Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila...Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.展开更多
Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequence...Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.展开更多
In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms(GMOs) and products,ensure bio-safety and reduce ecological risk in China,a real-time fluorescent quant...In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms(GMOs) and products,ensure bio-safety and reduce ecological risk in China,a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017.The established method was evaluated based on the specificity,sensitivity,accuracy and measurement uncertainty.The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017.1.50%MON88017 sample was detected with 29 replications.The average measured value(1.541%) was close to the actual value(1.50%) and the relative deviation was 2.70%.The variation coefficient of the measured value was 0.110 4;the recovery was 100.00%and the measurement uncertainty was 0.096.The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5%confidence level.Thus,the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity,accuracy and sensitivity,which could provide technical support for the safety supervision of genetically modified organisms and products in China.展开更多
Objective:To compare the effects of clinical application of chemiluminescence and real-time,fluorescence-based quantitative PCR in the detection Epstein-Barr virus(EBV).Methods:The data of chemiluminescence and real-t...Objective:To compare the effects of clinical application of chemiluminescence and real-time,fluorescence-based quantitative PCR in the detection Epstein-Barr virus(EBV).Methods:The data of chemiluminescence and real-time fluorescent quantitative PCR.fromipaEsfwo were suspected of being infectea w1tn rito1 roro January 2016 to January 2019 in our hospital were analyzed.The specific stage of EBV infection was analyzed,and the differences in results of the two detection methods were compared.Results:Chemiluminescence method was used to detect EBV infection during the active phase.The sensitivity of the chemiluminescence method was 76.7%(56/73)and the real-time quantitative PCRmethod was 90.4%(66/73).There was a statistical difference between the two detection methods(P<0.05).Conclusion:There was no statistical difference in positive predictive values between the chemiluminescence method and the real-time,fluorescence-based quantitative PCR method in the detection of EBV infection,but the sensitivity of chemiluminescence method is slightly lower than the real-time quantitative PCRmethod.It is noteworthy that chemiluminescence method is convenient and fast while the real-time,fluorescence-based quantitative PCR method is more accurate,which can provide a more accurate reference for clinical treatment.展开更多
Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in dise...Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.展开更多
Background: Protein kinase B (AKT/PKB) family is frequently amplified in ovarian cancer (OC). To the greatest of our knowledge, there is a lack of published reports about the amplification of the genes belonging to th...Background: Protein kinase B (AKT/PKB) family is frequently amplified in ovarian cancer (OC). To the greatest of our knowledge, there is a lack of published reports about the amplification of the genes belonging to the AKT family among Sudanese women with OC. The present study was conducted to detect the AKT1 gene amplification and its association with tumour types, grades, and ages among Sudanese women with OC, bearing in mind the ethnic variation. Methods: This institution-based study included 79 cases of women diagnosed with ovarian cancer (OC) at Omdurman Maternity Hospital in the period 2013-2018. Formalin-fixed, paraffin-embedded (FFPE) tissue sections were used to extract RNA. AKT1 gene amplification was assessed using quantitative real-time PCR. Results: The mean age (±SD) of included women was 49.29 (±13.612). The amplification of AKT1 gene was observed in 18/79 (22.8%) of OC women, with a high frequency in women with undifferentiated 1/2 (50%), clear cell 2/6 (33.3%), mucinous 3/11 (27.3%), endometrioid 3/17 (17.6%), and serous carcinomas 5/30 OC (16.7%). High frequency was seen in women with low (26.3%;n = 10/28) rather than in higher (19.5%;n = 8/33) grade carcinoma, and in older (25.8%;n = 8/23) rather than younger (18.2%;n = 2/9) women. No significant association between AKT1 gene amplification and tumour types, grades, and ages of women was observed (Fisher’s Exact test: p = 0.405, 0.593 and 0.851, respectively). Conclusion: AKT1 gene amplification arises in around one-fifth of Sudanese women with ovarian cancer (OC). It is seen more in undifferentiated, clear cell, and mucinous tumours types, and more frequently in low tumour grade and older women, but not to a statistically significant level. These outcomes sustenance previous studies suggesting that activated AKT genes have a vital role in OC progression and may offer a plan for targeted therapy and prognostic evaluation.展开更多
<strong>Objective: </strong><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">To investigate the prote...<strong>Objective: </strong><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">To investigate the protein expression and clinicopathological characteristics of ABCG2</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">, </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">Oct4</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">,</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> Nanog in laryngeal cancer tissues, and to seek new molecular markers for the diagnosis of laryngeal cancer.</span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><b><span style="font-family:Verdana;">Methods: </span></b></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">The laryngeal cancer tissues and paracancerous tissues of 87 patients with laryngeal carcinoma diagnosed in the department of otorhinolaryngology and head and neck surgery in our hospital from April 2016 to April 2018 were selected as the subjects. QRT-PCR, Real-time PcR, Western blot and immunohistochemical staining were used to detect the expression of ABCG2, Oct4 and Nanog in (Tumor Tissue) and (Adjacent Tissue) in tumor tissue and paracancerous tissue.</span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><b><span style="font-family:Verdana;">Results: </span></b></span></span><span><span><span style="font-family:;" "=""><span style="font-family:Verdana;">The results of RT-PCR showed that the positive rates of ABCG2, Oct4 and Nanog in laryngeal carcinoma tissues were 49.30%, 45.07% and 52.11%, respectively, while those in paracancerous tissues were </span><span style="font-family:Verdana;">22.54%, 21.13% and 15.49%, respectively (P < 0.01). The expression of ABCG2,</span><span style="font-family:Verdana;"> Oct4 and Nanog in laryngeal carcinoma was correlated with tumor differentiation, depth of invasion, age and sex (P < 0.05), but not with tumor size and TNM stage.</span></span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><b><span style="font-family:Verdana;">Conclusion: </span></b></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">The expressions of ABCG2, Oct4 and Nanog in cancer tissues are related to tumor differentiation status, and they can be used as new molecular markers for the diagnosis of laryngeal cancer</span></span></span><span style="font-family:Verdana;">.</span>展开更多
Toll like receptors(TLRs)are the main innate immune‘pattern recognition receptors’of animals,which play a central role in host cell recognition and responses to invasive pathogens,particularly common structures of m...Toll like receptors(TLRs)are the main innate immune‘pattern recognition receptors’of animals,which play a central role in host cell recognition and responses to invasive pathogens,particularly common structures of microbial pathogens.In this study,the gene expression profiles of TLRs in the spleen,head kidney,gill,small intestine,liver,muscle,and heart of healthy Paralichthys olivaceus were detected by real-time quantitative PCR(qPCR).The TLR family members were widely expressed in different tissues with different basic expression profiles.The highest expressions of TLR1,5m,7,8,9,14,and 21 were found in the spleen;the highest expressions of TLR3 and TLR21 were found in the gill;the highest expressions of TLR2 and 5s were found in the small intestine.The second highest expressions of TLR3,7,and 8 were found in small intestine.The gene expression profiles of TLRs stimulated with Edwardsiella tarda DNA,RNA,and lipopolysaccharide(LPS)were also detected in spleen,head kidney and gill.TLR9 and TLR21 were sensitive to E.tarda DNA;TLR 8 and TLR21 were sensitive to E.tarda RNA;and TLR1 and TLR14 were sensitive to E.tarda LPS.The expressions of the other TLR genes showed no significant changes.The results imply that the expressions of these TLR genes in P.olivaceus are differently regulated in the whole body and play important roles in the immune response against E.tarda infection.展开更多
BACKGROUND Cytomegalovirus(CMV)infections in the population are mostly subclinical,inapparent,or latent.However,it is rare in brain tissue.Most reported CMV encephalitis cases were patients with immunodeficiency.The d...BACKGROUND Cytomegalovirus(CMV)infections in the population are mostly subclinical,inapparent,or latent.However,it is rare in brain tissue.Most reported CMV encephalitis cases were patients with immunodeficiency.The diagnosis and detection rate of CMV encephalitis in patients with normal immune function needs to be further improved.CASE SUMMARY An 86-year-old male was admitted due to a sudden onset of unconsciousness for 3 h.The patient developed status epilepticus and was relieved after antiepileptic treatment.Encephalitis was considered due to the high signals of diffusionweighted imaging sequences in the right central region by magnetic resonance imaging.Metagenomic next-generation sequencing(mNGS)of blood and cerebrospinal fluid revealed CMV,with unique reads number being 614 and 1,respectively.Simultaneous quantitative PCR results showed CMV positive in blood samples and negative in cerebrospinal fluid samples.The patient was finally diagnosed as CMV encephalitis with status epilepticus.After the antiviral,hormonal,andγ-globulin pulse therapy,the patient’s condition improved,and he was finally discharged.CONCLUSION mNGS could be a reliable approach for the diagnosis of CMV encephalitis,with high efficiency,sensitivity,and specificity.展开更多
Antibiotic resistance genes(ARGs)have been considered as emerging contaminants in nature owing to their wide distribution and human health risk.Anthropogenic activities can increase the diversity and abundance of ARGs...Antibiotic resistance genes(ARGs)have been considered as emerging contaminants in nature owing to their wide distribution and human health risk.Anthropogenic activities can increase the diversity and abundance of ARGs and promote their spread in environment.Offshore environment is affected by multiple types of anthropogenic activities,of which excessive accumulation of petroleum substances poses a serious threat.Our previous experimental study has demonstrated that petroleum can increase the abundance of sulfonamide resistance genes(SRGs)in the seawater through horizontal gene transfer.However,the influence of petroleum substances on SRGs in offshore environment,especially adjacent the petroleum exploitation platform,is still unclear.Therefore,the effect of offshore oil exploitation on SRGs was investigated in the surface sediments collected from the Liaodong Bay,north China.The genes of sul1 and sul2 were present in all of the collected samples,while the sul3 gene was not detected in any sediments.The absolute abundance of sul2 gene in each sample was higher than sul1 gene.Class 1 integrons enhanced the maintenance and propagation of sul1 gene but not sul2 gene.More importantly,the results indicate that the absolute abundance of sul2 gene present in the offshore sediments that affected by petroleum exploitation was significantly higher than those in control.These findings provided direct evidence that offshore oil exploitation can influence the propagation of SRGs and implied that a more comprehensive risk assessment of petroleum substances to public health risks should be conducted.展开更多
Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud ...Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud elongation after removal of apical dominance in chrysanthemum,label-free quantification analysis was used to analyze the proteome changes after apical bud removal.Quantitative real-time PCR(qPCR)was used to analyze the changes in the expression of three plant hormone-related genes.A total of 440 differentially expressed proteins were successfully identified at three time points during the lateral bud elongation.The number of differentially expressed proteins in the three stages(24 h/0 h,48 h/0 h,48 h/24 h)were 219,332,and 97,respectively.The difference in expressed proteins in the three comparison stages mainly involves RNA processing and modification;translation,ribosomal structure and biogenesis;Posttranslational modification,protein turnover,and chaperones.Path analysis showed that there was various physiological activities in the process of lateral bud dormancy breaking and elongation,which involved energy metabolism,biosynthesis,signal transduction and stress response in the growth process of lateral buds.qPCR indicated that the expression of cytokinin synthesis related gene was significantly increased after the removal of apical dominance,while the expression of strigolactones synthesis related gene experiences a dramatic fall to promote the development of the lateral buds.However,there was a drop before a slight increase in the expression of the auxin synthesis related gene,which was mainly due to the removal of apical dominance that led to the loss of indoleacetic acid in the main stem.However,with formation of the new apical source,indoleacetic acid can be released again.展开更多
BACKGROUND Cytomegalovirus(CMV)is the most common viral pathogen after liver transplantation(LT).Although reactivation of CMV infection is generally described in the context of immunosuppression,it has also been descr...BACKGROUND Cytomegalovirus(CMV)is the most common viral pathogen after liver transplantation(LT).Although reactivation of CMV infection is generally described in the context of immunosuppression,it has also been described in critically ill immunocompetent patients including cirrhotic patients.AIM To determine the incidence of reactivated CMV prior to LT.METHODS This was a prospective cohort study evaluating adult patients who underwent LT between 2014 and 2016.A plasma sample was obtained from all patients for CMV quantitative real-time PCR testing right before transplantation.Patients were followed for at least 1 year to assess the following outcomes:Incidence of CMV infection,organ rejection and overall mortality.RESULTS A total of 72 patients were enrolled.Four patients died before transplantation,thus 68 patients were followed up for a median of 44 mo(20-50 mo).In 23/72 patients(31.9%)CMV was reactivated before transplantation.Post-transplantation,16/68(23.5%)patients had CMV infection and that was significantly associated with the recipient being CMV negative and a CMV-positive donor.Pretransplant CMV reactivation was not associated with overall mortality(log rank:0.9).CONCLUSION This study shows that CMV infection is common in patients with chronic liver disease just before LT,but the clinical impact of this infection seems to be negligible.展开更多
To correctly assess and properly manage the public health risks associated with exposure to contaminated water,it is necessary to identify the source of fecal pollution in a watershed.In this study,we evaluated the ef...To correctly assess and properly manage the public health risks associated with exposure to contaminated water,it is necessary to identify the source of fecal pollution in a watershed.In this study,we evaluated the efficacy of our two previously developed real time-quantitative PCR(qPCR)assays for the detection of swine-associated Bacteroidales genetic markers(gene 1-38,gene 3-53)in the Yangtze Delta watershed of southeastern China.The results indicated that the gene 1-38 and 3-53 markers exhibited high accuracy(92.5%,91.7%conditional probability,respectively)in detecting Bacteroidales spp.in water samples.According to binary logistic regression(BLR),these two swine-associated markers were well correlated(P<0.05)with fecal indicators(Escherichia coli and Enterococci spp.)and zoonotic pathogens(E.coli O157:H7,Salmonella spp.and Campylobacter spp.)in water samples.In contrast,concentrations of conventional fecal indicator bacteria(FIB)were not correlated with zoonotic pathogens,suggesting that they are noneffective at detecting fecal pollution events.Collectively,the results obtained in this study demonstrated that a swinetargeted qPCR assay based on two Bacteroidales genes markers(gene 1-38,gene 3-53)could be a useful tool in determining the swine-associated impacts of fecal contamination in a watershed.展开更多
Objective To observe the regulation of Fengxiaofang on the expression of gene Gob-5 in ovalbumin-challenged asthmatic mice.Methods The murine model of allergic asthma established by ovalbumin was used.The model mice w...Objective To observe the regulation of Fengxiaofang on the expression of gene Gob-5 in ovalbumin-challenged asthmatic mice.Methods The murine model of allergic asthma established by ovalbumin was used.The model mice were treated experimentally with Fengxiaofang and Dexamethasone as a positive control.The quantitive expression of gene Gob-5 in lung of mice was detected by real-time quantitative PCR.Results The expression of gene Gob-5 in lung of mice was down-regulated by Fengxiaofang.In the experiment,there were no significant differences between the one-fold or ten-fold dosage groups and the normal control group.Gene Gob-5 expression in five-fold dosage was significantly lower than that in the normal control group.Conclusion Fengxiaofang has the effect of lowering the expression of gene Gob-5 which maybe related to the potency on asthmatic disease.展开更多
To screen for effective small interference RNA(siRNA),a simple and visualized method was developed using the green fluorescence protein(GFP)as a reporter.Candidate siRNAs targeting macrophage migration inhibition fact...To screen for effective small interference RNA(siRNA),a simple and visualized method was developed using the green fluorescence protein(GFP)as a reporter.Candidate siRNAs targeting macrophage migration inhibition factor genes(MIF)were identified.By using the pEGFP-N3 vector,the MIF-GFP expression plasmid,pEGFP-MIF,was constructed with the same Kozak consensus translation initiation site and start code ATG for the MIF-EGFP coding sequence.Based on the siRNA expression vector pSilencer-4.1,3 candidate MIF siRNA expression plasmids were constructed and co-transfected with the pEGFP-MIF into the HEK293 cells,respectively.The GFP expression in HEK293 cells could be viewed by fluorescence microscopy and the MIF mRNA expressions were determined by real-time quantitative PCR.The 3 candidate MIF siRNA expression plasmids were also co-transfected with the MIF expression plasmid into the HEK293 cells,respectively,and the MIF mRNA expressions were determined by real-time quantitative PCR.The results show that the down-regulated expression of the MIF mRNA was consistent with the GFP expression and the same effective MIF siRNAs were screened by using the pEGFP-MIF or MIF expression plasmid with the candidate MIF siRNAs expression plasmids.Therefore,by using the GFP as a reporter,a useful method was provided to screen for effective siRNAs targeting specific genes co-expressed with the GFP.This may be a good strategy for screening for effective siRNAs targeting different genes.展开更多
基金supported by research grants from the Science and Technology Innovation Program of the Laoshan Laboratory(No.LSKJ202203803)the National Natural Science Foundation of China(No.32273107)+2 种基金supported by the Central Public-Interest Scientific Institution Basal Research Fund,Yellow Sea Fisheries Research Institute,CAFS(No.20603022022001)the project of Putian Science and Technology Department(No.2021NJJ002)the Shinan District Science and Technology Plan Project(No.2022-2-026-ZH).
文摘Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.
基金supported by a grant from Beijing Municipal Natural Science Foundation [L212011]National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention [131031102000210003&102393230020020000002]。
文摘Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.
基金Supported by Project of Standardization Technical System from the Administration of Quality and Technology Supervision of Sichuan Province(ZYBZ2013-39)
文摘In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms(GMOs) and products,ensure bio-safety and reduce ecological risk in China,a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017.The established method was evaluated based on the specificity,sensitivity,accuracy and measurement uncertainty.The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017.1.50%MON88017 sample was detected with 29 replications.The average measured value(1.541%) was close to the actual value(1.50%) and the relative deviation was 2.70%.The variation coefficient of the measured value was 0.110 4;the recovery was 100.00%and the measurement uncertainty was 0.096.The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5%confidence level.Thus,the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity,accuracy and sensitivity,which could provide technical support for the safety supervision of genetically modified organisms and products in China.
文摘Objective:To compare the effects of clinical application of chemiluminescence and real-time,fluorescence-based quantitative PCR in the detection Epstein-Barr virus(EBV).Methods:The data of chemiluminescence and real-time fluorescent quantitative PCR.fromipaEsfwo were suspected of being infectea w1tn rito1 roro January 2016 to January 2019 in our hospital were analyzed.The specific stage of EBV infection was analyzed,and the differences in results of the two detection methods were compared.Results:Chemiluminescence method was used to detect EBV infection during the active phase.The sensitivity of the chemiluminescence method was 76.7%(56/73)and the real-time quantitative PCRmethod was 90.4%(66/73).There was a statistical difference between the two detection methods(P<0.05).Conclusion:There was no statistical difference in positive predictive values between the chemiluminescence method and the real-time,fluorescence-based quantitative PCR method in the detection of EBV infection,but the sensitivity of chemiluminescence method is slightly lower than the real-time quantitative PCRmethod.It is noteworthy that chemiluminescence method is convenient and fast while the real-time,fluorescence-based quantitative PCR method is more accurate,which can provide a more accurate reference for clinical treatment.
基金supported by the the National Key Research and Development Program of China (2017YFD0201602)the National Natural Science Foundation of China (31401704)the Beijing Academy of Agriculture and Forestry Foundation, China (KJCX20180203)
文摘Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.
文摘Background: Protein kinase B (AKT/PKB) family is frequently amplified in ovarian cancer (OC). To the greatest of our knowledge, there is a lack of published reports about the amplification of the genes belonging to the AKT family among Sudanese women with OC. The present study was conducted to detect the AKT1 gene amplification and its association with tumour types, grades, and ages among Sudanese women with OC, bearing in mind the ethnic variation. Methods: This institution-based study included 79 cases of women diagnosed with ovarian cancer (OC) at Omdurman Maternity Hospital in the period 2013-2018. Formalin-fixed, paraffin-embedded (FFPE) tissue sections were used to extract RNA. AKT1 gene amplification was assessed using quantitative real-time PCR. Results: The mean age (±SD) of included women was 49.29 (±13.612). The amplification of AKT1 gene was observed in 18/79 (22.8%) of OC women, with a high frequency in women with undifferentiated 1/2 (50%), clear cell 2/6 (33.3%), mucinous 3/11 (27.3%), endometrioid 3/17 (17.6%), and serous carcinomas 5/30 OC (16.7%). High frequency was seen in women with low (26.3%;n = 10/28) rather than in higher (19.5%;n = 8/33) grade carcinoma, and in older (25.8%;n = 8/23) rather than younger (18.2%;n = 2/9) women. No significant association between AKT1 gene amplification and tumour types, grades, and ages of women was observed (Fisher’s Exact test: p = 0.405, 0.593 and 0.851, respectively). Conclusion: AKT1 gene amplification arises in around one-fifth of Sudanese women with ovarian cancer (OC). It is seen more in undifferentiated, clear cell, and mucinous tumours types, and more frequently in low tumour grade and older women, but not to a statistically significant level. These outcomes sustenance previous studies suggesting that activated AKT genes have a vital role in OC progression and may offer a plan for targeted therapy and prognostic evaluation.
文摘<strong>Objective: </strong><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">To investigate the protein expression and clinicopathological characteristics of ABCG2</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">, </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">Oct4</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">,</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> Nanog in laryngeal cancer tissues, and to seek new molecular markers for the diagnosis of laryngeal cancer.</span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><b><span style="font-family:Verdana;">Methods: </span></b></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">The laryngeal cancer tissues and paracancerous tissues of 87 patients with laryngeal carcinoma diagnosed in the department of otorhinolaryngology and head and neck surgery in our hospital from April 2016 to April 2018 were selected as the subjects. QRT-PCR, Real-time PcR, Western blot and immunohistochemical staining were used to detect the expression of ABCG2, Oct4 and Nanog in (Tumor Tissue) and (Adjacent Tissue) in tumor tissue and paracancerous tissue.</span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><b><span style="font-family:Verdana;">Results: </span></b></span></span><span><span><span style="font-family:;" "=""><span style="font-family:Verdana;">The results of RT-PCR showed that the positive rates of ABCG2, Oct4 and Nanog in laryngeal carcinoma tissues were 49.30%, 45.07% and 52.11%, respectively, while those in paracancerous tissues were </span><span style="font-family:Verdana;">22.54%, 21.13% and 15.49%, respectively (P < 0.01). The expression of ABCG2,</span><span style="font-family:Verdana;"> Oct4 and Nanog in laryngeal carcinoma was correlated with tumor differentiation, depth of invasion, age and sex (P < 0.05), but not with tumor size and TNM stage.</span></span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><b><span style="font-family:Verdana;">Conclusion: </span></b></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">The expressions of ABCG2, Oct4 and Nanog in cancer tissues are related to tumor differentiation status, and they can be used as new molecular markers for the diagnosis of laryngeal cancer</span></span></span><span style="font-family:Verdana;">.</span>
基金supported by the Applied Basic Research Programs of the Science and Technology Commission Foundation of Tianjin, China (Nos. 19JCZDJC34300, 14JCZDJC34200 and 18JCYBJC96100)
文摘Toll like receptors(TLRs)are the main innate immune‘pattern recognition receptors’of animals,which play a central role in host cell recognition and responses to invasive pathogens,particularly common structures of microbial pathogens.In this study,the gene expression profiles of TLRs in the spleen,head kidney,gill,small intestine,liver,muscle,and heart of healthy Paralichthys olivaceus were detected by real-time quantitative PCR(qPCR).The TLR family members were widely expressed in different tissues with different basic expression profiles.The highest expressions of TLR1,5m,7,8,9,14,and 21 were found in the spleen;the highest expressions of TLR3 and TLR21 were found in the gill;the highest expressions of TLR2 and 5s were found in the small intestine.The second highest expressions of TLR3,7,and 8 were found in small intestine.The gene expression profiles of TLRs stimulated with Edwardsiella tarda DNA,RNA,and lipopolysaccharide(LPS)were also detected in spleen,head kidney and gill.TLR9 and TLR21 were sensitive to E.tarda DNA;TLR 8 and TLR21 were sensitive to E.tarda RNA;and TLR1 and TLR14 were sensitive to E.tarda LPS.The expressions of the other TLR genes showed no significant changes.The results imply that the expressions of these TLR genes in P.olivaceus are differently regulated in the whole body and play important roles in the immune response against E.tarda infection.
文摘BACKGROUND Cytomegalovirus(CMV)infections in the population are mostly subclinical,inapparent,or latent.However,it is rare in brain tissue.Most reported CMV encephalitis cases were patients with immunodeficiency.The diagnosis and detection rate of CMV encephalitis in patients with normal immune function needs to be further improved.CASE SUMMARY An 86-year-old male was admitted due to a sudden onset of unconsciousness for 3 h.The patient developed status epilepticus and was relieved after antiepileptic treatment.Encephalitis was considered due to the high signals of diffusionweighted imaging sequences in the right central region by magnetic resonance imaging.Metagenomic next-generation sequencing(mNGS)of blood and cerebrospinal fluid revealed CMV,with unique reads number being 614 and 1,respectively.Simultaneous quantitative PCR results showed CMV positive in blood samples and negative in cerebrospinal fluid samples.The patient was finally diagnosed as CMV encephalitis with status epilepticus.After the antiviral,hormonal,andγ-globulin pulse therapy,the patient’s condition improved,and he was finally discharged.CONCLUSION mNGS could be a reliable approach for the diagnosis of CMV encephalitis,with high efficiency,sensitivity,and specificity.
文摘Antibiotic resistance genes(ARGs)have been considered as emerging contaminants in nature owing to their wide distribution and human health risk.Anthropogenic activities can increase the diversity and abundance of ARGs and promote their spread in environment.Offshore environment is affected by multiple types of anthropogenic activities,of which excessive accumulation of petroleum substances poses a serious threat.Our previous experimental study has demonstrated that petroleum can increase the abundance of sulfonamide resistance genes(SRGs)in the seawater through horizontal gene transfer.However,the influence of petroleum substances on SRGs in offshore environment,especially adjacent the petroleum exploitation platform,is still unclear.Therefore,the effect of offshore oil exploitation on SRGs was investigated in the surface sediments collected from the Liaodong Bay,north China.The genes of sul1 and sul2 were present in all of the collected samples,while the sul3 gene was not detected in any sediments.The absolute abundance of sul2 gene in each sample was higher than sul1 gene.Class 1 integrons enhanced the maintenance and propagation of sul1 gene but not sul2 gene.More importantly,the results indicate that the absolute abundance of sul2 gene present in the offshore sediments that affected by petroleum exploitation was significantly higher than those in control.These findings provided direct evidence that offshore oil exploitation can influence the propagation of SRGs and implied that a more comprehensive risk assessment of petroleum substances to public health risks should be conducted.
基金This work was supported by grants from the National Natural Science Foundation of China(Grant No.31800601).
文摘Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud elongation after removal of apical dominance in chrysanthemum,label-free quantification analysis was used to analyze the proteome changes after apical bud removal.Quantitative real-time PCR(qPCR)was used to analyze the changes in the expression of three plant hormone-related genes.A total of 440 differentially expressed proteins were successfully identified at three time points during the lateral bud elongation.The number of differentially expressed proteins in the three stages(24 h/0 h,48 h/0 h,48 h/24 h)were 219,332,and 97,respectively.The difference in expressed proteins in the three comparison stages mainly involves RNA processing and modification;translation,ribosomal structure and biogenesis;Posttranslational modification,protein turnover,and chaperones.Path analysis showed that there was various physiological activities in the process of lateral bud dormancy breaking and elongation,which involved energy metabolism,biosynthesis,signal transduction and stress response in the growth process of lateral buds.qPCR indicated that the expression of cytokinin synthesis related gene was significantly increased after the removal of apical dominance,while the expression of strigolactones synthesis related gene experiences a dramatic fall to promote the development of the lateral buds.However,there was a drop before a slight increase in the expression of the auxin synthesis related gene,which was mainly due to the removal of apical dominance that led to the loss of indoleacetic acid in the main stem.However,with formation of the new apical source,indoleacetic acid can be released again.
基金Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq)–Brazil,No.479880/2012-3.
文摘BACKGROUND Cytomegalovirus(CMV)is the most common viral pathogen after liver transplantation(LT).Although reactivation of CMV infection is generally described in the context of immunosuppression,it has also been described in critically ill immunocompetent patients including cirrhotic patients.AIM To determine the incidence of reactivated CMV prior to LT.METHODS This was a prospective cohort study evaluating adult patients who underwent LT between 2014 and 2016.A plasma sample was obtained from all patients for CMV quantitative real-time PCR testing right before transplantation.Patients were followed for at least 1 year to assess the following outcomes:Incidence of CMV infection,organ rejection and overall mortality.RESULTS A total of 72 patients were enrolled.Four patients died before transplantation,thus 68 patients were followed up for a median of 44 mo(20-50 mo).In 23/72 patients(31.9%)CMV was reactivated before transplantation.Post-transplantation,16/68(23.5%)patients had CMV infection and that was significantly associated with the recipient being CMV negative and a CMV-positive donor.Pretransplant CMV reactivation was not associated with overall mortality(log rank:0.9).CONCLUSION This study shows that CMV infection is common in patients with chronic liver disease just before LT,but the clinical impact of this infection seems to be negligible.
基金supported by the National Key Research and Development Program of China(No.2016YFD0501105)the Science and Technology Department of Zhejiang Province(No.2015C02044)the National Natural Science Foundation of China(No.31301492)
文摘To correctly assess and properly manage the public health risks associated with exposure to contaminated water,it is necessary to identify the source of fecal pollution in a watershed.In this study,we evaluated the efficacy of our two previously developed real time-quantitative PCR(qPCR)assays for the detection of swine-associated Bacteroidales genetic markers(gene 1-38,gene 3-53)in the Yangtze Delta watershed of southeastern China.The results indicated that the gene 1-38 and 3-53 markers exhibited high accuracy(92.5%,91.7%conditional probability,respectively)in detecting Bacteroidales spp.in water samples.According to binary logistic regression(BLR),these two swine-associated markers were well correlated(P<0.05)with fecal indicators(Escherichia coli and Enterococci spp.)and zoonotic pathogens(E.coli O157:H7,Salmonella spp.and Campylobacter spp.)in water samples.In contrast,concentrations of conventional fecal indicator bacteria(FIB)were not correlated with zoonotic pathogens,suggesting that they are noneffective at detecting fecal pollution events.Collectively,the results obtained in this study demonstrated that a swinetargeted qPCR assay based on two Bacteroidales genes markers(gene 1-38,gene 3-53)could be a useful tool in determining the swine-associated impacts of fecal contamination in a watershed.
基金National Natural Science Foundation of China (No.c03050402)
文摘Objective To observe the regulation of Fengxiaofang on the expression of gene Gob-5 in ovalbumin-challenged asthmatic mice.Methods The murine model of allergic asthma established by ovalbumin was used.The model mice were treated experimentally with Fengxiaofang and Dexamethasone as a positive control.The quantitive expression of gene Gob-5 in lung of mice was detected by real-time quantitative PCR.Results The expression of gene Gob-5 in lung of mice was down-regulated by Fengxiaofang.In the experiment,there were no significant differences between the one-fold or ten-fold dosage groups and the normal control group.Gene Gob-5 expression in five-fold dosage was significantly lower than that in the normal control group.Conclusion Fengxiaofang has the effect of lowering the expression of gene Gob-5 which maybe related to the potency on asthmatic disease.
基金The project was supported by the National Natural Science Foundation of China(Grant Nos.30300421,30672077,and 30772142).
文摘To screen for effective small interference RNA(siRNA),a simple and visualized method was developed using the green fluorescence protein(GFP)as a reporter.Candidate siRNAs targeting macrophage migration inhibition factor genes(MIF)were identified.By using the pEGFP-N3 vector,the MIF-GFP expression plasmid,pEGFP-MIF,was constructed with the same Kozak consensus translation initiation site and start code ATG for the MIF-EGFP coding sequence.Based on the siRNA expression vector pSilencer-4.1,3 candidate MIF siRNA expression plasmids were constructed and co-transfected with the pEGFP-MIF into the HEK293 cells,respectively.The GFP expression in HEK293 cells could be viewed by fluorescence microscopy and the MIF mRNA expressions were determined by real-time quantitative PCR.The 3 candidate MIF siRNA expression plasmids were also co-transfected with the MIF expression plasmid into the HEK293 cells,respectively,and the MIF mRNA expressions were determined by real-time quantitative PCR.The results show that the down-regulated expression of the MIF mRNA was consistent with the GFP expression and the same effective MIF siRNAs were screened by using the pEGFP-MIF or MIF expression plasmid with the candidate MIF siRNAs expression plasmids.Therefore,by using the GFP as a reporter,a useful method was provided to screen for effective siRNAs targeting specific genes co-expressed with the GFP.This may be a good strategy for screening for effective siRNAs targeting different genes.
