The species distinctive PCR primer of Lactobacillus acidophilus ( L. acidophilus) was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of s...The species distinctive PCR primer of Lactobacillus acidophilus ( L. acidophilus) was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative PCR (RT-PCR). Analysis on RT-PCR results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-PCR method in the test. indicating that the established RT-PCR method could be aookued to the detection of L. acidophilus in fermented material.展开更多
Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagn...Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.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 ...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 replica- tions. 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 g ; 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 organ- isms and products in China.展开更多
[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction (PCR) method with SYBR Green I for the detection of porcine circovirus type 2 (PCV2). [Methods] Specific primers were desig...[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction (PCR) method with SYBR Green I for the detection of porcine circovirus type 2 (PCV2). [Methods] Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR. The ampli- fied gene was cloned into the vector of pMD 18-T and transformed into DHSct to screen positive clones. After being extracted and purified, the recombinant plasraids pMD 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2, and the PCR re- action conditions were optimized. [ Results] Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21 × 100 -4.16 × 108 copies/μL , the correlation coefficient was O. 998 8 and the slope was - 3.286. The method did not show any cress-reactions with the genomes of PRRSV, PCV1, CSFV, PRV, PPV and Escherichia coli. Sensitivity of this method was proved to be 3.21 × 10 copies/μL, which was 1 000 times higher as conventional PCR method. Variation coefficients of the repeated trims among same batch or different batches were both less than 3.00%. Positive rate of clinical samples detected by the established PCR method was 58.94%, which was significantly higher than the detection rate by conventional PCR. [ Conclusions ] A reM-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established, which was better for conducting the quan- titative analysis and the early diagnosis of PCV2 infection.展开更多
Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ - PCR) method was developed for q...Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ - PCR) method was developed for quantitative detection of T. rotula. The RFQ - PCR assay data showed that the results obtained with the RFQ - PCR quite good agreement with those with the light microscope (LM) counting method, which suggested that the RFQ - PCR could be a useful method for red tide alga detection.展开更多
This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease (RSD) in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from s...This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease (RSD) in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from six plants of sugarcane ROC22, which had been confirmed RSD-positive by detecting the sugarcane juice, by employing the sugarcane seedlings production protocol. Real-time fluorescence quantitative PCR was used to detect RSD pathogens in tissue culture sam- pies. The results showed that target fragment of RSD pathogens was not found in all 10 samples in real-time fluorescence quantitative PCR, with the Ct values of 37 - 39. The healthy tissue culture sugarcane seedlings do not carry RSD pathogens, indicating that adopting healthy seedcane seedlings production technique could thoroughly get rid of RSD pathogens.展开更多
According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 (NC001718) available in GenBank (NC_001718), a pair of specific primer was designed, and the target fragment of 431 bp was obtained by P...According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 (NC001718) available in GenBank (NC_001718), a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.展开更多
[ Objective ] This study aimed to establish a rapid and effective quarantine method of Koi herpes virus. [ Method] Primers and corresponding TaqMan probe were designed based on the conserved sequence of Koi herpes vir...[ Objective ] This study aimed to establish a rapid and effective quarantine method of Koi herpes virus. [ Method] Primers and corresponding TaqMan probe were designed based on the conserved sequence of Koi herpes virus (KHV) pol-ymerase gene (Sph) to establish a rapid and effective fluorescence quantitative PCR method for Koi herpes virus detection. The cell cultures were detected by using the established fluorescence quantitative PCR assay, and the results were com- pared with that of conventional PCR. [ Result] The sensitivity of fluorescence quantitative PCR was higher than that of conventional PCR. The minimum copy num- ber that could be detected was 1.6 - 102 copies/p.1. The established method was adopted for sample detection, and a reliable diagnostic result could be obtained within 4 h. [Conclusion] The established method is rapid, sensitive, specific and repeatable, which is conducive to the rapid detection of Koi herpes virus. Key words Koi herpes virus; Fluorescence quantitative PCR; Detection展开更多
Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitativ...Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.展开更多
The mechanism involved in neural regeneration after spinal cord injury is unclear. The my-elin-derived protein Nogo-A, which is speciifc to the central nervous system, has been identiifed to negatively affect the cyto...The mechanism involved in neural regeneration after spinal cord injury is unclear. The my-elin-derived protein Nogo-A, which is speciifc to the central nervous system, has been identiifed to negatively affect the cytoskeleton and growth program of axotomized neurons. Studies have shown that Nogo-A exerts immediate and chronic inhibitory effects on neurite outgrowth.In vivo, inhibitors of Nogo-A have been shown to lead to a marked enhancement of regenerative axon extension. We established a spinal cord injury model in rats using a free-falling weight drop device to subsequently investigate Nogo-A expression. Nogo-A mRNA and protein expression and immunoreactivity were detected in spinal cord tissue using real-time quantitative PCR, immu-nohistochemistry and western blot analysis. At 24 hours after spinal cord injury, Nogo-A protein and mRNA expression was low in the injured group compared with control and sham-operated groups. The levels then continued to drop further and were at their lowest at 3 days, rapidly rose to a peak after 7 days, and then gradually declined again after 14 days. These changes were observed at both the mRNA and protein level. The transient decrease observed early after injury followed by high levels for a few days indicates Nogo-A expression is time dependent. This may contribute to the lack of regeneration in the central nervous system after spinal cord injury. The dynamic varia-tion of Nogo-A should be taken into account in the treatment of spinal cord injury.展开更多
Objective To study the oncogenic potential of mouse translation initiation factor 3 (TIF3) and elongation factor-1δ (TEF-1δ) in malignant transformed human bronchial epithelial cells induced by crystalline nicke...Objective To study the oncogenic potential of mouse translation initiation factor 3 (TIF3) and elongation factor-1δ (TEF-1δ) in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide (NiS). Methods Abnormal expressions of human TIF3 and TEF-1δ genes in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were investigated and analyzed by the reverse transcript polymerase chain reaction (RT-PCR) and fluorescent quantitative polymerase chain reaction (FQ-PCR), respectively. Results RT-PCR analysis primarily showed that both human TIF3 and TEF-1δ mRNA expressions in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were increased as compared with controls. FQ-PCR assay showed that the levels of TIF3 expressions in the transformed cells and tumorigenic cells were 3 and 4 times higher respectively, and the elevated expressions of TEF-16 eDNA copies were 2.7- to 3.5-fold in transformed cells and 4.1- to 5.2-fold in tumorigenic cells when compared with non-transformed cells, indicating that the over-expressions of human TIF3 and TEF-1δ genes were related to malignant degree of the cells induced by nickel. Conclusions These findings demonstrate that there are markedly abnormal expressions of TIF3 and TEF-1δ genes during malignant transformation of human bronchial epithelial cell lines induced by crystalline NiS. They seem to be the molecular mechanisms potentially responsible for human carcinogensis due to nickel.展开更多
An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures (32℃ for high temperature and 22℃ for opt...An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures (32℃ for high temperature and 22℃ for optimum temperature) at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes (SBEI, SBEIII and SBE/V) encoding starch branching enzyme (SBE) in the endosperms were studied by the real-time fluorescence quantitative PCR (FQ-PCR) method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.展开更多
The relationship between myocardial cell damages and HSPs mRNA transcription in heat stressed broilers was studied using a spectrophotometer, the histopathological technique, and fluorescence quantitative reverse tran...The relationship between myocardial cell damages and HSPs mRNA transcription in heat stressed broilers was studied using a spectrophotometer, the histopathological technique, and fluorescence quantitative reverse transcription PCR (FQ RT-PCR). The results showed that the activities of creatine kinase (CK) and glutamic-pyruvic transaminase (GPT) were induction during the persistent heat stress. The major lesions of the myocardial fibers were granular degeneration and necrosis. The transcription of constitutive or cognate heat shock protein 70 (HSC70) mRNA was changeable. The transcription of heat shock protein 70 (HSPT0) mRNA was increased obviously in the course of persistent heat stress. The results showed that the change of HSC70 mRNA transcription was contrary to the activity of CK, and the level of HSC70 mRNA transcription must be used as a symbol of the myocardial cell damages in the course of persistent heat stress.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
Partial sequence of Skeletonema costatum proliferating cell nuclear antigen (PCNA) gene was obtained by reverse transcriptase PCR(RT-PCR) and 3' rapid amplification of cDNA ends (3'-RACE) techniques. Based on ...Partial sequence of Skeletonema costatum proliferating cell nuclear antigen (PCNA) gene was obtained by reverse transcriptase PCR(RT-PCR) and 3' rapid amplification of cDNA ends (3'-RACE) techniques. Based on the obtained PCNA and cytochrome b gene( Cyt b gene) sequences, a real-time fluorescent quantitative PCR (FQ-PCR) method was developed to detect the expression S. costatum PCNA gene, and this method was applied to study the relationship between the growth rate of S. costatum and the average expression amount of PCNA gene in a single cell. The expression amount of PCNA gene had large variation in cells collected at different culture phases, and the trend was well consistent with the growth rate, which suggested that the expression amount of PCNA gene correlated well with the cell division, and the PCNA could be a promising indicator for the S. costatum cell proliferation. Furthermore, using the PCNA gene as the objective gene and the Cyt b gene as the house-keeping gene, a new method for estimating the in situ growth rate of S. costatum was established by analysis of the relative expression quantity (REQ) of the PCNA gene.展开更多
A total of 151 infant formula powder samples were tested for the screening of the Enterobacteriaceae bacteria, and it had been contaminated by the conditioned pathogen like Enterobacter sakazakii, Klebsiella pneumonia...A total of 151 infant formula powder samples were tested for the screening of the Enterobacteriaceae bacteria, and it had been contaminated by the conditioned pathogen like Enterobacter sakazakii, Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae. The results showed that six samples were separated Enterobacter sakazakii, 24 samples were separated Enterobacteriaceae by using the traditional validation and real-time fluorescence quantitative PCR. Then the result identified by API20E biochemistry test and VITEC2 identification system. Through the study we concluded the potential safety hazard of the current infant formula powder and should regard Enterobacteriaceae as an indicator to carry out effective management, the neglected mild and discontinuity contamination could be easily found, thus to guarantee the safety of the special group.展开更多
[ Objective] The paper was to study the differential expression of growth hormone (GH) gene in different ages and different tissues of Tan sheep. [ Method] Using real-time fluorescent quantitative PCR (RT-PCR) ass...[ Objective] The paper was to study the differential expression of growth hormone (GH) gene in different ages and different tissues of Tan sheep. [ Method] Using real-time fluorescent quantitative PCR (RT-PCR) assay, specific primers were designed according to the GH gene sequence published on Gen- Bank. With the total RNA of Tan sheep tissue as the template, the expression levels of GH gene in pectoral muscle, leg muscle and longissimus dorsi muscle at dif- ferent ages were analyzed. [ Result] In three types of muscle tissues, the expression patterns of GH gene in different tissues at the same month of age were pectoral muscle 〉 leg muscle 〉 longissimus dorsi muscle. The expression levels of GH gene were positively correlated with live weight before slaughter, carcass weight and net meat weight, and had significant positive correlation only with net meat weight ( P 〈 0.05 ). [ Conclusion ] The content of GH gene in different tissues gradually increased with the increasing months of age, and there were extremely significant differences in expression level of GH gene among different months of age or different sites (P〈0. 01).展开更多
High concentrations of ammonium nitrogen released from tannery sludge during storage in open air may cause nitrogen pollution to soil and groundwater.To study the transformation mechanism of NH_(4)^(+)-N by nitrifying...High concentrations of ammonium nitrogen released from tannery sludge during storage in open air may cause nitrogen pollution to soil and groundwater.To study the transformation mechanism of NH_(4)^(+)-N by nitrifying functional bacteria in tannery sludge contaminated soils,a series of contaminated soil culture experiments were conducted in this study.The contents of ammonium nitrogen(as NH_(4)^(+)-N),nitrite nitrogen(as NO_(2)^(−)-N)and nitrate nitrogen(as NO_(3)^(−)-N)were analyzed during the culture period under different conditions of pollution load,soil particle and redox environment.Sigmodial equation was used to interpret the change of NO_(3)^(−)-N with time in contaminated soils.The abundance variations of nitrifying functional genes(amoA and nxrA)were also detected using the real-time quantitative fluorescence PCR method.The results show that the nitrification of NH_(4)^(+)-N was aggravated in the contaminated silt soil and fine sand under the condition of lower pollution load,finer particle size and more oxidizing environment.The sigmodial equation well fitted the dynamic accumulation curve of the NO_(3)^(−)-N content in the tannery sludge contaminated soils.The Cr(III)content increased with increasing pollution load,which inhibited the reproduction and activity of nitrifying bacteria in the soils,especially in coarse-grained soil.The accumulation of NO_(2)^(−)-N contents became more obvious with the increase of pollution load in the fine sand,and only 41.5%of the NH_(4)^(+)-N was transformed to NO_(3)^(−)-N.The redox environment was the main factor affecting nitrification process in the soil.Compared to the aerobic soil environment,the transformation of NH_(4)^(+)-N was significantly inhibited under anaerobic incubation condition,and the NO_(3)^(−)-N contents decreased by 37.2%,61.9%and 91.9%under low,medium and high pollution loads,respectively.Nitrification was stronger in the silt soil since its copy number of amoA and nxrA genes was two times larger than that of fine sand.Moreover,the copy numbers of amoA and nxrA genes in the silt soil under the aerobic environment were 2.7 times and 2.2 times larger than those in the anaerobic environment.The abundance changes of the amoA and nxrA functional genes have a positive correlation with the nitrification intensity in the tannery sludge-contaminated soil.展开更多
Gene amplification is a common mechanism of oncogene activation and contributes to tumor progression. Analysis of such genetic alterations are relevant to the understanding of tumor genetics and could provide prognost...Gene amplification is a common mechanism of oncogene activation and contributes to tumor progression. Analysis of such genetic alterations are relevant to the understanding of tumor genetics and could provide prognostic information for the individual patient. Standard analytical approaches using Southern blot and slot blot require a large amount of good展开更多
文摘The species distinctive PCR primer of Lactobacillus acidophilus ( L. acidophilus) was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative PCR (RT-PCR). Analysis on RT-PCR results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-PCR method in the test. indicating that the established RT-PCR method could be aookued to the detection of L. acidophilus in fermented material.
基金Supported by National Natural Science Foundation of China ( 30800885,30871726)
文摘Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.
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 replica- tions. 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 g ; 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 organ- isms and products in China.
基金Supported by Shandong Province Natural Science Fund Project
文摘[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction (PCR) method with SYBR Green I for the detection of porcine circovirus type 2 (PCV2). [Methods] Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR. The ampli- fied gene was cloned into the vector of pMD 18-T and transformed into DHSct to screen positive clones. After being extracted and purified, the recombinant plasraids pMD 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2, and the PCR re- action conditions were optimized. [ Results] Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21 × 100 -4.16 × 108 copies/μL , the correlation coefficient was O. 998 8 and the slope was - 3.286. The method did not show any cress-reactions with the genomes of PRRSV, PCV1, CSFV, PRV, PPV and Escherichia coli. Sensitivity of this method was proved to be 3.21 × 10 copies/μL, which was 1 000 times higher as conventional PCR method. Variation coefficients of the repeated trims among same batch or different batches were both less than 3.00%. Positive rate of clinical samples detected by the established PCR method was 58.94%, which was significantly higher than the detection rate by conventional PCR. [ Conclusions ] A reM-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established, which was better for conducting the quan- titative analysis and the early diagnosis of PCV2 infection.
基金The research was supported by the National Natural Science Foundation of China under contract No.40406028the National High Technology Research and Development Program of China under contract No.2001AA635090.
文摘Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ - PCR) method was developed for quantitative detection of T. rotula. The RFQ - PCR assay data showed that the results obtained with the RFQ - PCR quite good agreement with those with the light microscope (LM) counting method, which suggested that the RFQ - PCR could be a useful method for red tide alga detection.
基金Supported by Special Funds for Basic Scientific Research of Guangxi Sugarcane Research Institute(G2009006,G2010006,G2009015)Sci-tech Research and Development Program of Guangxi Academy of Agricultural Sciences(200805)
文摘This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease (RSD) in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from six plants of sugarcane ROC22, which had been confirmed RSD-positive by detecting the sugarcane juice, by employing the sugarcane seedlings production protocol. Real-time fluorescence quantitative PCR was used to detect RSD pathogens in tissue culture sam- pies. The results showed that target fragment of RSD pathogens was not found in all 10 samples in real-time fluorescence quantitative PCR, with the Ct values of 37 - 39. The healthy tissue culture sugarcane seedlings do not carry RSD pathogens, indicating that adopting healthy seedcane seedlings production technique could thoroughly get rid of RSD pathogens.
文摘According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 (NC001718) available in GenBank (NC_001718), a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.
基金Supported by Project of Jilin Province Science and Technology Commission(20080218)
文摘[ Objective ] This study aimed to establish a rapid and effective quarantine method of Koi herpes virus. [ Method] Primers and corresponding TaqMan probe were designed based on the conserved sequence of Koi herpes virus (KHV) pol-ymerase gene (Sph) to establish a rapid and effective fluorescence quantitative PCR method for Koi herpes virus detection. The cell cultures were detected by using the established fluorescence quantitative PCR assay, and the results were com- pared with that of conventional PCR. [ Result] The sensitivity of fluorescence quantitative PCR was higher than that of conventional PCR. The minimum copy num- ber that could be detected was 1.6 - 102 copies/p.1. The established method was adopted for sample detection, and a reliable diagnostic result could be obtained within 4 h. [Conclusion] The established method is rapid, sensitive, specific and repeatable, which is conducive to the rapid detection of Koi herpes virus. Key words Koi herpes virus; Fluorescence quantitative PCR; Detection
文摘Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.
基金financially supported by a grant from the Natural Science Foundation of Jiangsu Province,No.BK2011180Ordinary University Graduate Student Scientific Research Innovation Projects of Jiangsu Province,No.CXZZ13-0614,CXZZ12-0609
文摘The mechanism involved in neural regeneration after spinal cord injury is unclear. The my-elin-derived protein Nogo-A, which is speciifc to the central nervous system, has been identiifed to negatively affect the cytoskeleton and growth program of axotomized neurons. Studies have shown that Nogo-A exerts immediate and chronic inhibitory effects on neurite outgrowth.In vivo, inhibitors of Nogo-A have been shown to lead to a marked enhancement of regenerative axon extension. We established a spinal cord injury model in rats using a free-falling weight drop device to subsequently investigate Nogo-A expression. Nogo-A mRNA and protein expression and immunoreactivity were detected in spinal cord tissue using real-time quantitative PCR, immu-nohistochemistry and western blot analysis. At 24 hours after spinal cord injury, Nogo-A protein and mRNA expression was low in the injured group compared with control and sham-operated groups. The levels then continued to drop further and were at their lowest at 3 days, rapidly rose to a peak after 7 days, and then gradually declined again after 14 days. These changes were observed at both the mRNA and protein level. The transient decrease observed early after injury followed by high levels for a few days indicates Nogo-A expression is time dependent. This may contribute to the lack of regeneration in the central nervous system after spinal cord injury. The dynamic varia-tion of Nogo-A should be taken into account in the treatment of spinal cord injury.
基金This research was supported by the National Natural Science Foundation of China (No. 30371195), Natural Science Foundation ofGuangdong Province (No. 031756), Guangzhou Science and Technology Foundation (No. 2003Z2-E0191/E0192), and Department ofGuangzhou Education (No. 1002).
文摘Objective To study the oncogenic potential of mouse translation initiation factor 3 (TIF3) and elongation factor-1δ (TEF-1δ) in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide (NiS). Methods Abnormal expressions of human TIF3 and TEF-1δ genes in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were investigated and analyzed by the reverse transcript polymerase chain reaction (RT-PCR) and fluorescent quantitative polymerase chain reaction (FQ-PCR), respectively. Results RT-PCR analysis primarily showed that both human TIF3 and TEF-1δ mRNA expressions in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were increased as compared with controls. FQ-PCR assay showed that the levels of TIF3 expressions in the transformed cells and tumorigenic cells were 3 and 4 times higher respectively, and the elevated expressions of TEF-16 eDNA copies were 2.7- to 3.5-fold in transformed cells and 4.1- to 5.2-fold in tumorigenic cells when compared with non-transformed cells, indicating that the over-expressions of human TIF3 and TEF-1δ genes were related to malignant degree of the cells induced by nickel. Conclusions These findings demonstrate that there are markedly abnormal expressions of TIF3 and TEF-1δ genes during malignant transformation of human bronchial epithelial cell lines induced by crystalline NiS. They seem to be the molecular mechanisms potentially responsible for human carcinogensis due to nickel.
文摘An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures (32℃ for high temperature and 22℃ for optimum temperature) at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes (SBEI, SBEIII and SBE/V) encoding starch branching enzyme (SBE) in the endosperms were studied by the real-time fluorescence quantitative PCR (FQ-PCR) method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.
基金This work was financially supported by the National Natural Science Foundation of China (30170682, 30571400) the Specialized Research Fund for the Doctoral Program of Higher Education, China (20050307008).
文摘The relationship between myocardial cell damages and HSPs mRNA transcription in heat stressed broilers was studied using a spectrophotometer, the histopathological technique, and fluorescence quantitative reverse transcription PCR (FQ RT-PCR). The results showed that the activities of creatine kinase (CK) and glutamic-pyruvic transaminase (GPT) were induction during the persistent heat stress. The major lesions of the myocardial fibers were granular degeneration and necrosis. The transcription of constitutive or cognate heat shock protein 70 (HSC70) mRNA was changeable. The transcription of heat shock protein 70 (HSPT0) mRNA was increased obviously in the course of persistent heat stress. The results showed that the change of HSC70 mRNA transcription was contrary to the activity of CK, and the level of HSC70 mRNA transcription must be used as a symbol of the myocardial cell damages in the course of persistent heat stress.
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.
基金The Zhejiang Provincial Natural Science Foundation of China under contract No.Y5080149 the National Natural Scientifie Foundation of China under contract No.40406028
文摘Partial sequence of Skeletonema costatum proliferating cell nuclear antigen (PCNA) gene was obtained by reverse transcriptase PCR(RT-PCR) and 3' rapid amplification of cDNA ends (3'-RACE) techniques. Based on the obtained PCNA and cytochrome b gene( Cyt b gene) sequences, a real-time fluorescent quantitative PCR (FQ-PCR) method was developed to detect the expression S. costatum PCNA gene, and this method was applied to study the relationship between the growth rate of S. costatum and the average expression amount of PCNA gene in a single cell. The expression amount of PCNA gene had large variation in cells collected at different culture phases, and the trend was well consistent with the growth rate, which suggested that the expression amount of PCNA gene correlated well with the cell division, and the PCNA could be a promising indicator for the S. costatum cell proliferation. Furthermore, using the PCNA gene as the objective gene and the Cyt b gene as the house-keeping gene, a new method for estimating the in situ growth rate of S. costatum was established by analysis of the relative expression quantity (REQ) of the PCNA gene.
文摘A total of 151 infant formula powder samples were tested for the screening of the Enterobacteriaceae bacteria, and it had been contaminated by the conditioned pathogen like Enterobacter sakazakii, Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae. The results showed that six samples were separated Enterobacter sakazakii, 24 samples were separated Enterobacteriaceae by using the traditional validation and real-time fluorescence quantitative PCR. Then the result identified by API20E biochemistry test and VITEC2 identification system. Through the study we concluded the potential safety hazard of the current infant formula powder and should regard Enterobacteriaceae as an indicator to carry out effective management, the neglected mild and discontinuity contamination could be easily found, thus to guarantee the safety of the special group.
基金Supported by Agricultural Breeding Special Project of Ningxia Autonomous Region(2013NYYZ0404)
文摘[ Objective] The paper was to study the differential expression of growth hormone (GH) gene in different ages and different tissues of Tan sheep. [ Method] Using real-time fluorescent quantitative PCR (RT-PCR) assay, specific primers were designed according to the GH gene sequence published on Gen- Bank. With the total RNA of Tan sheep tissue as the template, the expression levels of GH gene in pectoral muscle, leg muscle and longissimus dorsi muscle at dif- ferent ages were analyzed. [ Result] In three types of muscle tissues, the expression patterns of GH gene in different tissues at the same month of age were pectoral muscle 〉 leg muscle 〉 longissimus dorsi muscle. The expression levels of GH gene were positively correlated with live weight before slaughter, carcass weight and net meat weight, and had significant positive correlation only with net meat weight ( P 〈 0.05 ). [ Conclusion ] The content of GH gene in different tissues gradually increased with the increasing months of age, and there were extremely significant differences in expression level of GH gene among different months of age or different sites (P〈0. 01).
基金supported jointly by Natural Science Foundation of Hebei Province(D2020504003)National Key Research and Development Program of China(No.2019YFC1805300).
文摘High concentrations of ammonium nitrogen released from tannery sludge during storage in open air may cause nitrogen pollution to soil and groundwater.To study the transformation mechanism of NH_(4)^(+)-N by nitrifying functional bacteria in tannery sludge contaminated soils,a series of contaminated soil culture experiments were conducted in this study.The contents of ammonium nitrogen(as NH_(4)^(+)-N),nitrite nitrogen(as NO_(2)^(−)-N)and nitrate nitrogen(as NO_(3)^(−)-N)were analyzed during the culture period under different conditions of pollution load,soil particle and redox environment.Sigmodial equation was used to interpret the change of NO_(3)^(−)-N with time in contaminated soils.The abundance variations of nitrifying functional genes(amoA and nxrA)were also detected using the real-time quantitative fluorescence PCR method.The results show that the nitrification of NH_(4)^(+)-N was aggravated in the contaminated silt soil and fine sand under the condition of lower pollution load,finer particle size and more oxidizing environment.The sigmodial equation well fitted the dynamic accumulation curve of the NO_(3)^(−)-N content in the tannery sludge contaminated soils.The Cr(III)content increased with increasing pollution load,which inhibited the reproduction and activity of nitrifying bacteria in the soils,especially in coarse-grained soil.The accumulation of NO_(2)^(−)-N contents became more obvious with the increase of pollution load in the fine sand,and only 41.5%of the NH_(4)^(+)-N was transformed to NO_(3)^(−)-N.The redox environment was the main factor affecting nitrification process in the soil.Compared to the aerobic soil environment,the transformation of NH_(4)^(+)-N was significantly inhibited under anaerobic incubation condition,and the NO_(3)^(−)-N contents decreased by 37.2%,61.9%and 91.9%under low,medium and high pollution loads,respectively.Nitrification was stronger in the silt soil since its copy number of amoA and nxrA genes was two times larger than that of fine sand.Moreover,the copy numbers of amoA and nxrA genes in the silt soil under the aerobic environment were 2.7 times and 2.2 times larger than those in the anaerobic environment.The abundance changes of the amoA and nxrA functional genes have a positive correlation with the nitrification intensity in the tannery sludge-contaminated soil.
文摘Gene amplification is a common mechanism of oncogene activation and contributes to tumor progression. Analysis of such genetic alterations are relevant to the understanding of tumor genetics and could provide prognostic information for the individual patient. Standard analytical approaches using Southern blot and slot blot require a large amount of good