotic properties.Additionally,the genome of strain Q35 harbors three truncated oxaloyl-CoA decarboxylase genes(oxc1,oxc2 and oxc3),overexpression of which resulted in a significant increase in ammonium oxalate degradation from 29.5%to 48.8%.These findings highlight that L.reuteri Q35 exhibits both favorable safety characteristics alongside beneficial properties,making it a promising candidate for treating metabolic disorders such as hyperoxaluria.

Genetic Analysis of a Biomass Mutant in Oryza sativa

[ Objective ] The study aimed to reveal the genetic model of a biomass mutant in Oryza sativa. [ Method ] In the process of screening and identification of Bar-transgenic rice, a biomass mutant was found in 10 lines of T1 progenies. The mutant was investigated for genetic analysis and agronomic traits by herbicide spraying and PCR amplification. [ Result] The segregation ratio is consistent with mendelian law（3：1）. The mutant assumed not only higher plant height, wider straw and earlier florescence, but also more tillers, bigger spikes and resultantly higher biomass. PCR detections indicated that no co-segregation was observed between mutant traits and target gene（Bar） in the T-DNA inserted, proving that the mutant is not caused by the insertion of T-DNA containing target gene （Bar）. [ Conclusion] Our study may avail to understand the cloning of mutant gene and the mechanism of the mutant gene on biomass.

Determination and Analysis of Mitochondrial ND2 Gene Sequence of Anas platyrhynchos

[Objective] The study was to analyze the phylogenesis of Anas platyrhynchos. [Method] Complete sequence of mitochondrial ND2 gene of 4 Anas platyrhynchos was determined by direct DNA sequencing based on PCR products. Combined with ND2 gene sequences of the Anas Linnaeus accessed in GenBank, phylogenetic tree was constructed by Neighbor-joining and maximum parsimony methods. [Result] The ND2 gene sequences of 4 Anas platyrhynchos were identical(1 041 bp in length; the nucleotide contents of A, G, T, and C were 28.91%, 13.35%, 20.75% and 36.98% respectively; A+T content approximated to that of C+G). Sequences of ND2 gene of mallard were same as spotbill duck, and had high homology with others. The phylogenetic trees indicated mallard and spotbilled duck were close in genetic relationship, both shared a haplotype; then Philippine duck, green-winged teal and northern pintail fell into branch ''A". [Conclusion] The domestic duck may be domesticated from mallard and spotbilled duck.

Detection and Sequence Analysis of Escherichia coli Virulence Genes

[Objective] This study aimed to explore the pathogenic mechanism of E., cherichia coll. [Method] An E. coil strain isolated from raccoon dogs in vivo was studied, which had been identified, PCR was used to detect the gene of irp2 （301 bp） and fyuA （953 bp） related to E. coil virulence and PCR products were s, quenced. [Result] The genes of irp2 and fyuA were successfully amplified in boi strains isolated from raccoon dogs. Compared with the GenBank, the identity of tTr irp2 gene sequence and the fyuA gene sequence of the strain reached 98.5% 99.2% and 98.9%-100% respectively. Compared with each other, the identity of tt- two gene sequences of irp2 was 99.3%, and that between the two fyuA gene se quences was 98.9%. [Conclusion] This study provided scientific experimental data fi E. coil pathogenicity, prevention, diagnosis and treatment.

Sequencing and Analysis of Porcine Intrleukin-18 Gene

[ Objective] To clone the porcine interleukin-18（/L-18） cDNA and explore the immunological effectiveness of porcine IL-18 as an adjuvant of genetic vaccine. [ Method] The spleen lymphccytes were isolated from Henan three-way cross-breeding pigs. According to the porcine IL-18 gene in GenBank, a pair of specific primers was designed. The full length cDNA of porcine IL-18 was amplified by RT-PCR. Subsequently, porcine IL-18 cDNA was cloned into pGEM-T vector and sequenced and analyzed. [ Result] The porcine IL-18 gene demonstrated an open reading frame of 579 bp encoding an inactive precursor protein with 192 amino acids. The precursor protein had no typical hydrophobic signal peptide and cleaved by interleukin-1 beta （IL-1β） converting enzyme（ICE） in caspase-1 splice site; the porcine mature protein had biological activity： After comparing with other porcine IL-18 genes, the nucleotide sequence homology was over 96% and the deduced amino acid homology was more than 98%. [ Conclusion] A full length procine IL-18 gene was gained. It lays the foundation for porcine IL-18 as an adjuvant of genetic vaccine.

Sequence Analysis of Mitochondrial DNA D-loop Region in Xinjiang Goose

[Objective] The sequences of mitochondrial DNA D-loop region of Xinjiang Goose with three different colors of plumage were analyzed in order to study the genetic diversity of Xinjiang Goose, as well as the phylogeny and evolution. [Method] Ten geese were selected randomly from the core populations of grey-, mosaic- and white-plumaged Xinjiang Goose respectively with a total number of thirty as experi- mental materials, of which the blood samples were collected from the largest vein under the wing （brachial vein） for DNA extraction. Sequences of mitochondrial DNA D-loop regions were determined using DNA sequencing technology to analyze the polymorphism. In addition, the genetic distances among different populations were estimated through the comparison with the reference sequences. [Resull] The con- tents of A, G, C and T nucleotides in the D-loop region of Xinjiang Goose were 28.85%, 17.05%, 25.38% and 28.72%, respectively. The average haplotype diversity and nucleotide diversity of Xinjiang Goose were 0.583 and 0.056. Xinjiang Goose and Greylag Goose were clustered into the same group. [Conclusion] The results showed that Xinjiang Geese with three different colors of plumage all descend from Greylag Goose （Anser anser）.

Current blockade mechanism for DNA translocation through solid-state nanopore with different membrane thickness

The current blockade mechanism for λ -DNA translocation under electrical field is investigated through solid-state nanopores with different pore thicknesses. The conductance of a nanopore system mainly consists of the contribution of the pore and access region, and the latter becomes dominant when the nanopore thickness gradually decreases to atomic layer thickness. Based on the existing model of nanopore resistance, a simplified model which describes the relative current blockade during the X-DNA translocation through the nanopores is deduced to quantitatively present the relationship between nanopore thickness and relative current blockade. Results show that the relative current blockade is effectively increased by reducing the nanopore diameter but it decreases with the decreasing nanopore thickness. A two-stage schematic is proposed to increase the relative current blockade by setting a much smaller resistance region. Experimental results show a 21. 9% increase in the relative current blockade with the proposed schematic.

Bacterial diversity and community structure in the East China Sea by 454 sequencing of the 16S rRNA gene

The 454 sequencing method was used to detect bacterial diversity and community structure in the East China Sea. Overall, 149 067 optimized reads with an average length of 454 nucleotides were obtained from 17 seawater samples and fi ve sediment samples sourced in May 2011. A total of 22 phyla, 34 classes, 74 orders, 146 families, and 333 genera were identifi ed in this study. Some of them were detected for the fi rst time from the East China Sea. The estimated richness and diversity indices were both higher in the sediment samples compared with in the seawater samples. All the samples were divided by their diversity indices into four regions. Similarity analysis showed that the seawater samples could be classifi ed into six groups. The groups differed from each other and had unique community structure characteristics. It was found that different water masses in the sampling areas may have had some infl uence on the bacterial community structure. A canonical correspondence analysis revealed that seawater samples in different areas and at different depths were affected by different environmental parameters. This study will lay the foundation for future research on microbiology in the East China Sea.

Genetic Characteristics of 2009 Pandemic H1N1 Influenza A Viruses Isolated from China's Mainland

A total of 100 H1N1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang, Hubei and Guangdong between June and November 2009, were provided by local CDC laboratories. After MDCK cell culture, 57 Influenza A Pandemic （H1N1） viruses were isolated and submitted for whole genome sequencing. A total of 39 HA sequences, 52 NA sequences, 36 PB2 sequences, 31 PB1 sequences, 40 PA sequences, 48 NP sequences, 51 MP sequences and 36 NS sequences were obtained, including 20 whole genome sequences. Sequence comparison revealed they shared a high degree of homology （96%-99%） with known epidemic strains （A/Califomia/04/2009（H1N1）. Phylogenetic analysis showed that although the sequences were highly conserved, they clustered into a small number of groups with only a few distinct strains. Site analysis revealed three substitutions at loop 220 （221-228） of the HA receptor binding site in the 39 HA sequences： A/Hubei/86/2009 PKVRDQEG→PKVRDQEA, A/Zhejiang/08/2009 PKVRDQEG→PKVRDQER, A/Hubei/75/2009 PKVRDQEG→PKVRDQGG, the A/Hubei/75/2009 was isolated from an acute case, while the other two were from patients with mild symptoms. Other key sites such as 119, 274, 292 and 294 amino acids of NA protein,627 of PB2 protein were conserved. Meanwhile, all the M2 protein sequences possessed the Ser32Asn mutation, suggesting that these viruses were resistant to adamantanes. Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.

Study of apolipoprotein E genetic polymorphism in patients with atherosclerotic cerebral infarction

Objective: To explore the frequency and significance of ApoE gene polymorphisms in Chinese patients with atherosclerotic cerebral infarction (ACI). Methods: Polymerase chain reaction and gene sequencing, single nucleotide polymorphisms of ApoE gene were used to analyze 33 cases of patients with ACI and 35 controls. Results: The frequencies of ApoE gene single nucleotide polymorphisms 465C/G, 462C/G and 451delC in the ACI group were significantly higher than those in the control group (P<0.05). The prevalence of polymorphism 486G/T in the control group was significantly higher than that in the ACI group ( P = 0.011) . Conclusions: 465C/G,462C/G and 451delC polymorphisms might be associated with ACI.486GT allele might have protective effect on the pathogenesis of ACI.

High-throughput sequencing exclusively identified a novel Torque teno virus genotype in serum of a patient with fatal fever

Torque teno virus(TTV) has been found to be prevalent world-wide in healthy populations and in patients with various diseases, but its etiological role has not yet been determined. Using high-throughput unbiased sequencing to screen for viruses in the serum of a patient with persistent high fever who died of suspected viral infection and prolonged weakness, we identified the complete genome sequence of a TTV(isolate Hebei-1). The genome of TTV-Hebei-1 is 3649 bp in length, encoding four putative open reading frames, and it has a G+C content of 49%. Genomic comparison and a BLASTN search revealed that the assembled genome of TTV-Hebei-1 represented a novel isolate, with a genome sequence that was highly heterologous to the sequences of other reported TTV strains. A phylogenetic tree constructed using the complete genome sequence showed that TTV-Hebei-1 and an uncharacterized Taiwan Residents strain, TW53A37, constitute a new TTV genotype. The patient was strongly suspected of carrying a viral infection and died eventually without any other possible causes being apparent. No virus other than the novel TTV was identified in his serum sample. Although a direct causal link between the novel TTV genotype infection and the patient's disease could not be confirmed, the findings suggest that surveillance of this novel TTV genotype is necessary and that its role in disease deserves to be explored.

Molecular Characterization of Avian-like H1N1 Swine Influenza A Viruses Isolated in Eastern China, 2011

Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide： H1N1, H3N2, and H1N2. European avian-Hke H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3＇ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation （S31N）, a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.

Preliminary Studies on Identification of Lycium Linn.Germplasm Resources by nrDNA ITS Sequencing

[Objective] The study aimed to identify woltberry （Lycium Linn.） germplasm resources at molecular level by analyzing the nrDNA ITS sequence. [Method] Genomic DNA from woltberry leaves extracted by modified CTAB method were regarded as templates for PCR amplification by specific primer, clone and sequencing. [Result] The nrDNA ITS sequences were obtained and then differentiated among three tested materials. [Conclusion] PCR amplification and sequencing on nrDNA ITS is a feasible approach to identify different woltberry germplasm resources.

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma

AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal esophageal epithelial cell line,primary ESCC tumor tissue,and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction(RTPCR).Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels.Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene.RESULTS:In the majority of ESCC cell lines,we found that PTX3 expression was down-regulated due to gene promoter hypermethylation,which was further confirmed by bisulphite genomic sequencing.Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines.Methylation was more common in tumor tissues(85%) than in adjacent nontumor tissues(25%)(P < 0.01).CONCLUSION:PTX3 is down-regulated through promoter hypermethylation in ESCC,and could potentially serve as a biomarker of ESCC.

Molecular Cloning of Diapause Hormone Analog in Helicoverpa assulta

Diapause hormone (DH) is a neurohormone which is secreted from suboesophageal ganglion and responsible for induction of embryonic diapause in Bombyx mori.Here,using bioassay,we present evidence that Has-SGNPⅠ,which clearly exhibited diapause egg inducing activity as does Bom-DH,is likely to be DH.We reported the molecular characterization of the Helicoverpa assulta diapause hormone analog by polymerase chain reaction.A 665?bp fragment containing the entire DH-like gene was isolated and characterized.DH-like gene comprised two exons interspersed by a single intron,and the consensus sequence for splicing junction is identified at the exon/intron boundary.The result shows that DH-like gene is localized in the genome of Helicoverpa assulta.

Genetic evaluation and testing for hereditary forms of cancer in the era of next-generation sequencing

The introduction of next-generation sequencing(NGS) technology in testing for hereditary cancer susceptibility allows testing of multiple cancer susceptibility genes simultaneously. While there are many potential benefits to utilizing this technology in the hereditary cancer clinic, including efficiency of time and cost, there are also important limitations that must be considered. The best panel for the given clinical situation should be selected to minimize the number of variants of unknown significance. The inclusion in panels of low penetrance or newly identified genes without specific actionability can be problematic for interpretation.Genetic counselors are an essential part of the hereditary cancer risk assessment team, helping the medical team select the most appropriate test and interpret the often complex results. Genetic counselors obtain an extended family history, counsel patients on the available tests and the potential implications of results for themselves and their family members(pre-test counseling), explain to patients the implications of the test results(post-test counseling), and assist in testing family members at risk.

Scanning electron microscopy coupled with energydispersive X-ray spectrometry for quick detection of sulfuroxidizing bacteria in environmental water samples

Detection of sulfur-oxidizing bacteria has largely been dependent on targeted gene sequencing technology or traditional cell cultivation, which usually takes from days to months to carry out. This clearly does not meet the requirements of analysis for time-sensitive samples and/or complicated environmental samples. Since energy-dispersive X-ray spectrometry(EDS) can be used to simultaneously detect multiple elements in a sample, including sulfur, with minimal sample treatment, this technology was applied to detect sulfur-oxidizing bacteria using their high sulfur content within the cell. This article describes the application of scanning electron microscopy imaging coupled with EDS mapping for quick detection of sulfur oxidizers in contaminated environmental water samples, with minimal sample handling. Scanning electron microscopy imaging revealed the existence of dense granules within the bacterial cells, while EDS identified large amounts of sulfur within them. EDS mapping localized the sulfur to these granules. Subsequent 16S rRNA gene sequencing showed that the bacteria detected in our samples belonged to the genus Chromatium, which are sulfur oxidizers. Thus, EDS mapping made it possible to identify sulfur oxidizers in environmental samples based on localized sulfur within their cells, within a short time(within 24 h of sampling). This technique has wide ranging applications for detection of sulfur bacteria in environmental water samples.

Characterization of Genome-Wide Microsatellites of Saccharina japonica Based on a Preliminary Assembly of Illumina Sequencing Reads

Microsatellites or simple sequence repeats(SSR) function widely and locate dependently in genome. However, their characteristics are often ignored due to the lack of genomic sequences of most species. Kelp(Saccharina japonica), a brown macroalga, is extensively cultured in China. In this study, the genome of S. japonica was surveyed using an Illumina sequencing platform, and its microsatellites were characterized. The preliminarily assembled genome was 469.4 Mb in size, with a scaffold N_(50) of 20529 bp. Among the 128370 identified microsatellites, 90671, 25726 and 11973 were found in intergenic regions, introns and exons, averaging 339.3, 178.8 and 205.4 microsatellites per Mb, respectively. These microsatellites distributed unevenly in S. japonica genome. Mononucleotide motifs were the most abundant in the genome, while trinucleotide ones were the most prevalent in exons. The microsatellite abundance decreased significantly with the increase of motif repeat numbers, and the microsatellites with a small number of repeats accounted for a higher proportion of the exons than those of the intergenic regions and introns. C/G-rich motifs were more common in exons than in intergenic regions and introns. These characteristics of microsatellites in S. japonica genome may associate with their functions, and ultimately their adaptation and evolution. Among the 120140 pairs of designed microsatellite primers, approximately 75% were predicted to be able to amplify S. japonica DNA. These microsatellite markers will be extremely useful for the genetic breeding and population evolution studies of kelp.

Molecular cloning and mRNA expression analysis of myosin heavy chain(MyHC)from fast skeletal muscle of grass carp,Ctenopharyngodon idella

The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.