Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the...Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.展开更多
CACNA1 S gene is the gene encoding L-type calcium channel αa-subunit. CACNA1 S gene mutations can cause hypokalemic periodic pa- ralysis （HOKPP）. The related research speculated that CACNA1 S gene was the candidate...CACNA1 S gene is the gene encoding L-type calcium channel αa-subunit. CACNA1 S gene mutations can cause hypokalemic periodic pa- ralysis （HOKPP）. The related research speculated that CACNA1 S gene was the candidate genes which affect meat quality traits. In the present ar- ticle, the biological characteristics of CACNA1 S gene, structure, genetic diseases and the research development were respectively reviewed so as to provide a reference for further research.展开更多
Chinese isolate of transmissible gastroenteritis virus(TGEV)was propagated and harvested in swine testicle(ST)cells.Two pairs of primers were designed according to the published sequence with Oligo 4.1 and DNasis soft...Chinese isolate of transmissible gastroenteritis virus(TGEV)was propagated and harvested in swine testicle(ST)cells.Two pairs of primers were designed according to the published sequence with Oligo 4.1 and DNasis softwares.The products of RT-PCR were named Sa and Sb,of 2.3kb and 2.1kb respectively.Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I sites of the same pUC18 plasmid.The recombinant designated pUC-S was verified and analyzed by corresponding restriction endonuclease(RE)and nested PCR on the basis of genetic sites of S gene and physical map of pUC18 plasmid,which was identified as S gene from Chinese isolate of TGEV.展开更多
A novel proventriculopathogic variant (isolate ZJ971) of infectious bronchitis virus (IBV) was identified from enlarged proeventriculus of the sick chickens in the study. The S gene cDNA segment with 3.6 kb in length ...A novel proventriculopathogic variant (isolate ZJ971) of infectious bronchitis virus (IBV) was identified from enlarged proeventriculus of the sick chickens in the study. The S gene cDNA segment with 3.6 kb in length was amplified by RT-PCR with special primers from the ZJ971 viral isolate of (IBV) and cloned into plasmid pBluescript SK( + ). The recombinants containing S gene of IBV-ZJ971 isolate were identified by digestion of restriction enzyme EcoRI, BamHI and PCR amplification. The cloned S gene from isolate IBV-7J971 was composed of 3492 bp in length encoding for a polypeptide of 1080 amino acids. Comparing the nucleotide of S gene of IBV isolate ZJ971 with that of reported IBV strains Beaudette, M41, Ark99 and CuT2, the homology was 97.3%, 97.5%, 88.6% and 85.6%, respectively; and the homology of the deduced amino acids of S protein of IBV isolate ZJ971 was 96%, 96.3%, 86.1% and 83.1% respectively; especially, the mutation of 3241st nucleotide of S gene of IBV isolate ZJ971 from G to T resulted in the translating termination of S protein at 3240th nucleotide site.展开更多
TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published...TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published sequence of TGEV'S gene cDNA with Oligo version 4.1 and DNasis software. The products of PCR were named Sa and Sb, of 2.3 kb and 2.1 kb respectively. Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I multiple cloning sites of the same pUC18 plasmid. The recombinant pUC-S plasmid was identified and analyzed by corresponding restriction endonuclease and nested PCR on the basis of the genetic sites of S gene and pUC18 plasmid, which was identified as S gene of TGEV. Recombinant pUC-S was sequenced and analyzed in comparison with the other strains. Gene sequence comparison indicated that TH-98 shared 99, 97, 98, 97 and 94% identities with Purdue-115(US), Miller(US), TO14(Japan), FS772(British), 96-1933(British), respectively, their deduced amino acid homology was 99, 97, 97, 96 and 93% correspondingly. In addition, the analysis report verified that pUC-S owned a complete open reading frame (ORF) including initiation codon, signal sequences, remaining sequences and termination codon as well. Therefore, the results affirmed that S gene of TGEV TH-98 was extremely conservative.展开更多
Objective: To study the relationship of the mutation of HBV preS/S gene in HBsAg carrying pregnant women and intrauterine transmission. Methods: Polymerase chain reaction (PCR) was used to amplify HBV preS/S gene from...Objective: To study the relationship of the mutation of HBV preS/S gene in HBsAg carrying pregnant women and intrauterine transmission. Methods: Polymerase chain reaction (PCR) was used to amplify HBV preS/S gene from sera of 8 HBsAg carrying pregnant women, 4 women's neonates infected with HBV, and the other's neonates non-infected with. The PCR products were cloned and 5 clones were chosen from every woman for DNA sequencing. Results: Heterogeneity of HBV preS/S gene in HBsAg carrying pregnant women having intrauterine transmission was much higher than that from having not intrauterine transmission, and the divergence rate of nucleotide sequences also higher strikingly. Conclusion: High heterogeneity of HBV preS/S gene of HBsAg positive pregnant women may be relative to high rate of intrauterine transmis-sion展开更多
Hypotrichs are one of the highly differentiated ciliated lineages which play important roles in ecological, environmental,evolutionary and basic biological studies. In the present study, we investigated the living cha...Hypotrichs are one of the highly differentiated ciliated lineages which play important roles in ecological, environmental,evolutionary and basic biological studies. In the present study, we investigated the living characteristics, infraciliature, nuclear apparatus, ontogenesis and phylogenetic position of a marine hypotrichous ciliate, Apokeronopsis wrighti Long et al., 2008, which was isolated from coastal waters in Shenzhen, China. The new isolate resembles the type population in terms of morphological characteristics, morphometrics, and SSU rRNA gene sequence that is with a 99.7% similarity. Ontogenesis of A. wrighti is characterized by oral primordium for the proter as well as marginal and dorsal kineties anlagen in both filial products formed de novo, and the cirral row arranged along the paroral and endoral arises from several anterior frontoventral-transverse cirral streaks. Phylogenetic analyses based on SSU and concatenated gene data suggest that five species of Apokeronopsis form a monophyletic clade, and the genus Apokeronopsis is closely related to Thigmokeronopsis and Metaurostylopsis.展开更多
Objective To assess the correlation between hepatitis B virus (HBV) surface gene mutant infection and hepatitis B (HB) vaccination failure Methods Using sera from 106 infants who were born to HBV carrier mothers a...Objective To assess the correlation between hepatitis B virus (HBV) surface gene mutant infection and hepatitis B (HB) vaccination failure Methods Using sera from 106 infants who were born to HBV carrier mothers and failed in HB immunoprophylaxis, HBV S gene was amplified by PCR, transferred to nylon membranes for Southern blots, and then hybridized with oligonucleotide probes Eleven of non hybridizing samples were used for DNA sequencing Results 93 4% (99/106) of the samples were HBV DNA positive, and 30 3% (30/99) failed to hybridize with at least one of the four probes DNA sequencing confirmed that 10 of the 11 samples had an S gene mutation with amino acid (aa) change The identified mutants included nucleotide (nt) 546T→A(aa131N→T), nt531T→C (aa126I→T), nt491A→C (aa113T→P), nt491T→A (aa113S→T), nt533C→A (aa127P→T), nt581T→A (aa143S→T), nt636A→T (aa161Y→F), and nt679A→C (aa175L→F) The sequence in one mother infant pair was completely the same, with mutations at aa131 and aa161 Conclusions The prevalence of HBV surface mutants is about 30% in the children failing in HB vaccination HBV mutants can infect infants by maternal infant transmission展开更多
Gold nanoparticles (nano Au)/titanium dioxide (TiO2) hollow microsphere membranes were prepared on the carbon paste electrode (CPE) for enhancing the sensitivity of DNA hybridization detection. The immobilization of n...Gold nanoparticles (nano Au)/titanium dioxide (TiO2) hollow microsphere membranes were prepared on the carbon paste electrode (CPE) for enhancing the sensitivity of DNA hybridization detection. The immobilization of nano Au and TiO2 microsphere was investigated with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored with EIS us-ing [Fe(CN)6]3-/4- as indicator. The sequence-specific DNA of the 35S promoter from cauliflower mosaic virus (CaMV35S) gene was detected with this DNA electrochemical sensor. The dynamic detection range was from 1.0×10-12 to 1.0×10-8 mol/L DNA and a detection limit of 2.3×10-13 mol/L could be ob-tained. The polymerase chain reaction (PCR) amplification of the terminator of nopaline synthase (NOS) gene from the real sample of a kind of transgenic soybean was also satisfactorily detected.展开更多
Staphylococcus aureus is a bacterial species responsible for food poisoning and outbreaks of opportunistic, nosocomial and community-acquired diseases. The aim of this study was to characterize S. aureus strains resis...Staphylococcus aureus is a bacterial species responsible for food poisoning and outbreaks of opportunistic, nosocomial and community-acquired diseases. The aim of this study was to characterize S. aureus strains resistant to methicillin. Seventy-five (75) samples of smoked fish, including Scomber scombrus, Trachurus trachurus, Thunnus spp., Cyprinus spp. and Sardinella spp., were studied. The Mueller-Hinton diffusion method was used to determine the phenotypic resistance profile. The coagulase test and thermonuclease detection were used to assess the enzymatic production potential of the strains. The methicillin resistance mecA gene was detected by PCR. With a contamination rate of 80%, the prevalence of S. aureus varied from 15% to 31.7% in animal products. S. aureus strains were DNase (91.7%) and coagulase (50%) producers. The resistance of these strains was 42.7% (cefoxitin), 37.8% (oxacillin) and 26.4% (cefuroxime sodium). They were resistant to tetracycline (62.4%), erythromycin (61.1%), vancomycin (34.6%), levofloxacin (33.3%) and imipenem (12.7%). The prevalence of the mecA gene in animal products ranged from 13.9% to 33.4%. The mecA gene induction showed different sensitivities with cefoxitin (100%) and oxacillin (56.7%). In addition, all tests showed a specificity of 100%. This work demonstrates the need to strengthen surveillance to prevent the spread of S. aureus epidemics in various environments.展开更多
Mitochondrial 12S and 16S ribosomal RNA genes sequences were sequenced using dye-labeled terminator on an ABI 377 automated sequencer in 11 individuals and 1 species' sequences were gained from GenBank,representin...Mitochondrial 12S and 16S ribosomal RNA genes sequences were sequenced using dye-labeled terminator on an ABI 377 automated sequencer in 11 individuals and 1 species' sequences were gained from GenBank,representing 6 genera of family Tetrigidae.The collated sequences were aligned using Clustal X version 1.81 and then,the sequence variability and heredity distances based on Kimura 2-parameter model were calculated using Mega 2.1.In obtained sequences (736 bp),the average A+T content is 73.9%,ranging from 71.2% to 77.5%;the overall G+C content is 26.1%,ranging from 22.5% to 28.8%.Based on alignment of the combined sequences,185 parsimony-informative sites were revealed in 755 available base pairs.Phylogenetic trees were reconstructed using NJ,MP and ML methods with Cylindraustralia kochii as outgroup.The results indicated that the monophyletic nature of Tetrix is questioned and suggest that T.tubercarina may be given tribal rank.Our results also show that Coptltettix huanjiangensis and C.gongshanensis are the same species,i.e.Coptltettix gongshanensis Zheng,and C.huanjiangensis is the synonyms of C.gongshanensis.展开更多
A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes s...A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes sequences. 402 bp Cytb genes were achieved by PCR-sequencing using DNA extracted from 8 case samples, and contrasted with 27 sequences of Cytb gene downloaded from GenBank database. The values of three nucleotide distance between three suspected samples and sika deer were identical （0.026±0.006）, which was smaller than the smallest nucleotide distance between eastern red deer and sika deer （0.036）. Furthermore, phylogenetic analysis of sika deer and red deer indicated that the evidences located within the same cluster as sika deer. The evidences were sika deer materials. As the same way, other three suspected samples were derived from red deer. The results were further confirmed by phylogenetic and nucleotide distance analysis of 387 bp 12s rRNA gene. The method was powerful and less time-consuming and helpful to reduce the related cases with wildlife.展开更多
Weaning of piglets is generally considered as a stressor which changes intestinal ecosystem and leads to clinical implications. Microbiota inhabiting in small intestine （especially ileum） are assumed to promote heal...Weaning of piglets is generally considered as a stressor which changes intestinal ecosystem and leads to clinical implications. Microbiota inhabiting in small intestine （especially ileum） are assumed to promote health, but their functional properties are yet poody dascdbed. As indicated by the 16S rRNA gene sequences of ileal micrebiota in nursing piglets （at the age of 21 and 28 d） and 28-day-old weaned piglets （weaned at 21 d of age）, the microbiota were mainly comprised of gram-positive bacteria. There were 40 operational taxonomic units （OTUs） （from 171 clones） in the ileum of nursing piglets aged 21 d, 61 OTUs （from 194 clones） in the ileum of nursing piglets aged 28 d, and 56 OTUs （from 171 clones） in the ileum of weaned piglets aged 28 d. The flea of nursing piglets aged 21 d were dominantly occupied by Lactobacilli （87.7%） as well as Streptococ cus （ 3.5 % ）. Lactobacillus amy/ovorus （41.5 % ）, Lactobaci/lus sp. （ 19.3 % ）, Lactobaci/lus reuteri （ 12.3 % ）, Lactobacillus salivarius （ 9.4 % ） and L. mucosae （4.7%） were the predominant species among Lactobacil/L Similar results were obtained in the nursing piglets at 28 d of age ex- cept that Lactobaci/li decreased to 71.1% and Streptococcus increased to 21.1% significantly. Lactobacillus （52.0%） and Streptococcus （26.3%） were the two major groups in the ileum of weaned piglets aged 28 d. Lactobacillus amylovorus （31.6%） and Lactobaci/lus reuteri （ 16.4% ） was the two most important species in Lactobacillus. Therefore, Lactobacilli were predominant in the ileum of nursing and weaned piglets, and they had the highest diversity, followed by Streptococcus. The diversity of ileal microbiota was not different remarkably between the nursing piglets and the weaned piglets, but the composition changed significantly. These findings are helpful to understand ileal bacterial ecophysiology and further develop nutritional regimes to prevent or counteract complications during the weaning transition.展开更多
[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing...[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.展开更多
S-locus genes were cloned from three Brassica napus and three B. campestris lines by using PCR walking and homologuesequence methods. A phylogenetic gene tree was constructed based on the six cloned genes and fifty-on...S-locus genes were cloned from three Brassica napus and three B. campestris lines by using PCR walking and homologuesequence methods. A phylogenetic gene tree was constructed based on the six cloned genes and fifty-one previouslyreported SLG/SRK genes of Brassica and Raphanus. The SLGs from R. sativus were dispersed in the phylogenetic treeintermingling with SLG/SRKs from B. oleracea, B. napus and B. campestris. The SLG/SRK genes of classⅡclusteredindependently in one group. The SLG/SRK genes of classⅠshowed to be more divergent than classⅡgenes. Theseresults suggested that the divergence of classⅠand classⅡ should have occurred before the differentiation of thegenera Brassica and Raphanus. In addition, SLG and SRK of the same S haplotypes belonged to the same class. Itsuggested that class-Ⅰ and class-Ⅱ group divergence occurred first, and then SLG and SRK diverged. The three SC SRKgenes from B. napus and B. campestris were grouped into one cluster, displaying difference from the SC SLG of B.oleracea. These three SC SRK genes were close to SI SRK of SI1300, SI271 and guanyou in phylogenetic relationships.These results indicated that SC and SI genes diverged more recently. It is not clear yet whether the differentiation of SCand SI genes was earlier than the differentiation of Brassica and Raphanus. Studies based on more genes are necessaryfor a comprehensive elucidation of the phylogenetic relationships in Brassicaceae.展开更多
[Objective] The aim was to select suitable gene for Chlorella identification and to identify the oil-producing microalgae.[Method] Four candidate gene sequences,the nuclear genomic rDNA of the 18S rRNA gene,internal t...[Objective] The aim was to select suitable gene for Chlorella identification and to identify the oil-producing microalgae.[Method] Four candidate gene sequences,the nuclear genomic rDNA of the 18S rRNA gene,internal transcribed spacer（ITS）,internal transcribed spacer Ⅱ（ITS Ⅱ）and the chloroplast rbcL gene,were selected for Chlorella molecular identification.Through these four candidate genes,the genetic variability and distinguish ability between intra-species and inter-species was analyzed to choose the right genes for identification of the high oil-content Chlorella.On this basis,application of these gene segments were classified and identified for five fresh-water isolated Chlorella,which oil-content is more than 30%.[Result] ITS gene was a suitable gene because of its high variation and short fragment length,meanwhile its genetic distance intra-species（0.439 6±0.135 9）was larger than inter-species（0.045 7±0.084 3）.Its sequence length varied between different species whereas highly conserved in the same species.By the application of ITS sequences,respectively,five high oil-content stains were identified as one C.vulgaris,two strains of C.sorokiniana and two strains of algae Chlorella sp.[Conclusion] This study had provided reference for the establishment of identification gene pool of Chlorella.展开更多
文摘Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.
文摘CACNA1 S gene is the gene encoding L-type calcium channel αa-subunit. CACNA1 S gene mutations can cause hypokalemic periodic pa- ralysis （HOKPP）. The related research speculated that CACNA1 S gene was the candidate genes which affect meat quality traits. In the present ar- ticle, the biological characteristics of CACNA1 S gene, structure, genetic diseases and the research development were respectively reviewed so as to provide a reference for further research.
文摘Chinese isolate of transmissible gastroenteritis virus(TGEV)was propagated and harvested in swine testicle(ST)cells.Two pairs of primers were designed according to the published sequence with Oligo 4.1 and DNasis softwares.The products of RT-PCR were named Sa and Sb,of 2.3kb and 2.1kb respectively.Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I sites of the same pUC18 plasmid.The recombinant designated pUC-S was verified and analyzed by corresponding restriction endonuclease(RE)and nested PCR on the basis of genetic sites of S gene and physical map of pUC18 plasmid,which was identified as S gene from Chinese isolate of TGEV.
基金the National NatureScience Foundation of China(30070570)the NatureScience Foundation of Zhejiang Province(399411) the Science and Technology Commission of Zhejiang Province(991102030).
文摘A novel proventriculopathogic variant (isolate ZJ971) of infectious bronchitis virus (IBV) was identified from enlarged proeventriculus of the sick chickens in the study. The S gene cDNA segment with 3.6 kb in length was amplified by RT-PCR with special primers from the ZJ971 viral isolate of (IBV) and cloned into plasmid pBluescript SK( + ). The recombinants containing S gene of IBV-ZJ971 isolate were identified by digestion of restriction enzyme EcoRI, BamHI and PCR amplification. The cloned S gene from isolate IBV-7J971 was composed of 3492 bp in length encoding for a polypeptide of 1080 amino acids. Comparing the nucleotide of S gene of IBV isolate ZJ971 with that of reported IBV strains Beaudette, M41, Ark99 and CuT2, the homology was 97.3%, 97.5%, 88.6% and 85.6%, respectively; and the homology of the deduced amino acids of S protein of IBV isolate ZJ971 was 96%, 96.3%, 86.1% and 83.1% respectively; especially, the mutation of 3241st nucleotide of S gene of IBV isolate ZJ971 from G to T resulted in the translating termination of S protein at 3240th nucleotide site.
文摘TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published sequence of TGEV'S gene cDNA with Oligo version 4.1 and DNasis software. The products of PCR were named Sa and Sb, of 2.3 kb and 2.1 kb respectively. Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I multiple cloning sites of the same pUC18 plasmid. The recombinant pUC-S plasmid was identified and analyzed by corresponding restriction endonuclease and nested PCR on the basis of the genetic sites of S gene and pUC18 plasmid, which was identified as S gene of TGEV. Recombinant pUC-S was sequenced and analyzed in comparison with the other strains. Gene sequence comparison indicated that TH-98 shared 99, 97, 98, 97 and 94% identities with Purdue-115(US), Miller(US), TO14(Japan), FS772(British), 96-1933(British), respectively, their deduced amino acid homology was 99, 97, 97, 96 and 93% correspondingly. In addition, the analysis report verified that pUC-S owned a complete open reading frame (ORF) including initiation codon, signal sequences, remaining sequences and termination codon as well. Therefore, the results affirmed that S gene of TGEV TH-98 was extremely conservative.
基金Supported by the National Natural Science Foundation of China (No. 39970652)
文摘Objective: To study the relationship of the mutation of HBV preS/S gene in HBsAg carrying pregnant women and intrauterine transmission. Methods: Polymerase chain reaction (PCR) was used to amplify HBV preS/S gene from sera of 8 HBsAg carrying pregnant women, 4 women's neonates infected with HBV, and the other's neonates non-infected with. The PCR products were cloned and 5 clones were chosen from every woman for DNA sequencing. Results: Heterogeneity of HBV preS/S gene in HBsAg carrying pregnant women having intrauterine transmission was much higher than that from having not intrauterine transmission, and the divergence rate of nucleotide sequences also higher strikingly. Conclusion: High heterogeneity of HBV preS/S gene of HBsAg positive pregnant women may be relative to high rate of intrauterine transmis-sion
基金supported by the Natural Science Foundation of Shaanxi Province(Nos.2023-JC-QN-0214,2023JC-QN-0185)the Postdoctoral Science Foundation of Shaanxi Province(No.2023BSHEDZZ199)the Fundamental Research Funds for the Central Universities(No.GK202207019)。
文摘Hypotrichs are one of the highly differentiated ciliated lineages which play important roles in ecological, environmental,evolutionary and basic biological studies. In the present study, we investigated the living characteristics, infraciliature, nuclear apparatus, ontogenesis and phylogenetic position of a marine hypotrichous ciliate, Apokeronopsis wrighti Long et al., 2008, which was isolated from coastal waters in Shenzhen, China. The new isolate resembles the type population in terms of morphological characteristics, morphometrics, and SSU rRNA gene sequence that is with a 99.7% similarity. Ontogenesis of A. wrighti is characterized by oral primordium for the proter as well as marginal and dorsal kineties anlagen in both filial products formed de novo, and the cirral row arranged along the paroral and endoral arises from several anterior frontoventral-transverse cirral streaks. Phylogenetic analyses based on SSU and concatenated gene data suggest that five species of Apokeronopsis form a monophyletic clade, and the genus Apokeronopsis is closely related to Thigmokeronopsis and Metaurostylopsis.
文摘Objective To assess the correlation between hepatitis B virus (HBV) surface gene mutant infection and hepatitis B (HB) vaccination failure Methods Using sera from 106 infants who were born to HBV carrier mothers and failed in HB immunoprophylaxis, HBV S gene was amplified by PCR, transferred to nylon membranes for Southern blots, and then hybridized with oligonucleotide probes Eleven of non hybridizing samples were used for DNA sequencing Results 93 4% (99/106) of the samples were HBV DNA positive, and 30 3% (30/99) failed to hybridize with at least one of the four probes DNA sequencing confirmed that 10 of the 11 samples had an S gene mutation with amino acid (aa) change The identified mutants included nucleotide (nt) 546T→A(aa131N→T), nt531T→C (aa126I→T), nt491A→C (aa113T→P), nt491T→A (aa113S→T), nt533C→A (aa127P→T), nt581T→A (aa143S→T), nt636A→T (aa161Y→F), and nt679A→C (aa175L→F) The sequence in one mother infant pair was completely the same, with mutations at aa131 and aa161 Conclusions The prevalence of HBV surface mutants is about 30% in the children failing in HB vaccination HBV mutants can infect infants by maternal infant transmission
基金the National Natural Science Foundation of China (Grant Nos. 20635020 and 20375020)Doctoral Foundation of the Ministry of Education of China (Grant No. 20060426001)Natural Science Foundation of Qingdao City (Grant No. 04-2-JZP-8)
文摘Gold nanoparticles (nano Au)/titanium dioxide (TiO2) hollow microsphere membranes were prepared on the carbon paste electrode (CPE) for enhancing the sensitivity of DNA hybridization detection. The immobilization of nano Au and TiO2 microsphere was investigated with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored with EIS us-ing [Fe(CN)6]3-/4- as indicator. The sequence-specific DNA of the 35S promoter from cauliflower mosaic virus (CaMV35S) gene was detected with this DNA electrochemical sensor. The dynamic detection range was from 1.0×10-12 to 1.0×10-8 mol/L DNA and a detection limit of 2.3×10-13 mol/L could be ob-tained. The polymerase chain reaction (PCR) amplification of the terminator of nopaline synthase (NOS) gene from the real sample of a kind of transgenic soybean was also satisfactorily detected.
文摘Staphylococcus aureus is a bacterial species responsible for food poisoning and outbreaks of opportunistic, nosocomial and community-acquired diseases. The aim of this study was to characterize S. aureus strains resistant to methicillin. Seventy-five (75) samples of smoked fish, including Scomber scombrus, Trachurus trachurus, Thunnus spp., Cyprinus spp. and Sardinella spp., were studied. The Mueller-Hinton diffusion method was used to determine the phenotypic resistance profile. The coagulase test and thermonuclease detection were used to assess the enzymatic production potential of the strains. The methicillin resistance mecA gene was detected by PCR. With a contamination rate of 80%, the prevalence of S. aureus varied from 15% to 31.7% in animal products. S. aureus strains were DNase (91.7%) and coagulase (50%) producers. The resistance of these strains was 42.7% (cefoxitin), 37.8% (oxacillin) and 26.4% (cefuroxime sodium). They were resistant to tetracycline (62.4%), erythromycin (61.1%), vancomycin (34.6%), levofloxacin (33.3%) and imipenem (12.7%). The prevalence of the mecA gene in animal products ranged from 13.9% to 33.4%. The mecA gene induction showed different sensitivities with cefoxitin (100%) and oxacillin (56.7%). In addition, all tests showed a specificity of 100%. This work demonstrates the need to strengthen surveillance to prevent the spread of S. aureus epidemics in various environments.
文摘Mitochondrial 12S and 16S ribosomal RNA genes sequences were sequenced using dye-labeled terminator on an ABI 377 automated sequencer in 11 individuals and 1 species' sequences were gained from GenBank,representing 6 genera of family Tetrigidae.The collated sequences were aligned using Clustal X version 1.81 and then,the sequence variability and heredity distances based on Kimura 2-parameter model were calculated using Mega 2.1.In obtained sequences (736 bp),the average A+T content is 73.9%,ranging from 71.2% to 77.5%;the overall G+C content is 26.1%,ranging from 22.5% to 28.8%.Based on alignment of the combined sequences,185 parsimony-informative sites were revealed in 755 available base pairs.Phylogenetic trees were reconstructed using NJ,MP and ML methods with Cylindraustralia kochii as outgroup.The results indicated that the monophyletic nature of Tetrix is questioned and suggest that T.tubercarina may be given tribal rank.Our results also show that Coptltettix huanjiangensis and C.gongshanensis are the same species,i.e.Coptltettix gongshanensis Zheng,and C.huanjiangensis is the synonyms of C.gongshanensis.
文摘A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes sequences. 402 bp Cytb genes were achieved by PCR-sequencing using DNA extracted from 8 case samples, and contrasted with 27 sequences of Cytb gene downloaded from GenBank database. The values of three nucleotide distance between three suspected samples and sika deer were identical （0.026±0.006）, which was smaller than the smallest nucleotide distance between eastern red deer and sika deer （0.036）. Furthermore, phylogenetic analysis of sika deer and red deer indicated that the evidences located within the same cluster as sika deer. The evidences were sika deer materials. As the same way, other three suspected samples were derived from red deer. The results were further confirmed by phylogenetic and nucleotide distance analysis of 387 bp 12s rRNA gene. The method was powerful and less time-consuming and helpful to reduce the related cases with wildlife.
基金Supported by grants from Knowledge Innovation Project of Chinese Academy of Sciences (KSCX2-YW-N-051 and SW-323)NSFC(30901040, 30901041, 30928018, 30828025, 30700581, and 30771558)+2 种基金National Basic Research Program of China(2009CB118800)National 863 project (2008AA10Z316)National Scientific and Technological Supporting Project(2007BAQ01047 and 2006BAD12B07)~~
文摘Weaning of piglets is generally considered as a stressor which changes intestinal ecosystem and leads to clinical implications. Microbiota inhabiting in small intestine （especially ileum） are assumed to promote health, but their functional properties are yet poody dascdbed. As indicated by the 16S rRNA gene sequences of ileal micrebiota in nursing piglets （at the age of 21 and 28 d） and 28-day-old weaned piglets （weaned at 21 d of age）, the microbiota were mainly comprised of gram-positive bacteria. There were 40 operational taxonomic units （OTUs） （from 171 clones） in the ileum of nursing piglets aged 21 d, 61 OTUs （from 194 clones） in the ileum of nursing piglets aged 28 d, and 56 OTUs （from 171 clones） in the ileum of weaned piglets aged 28 d. The flea of nursing piglets aged 21 d were dominantly occupied by Lactobacilli （87.7%） as well as Streptococ cus （ 3.5 % ）. Lactobacillus amy/ovorus （41.5 % ）, Lactobaci/lus sp. （ 19.3 % ）, Lactobaci/lus reuteri （ 12.3 % ）, Lactobacillus salivarius （ 9.4 % ） and L. mucosae （4.7%） were the predominant species among Lactobacil/L Similar results were obtained in the nursing piglets at 28 d of age ex- cept that Lactobaci/li decreased to 71.1% and Streptococcus increased to 21.1% significantly. Lactobacillus （52.0%） and Streptococcus （26.3%） were the two major groups in the ileum of weaned piglets aged 28 d. Lactobacillus amylovorus （31.6%） and Lactobaci/lus reuteri （ 16.4% ） was the two most important species in Lactobacillus. Therefore, Lactobacilli were predominant in the ileum of nursing and weaned piglets, and they had the highest diversity, followed by Streptococcus. The diversity of ileal microbiota was not different remarkably between the nursing piglets and the weaned piglets, but the composition changed significantly. These findings are helpful to understand ileal bacterial ecophysiology and further develop nutritional regimes to prevent or counteract complications during the weaning transition.
基金Supported by Agricultural Seed Project of Shandong Province~~
文摘[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.
基金supported by the Outstanding Youth Science Funds(39825117)Bohai University Science Funds(BJ2004001),P.R.China
文摘S-locus genes were cloned from three Brassica napus and three B. campestris lines by using PCR walking and homologuesequence methods. A phylogenetic gene tree was constructed based on the six cloned genes and fifty-one previouslyreported SLG/SRK genes of Brassica and Raphanus. The SLGs from R. sativus were dispersed in the phylogenetic treeintermingling with SLG/SRKs from B. oleracea, B. napus and B. campestris. The SLG/SRK genes of classⅡclusteredindependently in one group. The SLG/SRK genes of classⅠshowed to be more divergent than classⅡgenes. Theseresults suggested that the divergence of classⅠand classⅡ should have occurred before the differentiation of thegenera Brassica and Raphanus. In addition, SLG and SRK of the same S haplotypes belonged to the same class. Itsuggested that class-Ⅰ and class-Ⅱ group divergence occurred first, and then SLG and SRK diverged. The three SC SRKgenes from B. napus and B. campestris were grouped into one cluster, displaying difference from the SC SLG of B.oleracea. These three SC SRK genes were close to SI SRK of SI1300, SI271 and guanyou in phylogenetic relationships.These results indicated that SC and SI genes diverged more recently. It is not clear yet whether the differentiation of SCand SI genes was earlier than the differentiation of Brassica and Raphanus. Studies based on more genes are necessaryfor a comprehensive elucidation of the phylogenetic relationships in Brassicaceae.
基金Supported by Key Project of Knowledge Innovation Project of Chinese Academy of Sciences(KGCX2-YW-374-3)Scientific and Technological Project of Shandong Province(2008GG20007002)~~
文摘[Objective] The aim was to select suitable gene for Chlorella identification and to identify the oil-producing microalgae.[Method] Four candidate gene sequences,the nuclear genomic rDNA of the 18S rRNA gene,internal transcribed spacer（ITS）,internal transcribed spacer Ⅱ（ITS Ⅱ）and the chloroplast rbcL gene,were selected for Chlorella molecular identification.Through these four candidate genes,the genetic variability and distinguish ability between intra-species and inter-species was analyzed to choose the right genes for identification of the high oil-content Chlorella.On this basis,application of these gene segments were classified and identified for five fresh-water isolated Chlorella,which oil-content is more than 30%.[Result] ITS gene was a suitable gene because of its high variation and short fragment length,meanwhile its genetic distance intra-species（0.439 6±0.135 9）was larger than inter-species（0.045 7±0.084 3）.Its sequence length varied between different species whereas highly conserved in the same species.By the application of ITS sequences,respectively,five high oil-content stains were identified as one C.vulgaris,two strains of C.sorokiniana and two strains of algae Chlorella sp.[Conclusion] This study had provided reference for the establishment of identification gene pool of Chlorella.
