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
Inheritance of line Jinghe891-l resistant to pathotype of Puccinia striiformis in two patterns of temperature (Normal: day 18℃ /night 10℃ , High: day 24℃ /night 15℃ )was studied in this paper. The results showed t...Inheritance of line Jinghe891-l resistant to pathotype of Puccinia striiformis in two patterns of temperature (Normal: day 18℃ /night 10℃ , High: day 24℃ /night 15℃ )was studied in this paper. The results showed that there were at least two pairs of dominant major genes and one pair of recessive minor genes in Jinghe 891-1. The two pairs of major genes that conferred resistance to CY31 were allelic or linked closely with resistance gene in Jubilejna Ⅱ , Kangyin655 and T. spelta Album. They were novel resistance genes and were inherited in a repeated or independent mode. The minor genes, which could modify the major genes, were sensitive to temperature and conferred resistance to all pathotypes of Puccinia striiformis in China. It is recommended that this line can be used as an important resource stock.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
Summary: The relationship between glutathione S-transferases (GSTs) M1, T1 genotype and childhood acute lymphoblastic leukemia (ALL) was investigated. GSTM1 and GSTT1 genotypes in genomic DNA from 67 children with ALL...Summary: The relationship between glutathione S-transferases (GSTs) M1, T1 genotype and childhood acute lymphoblastic leukemia (ALL) was investigated. GSTM1 and GSTT1 genotypes in genomic DNA from 67 children with ALL and 146 healthy controls were analyzed by using the multiplex polymerase chain reaction (PCR). The frequencies of GSTM1, M1-T1 null genotypes in ALL children were significantly higher than in the healthy controls (76.12 % versus 52.74 %, OR=2.856, P<0.001; 50.74 % versus 24.66 %, OR=3.148, P<0.001, respectively). However, there was no significant relationship between GSTT1 null genotype and ALL of children (61.19 % versus 49.32 %, OR=1.621, P>0.05). It was suggested that GSTM1 null genotype might be a risk genotype of childhood ALL, while there as no correlation between GSTT1 null genotype and childhood ALL.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
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
The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene...The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction （PCR） from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- Nanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21（DE3） for expression. After induction by isopropyl-β-D-thiogalactoside （IPTG） at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.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.
基金supported by the Beijing Natural Science Foundation(6962006).
文摘Inheritance of line Jinghe891-l resistant to pathotype of Puccinia striiformis in two patterns of temperature (Normal: day 18℃ /night 10℃ , High: day 24℃ /night 15℃ )was studied in this paper. The results showed that there were at least two pairs of dominant major genes and one pair of recessive minor genes in Jinghe 891-1. The two pairs of major genes that conferred resistance to CY31 were allelic or linked closely with resistance gene in Jubilejna Ⅱ , Kangyin655 and T. spelta Album. They were novel resistance genes and were inherited in a repeated or independent mode. The minor genes, which could modify the major genes, were sensitive to temperature and conferred resistance to all pathotypes of Puccinia striiformis in China. It is recommended that this line can be used as an important resource stock.
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.
文摘Summary: The relationship between glutathione S-transferases (GSTs) M1, T1 genotype and childhood acute lymphoblastic leukemia (ALL) was investigated. GSTM1 and GSTT1 genotypes in genomic DNA from 67 children with ALL and 146 healthy controls were analyzed by using the multiplex polymerase chain reaction (PCR). The frequencies of GSTM1, M1-T1 null genotypes in ALL children were significantly higher than in the healthy controls (76.12 % versus 52.74 %, OR=2.856, P<0.001; 50.74 % versus 24.66 %, OR=3.148, P<0.001, respectively). However, there was no significant relationship between GSTT1 null genotype and ALL of children (61.19 % versus 49.32 %, OR=1.621, P>0.05). It was suggested that GSTM1 null genotype might be a risk genotype of childhood ALL, while there as no correlation between GSTT1 null genotype and childhood ALL.
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.
文摘The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction （PCR） from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- Nanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21（DE3） for expression. After induction by isopropyl-β-D-thiogalactoside （IPTG） at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.
