For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit ...For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor（TKI） therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded（FFPE） tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction（PCR） are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100（pH=10.0） was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs（bp）.However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.展开更多
[Objectives]This study was conducted for the purpose of widely using molecular biology techniques in the detection of seafood species.[Methods]In this study,such four kinds of seafood as fish,shrimps,crabs and shellfi...[Objectives]This study was conducted for the purpose of widely using molecular biology techniques in the detection of seafood species.[Methods]In this study,such four kinds of seafood as fish,shrimps,crabs and shellfish were used as samples to compare three DNA extraction methods,including Tiangen marine animal tissue genome extraction kit method(DP324)(a),SN/T 3589.6-2013/8.3(b)method and modified SN/T 3730.8-2013/8.1(c)method.The purity and concentrations of DNA and the amplification effects of real-time fluorescene PCR were compared.[Results]The three methods had different extraction effects while satisfying the real-time fluorescent PCR detection.The DNA templates extracted by the SN/T 3730.8-2013/8.1 standard method had higher purity and concentrations,and showed clearer bands in gel electrophoresis,indicating that the method has less effect on real-time fluorescent PCR and less inhibition,and can meet the detection needs of the four types of seafood,including fish,shrimps,crabs and shellfish.[Conclusions]This study provides a theoretical basis for the detection of seafood animal-derived components.展开更多
[ Objective] This study aimed to develop a simple and effective DNA extraction method for citrus Huanglongbing pathogen detection. [ Method] From aspects of preparing procedure, prepare time and the quality of DNA, ad...[ Objective] This study aimed to develop a simple and effective DNA extraction method for citrus Huanglongbing pathogen detection. [ Method] From aspects of preparing procedure, prepare time and the quality of DNA, advantages and disadvantages of three sample preparation methods were compared, include two Direet-PCR extraction methods and one universal genomic DNA extraction kit method. In addition, PCR amplification effect on specific primers for 16S rDNA of ＂Candidatua Libefibacter asiaticus＂ （CLas） had also been evaluated. [ Result] The results showed that RT-qPCR detected CLas by using DNA obtained from one of Direct-PCR extraction method as templates. Under improved Direct-PCR extraction method, 16S rDNA of CLas could also be amplified by routine PCR. [ Conclusion] A simple and effective DNA extraction method of citrus Huanglongbing pathogen have been established, which provides technical supports for prepa- ration of large number of samples for detection of CLas.展开更多
[Objective] The paper aimed to get a efficient, stable and low-priced DNA extraction method of mulberry leaves. [Method] With tender leaves of mulberry varieties in Anhui Province as materials, genomic DNA was extract...[Objective] The paper aimed to get a efficient, stable and low-priced DNA extraction method of mulberry leaves. [Method] With tender leaves of mulberry varieties in Anhui Province as materials, genomic DNA was extracted with CTAB method, and further identified by ultraviolet spectrophotometer, agarose gel electrophoresis and PCR methods. [ Result] The A260/A280 ratio of extracted DNA was 1.80 - 1.90, and the yield was 0. 187 -0.275μg/mg. With extracted DATA as the template, clear target bands were obtained by PCR. [Conclusion] Extracted DNA was good in quality, which could satisfy the needs of follow-up mo-lecular biological operation.展开更多
To ensure food safety, it's vital to accurately detect genetically modified(GM)ingredient adulteration in food products and effectively detect the adulteration of vegetable oils from GM organisms(GMO). Therefore, ...To ensure food safety, it's vital to accurately detect genetically modified(GM)ingredient adulteration in food products and effectively detect the adulteration of vegetable oils from GM organisms(GMO). Therefore, it's essential to establish efficient DNA isolation method from vegetable oil. Here, we evaluate 5 DNA isolation methods using 25 commercial vegetable oils produced from soybean, peanut, corn, sunflower, rapeseed as well as blended oils. Quality of isolated DNA was determined by Nanodrop 2000 spectrophotometry. Real-time PCR and universal gene tRNA-Leu was used to assess resolution of methods. Our results showed that only DNA samples isolated by modified emulsification method based on cetyl trimethylammonium bromide(CTAB) were able to amplify t RNA-Leu gene.Moreover, Ct values of species specific endogenous reference genes were greater than 36 in these samples. In summary, CTAB method showed the best resolution on GMO adulteration detection for commercial vegetable oils, especially in fully refined oils.展开更多
AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS: Three standards were prepared by cloning PCR products which targeted S, ...AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 104/mL, 6.3 × 102/mL and 1.6 × 103/ mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 109/mL, 2.08 × 106/mL and 4.40 × 107/mL respectively, the relative Ct valuewas 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 105/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.展开更多
利用磁珠法对甘蓝、白菜、芥菜、洋葱、黄瓜、番茄、辣椒等7种形状、大小不同的蔬菜单粒种子和幼苗进行DNA提取,采用内参基因Actin进行实时荧光定量PCR评价DNA质量,利用KASP(kompetitive allele specific PCR)标记检验DNA质量是否达到K...利用磁珠法对甘蓝、白菜、芥菜、洋葱、黄瓜、番茄、辣椒等7种形状、大小不同的蔬菜单粒种子和幼苗进行DNA提取,采用内参基因Actin进行实时荧光定量PCR评价DNA质量,利用KASP(kompetitive allele specific PCR)标记检验DNA质量是否达到KASP检测要求。结果显示,单粒种子磁珠法提取的各蔬菜DNA平均Ct值均处于19~24范围内。KASP检测进一步证实种子提取DNA的质量达到KASP标记分型的要求。综上所述,本研究建立了一种简单、经济、实用性强的单粒种子DNA提取方法,该方法对不同大小的蔬菜种子具有较好的稳定性,且大幅缩短了获得基因型数据的时间。展开更多
探究了不同DNA提取方法对人体肠道微生物菌群研究的影响,为今后研究人体肠道微生物时选择提取方法提供参考依据。选择市面上3种商业化肠道微生物DNA提取试剂盒和1种实验室改良方法对20份人类粪便样本中微生物DNA进行提取,根据DNA浓度、...探究了不同DNA提取方法对人体肠道微生物菌群研究的影响,为今后研究人体肠道微生物时选择提取方法提供参考依据。选择市面上3种商业化肠道微生物DNA提取试剂盒和1种实验室改良方法对20份人类粪便样本中微生物DNA进行提取,根据DNA浓度、纯度和微生物多样性比较4种DNA提取方法差异。结果表明:Q-试剂盒法(QIAamp PowerFecal Pro DNA Kit,Qiagen)、ET-试剂盒法(ET粪便DNA快速提取试剂盒,IDMBIO)、P-实验室改良法3种方法提取的DNA得率较高;方法 P获得的OTU数量最多;α多样性分析结果显示4种提取方法差异不显著;方法 P、方法 Q得到肠道微生物组成结构相似度较高,方法 ET、方法 Y(MolPure Stool DNA Kit,Yeasen)得到肠道微生物组成相似度较高;4种DNA提取方法所得微生物群落构成在门水平和属水平差异明显。每种DNA提取方法都有各自提取偏好性菌属,经过综合比较,方法 Q和方法 ET提取DNA的得率更高、速度更快。展开更多
Deoxyribonucleic acid (DNA) damage arising from radiations widely occurred along with the development of nuclear weapons and clinically wide application of computed tomography (CT) scan and nuclear medicine. All ioniz...Deoxyribonucleic acid (DNA) damage arising from radiations widely occurred along with the development of nuclear weapons and clinically wide application of computed tomography (CT) scan and nuclear medicine. All ionizing radiations (X-rays, γ-rays, alpha particles, etc.) and ultraviolet (UV) radiation lead to the DNA damage. Polymerase chain reaction (PCR) is one of the most wildly used techniques for detecting DNA damage as the amplification stops at the site of the damage. Improvements to enhance the efficiency of PCR are always required and remain a great challenge. Here we establish a multiplex PCR assay system (MPAS) that is served as a robust and efficient method for direct detection of target DNA sequences in genomic DNA. The establishment of the system is performed by adding a combination of PCR enhancers to standard PCR buffer. The performance of MPAS was demonstrated by carrying out the direct PCR amplification on 1.2 mm human blood punch using commercially available primer sets which include multiple primer pairs. The optimized PCR system resulted in high quality genotyping results without any inhibitory effect indicated and led to a full-profile success rate of 98.13%. Our studies demonstrate that the MPAS provides an efficient and robust method for obtaining sensitive, reliable and reproducible PCR results from human blood samples.展开更多
We examined 50 Escherichia coli (E. coli) strains isolated from broiler chickens between January 2013 to March 2014 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Jordan by m...We examined 50 Escherichia coli (E. coli) strains isolated from broiler chickens between January 2013 to March 2014 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Jordan by multiplex PCR and random amplification of polymorphic DNA (RAPD) tests. The multiplex polymerase chain reaction (PCR) which was used as tentative criteria of APEC targets 8 virulence associated genes;enteroaggregative toxin (astA), Type 1 fimbria adhesion (fimH), iron-repressible protein (irp2), P fimbriae (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon (cva/cvi) genes. The number of detected genes could be used as a reliable index of their virulence. E. coli strains already typed as an APEC always harbor 5 to 8 genes, but non-APEC strains harbor less than 4 genes. Assuming the criteria of an APEC is possession of 5 or more virulence associated genes;we found that all 50 E. coli strains were classified as APEC strains. The RAPD analysis showed that the E. coli strains could be grouped into 35 of RAPD types by using these two different RAPD primer sets, RAPD analysis primer 4 5'AAGAGCCCGT5', and RAPD analysis primer 6 5'CCCGTCAGCA3'. The current study confirmed the endemic nature of APEC in broiler flocks in Jordan. It is essential that the biosecurity on poultry farms should be improved to prevent the introduction and dissemination of APEC and other agents. Furthermore, farmers need to be educated about the signs, lesions, and the importance of this agent.展开更多
[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel rea...[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses （WSSV, IHHNV, TSV and YHV ）. [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies （E = 1.06, 1.07, 0.92 and 0.92, respectively）, good hnear relationship （ r = 1 ）, uniform repeatability （ standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ） and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR （average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ）. The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.展开更多
Polymerase chain reaction （PCR） was used to amplify a 600-base pair （bp） sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil （Alfisol） and Red soil （Ultisol）, and three different ...Polymerase chain reaction （PCR） was used to amplify a 600-base pair （bp） sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil （Alfisol） and Red soil （Ultisol）, and three different minerals （goethite, kaolinite, montmorillonite）. DNA bound on soil colloids, kaolinite, and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted, 10- and 20-fold. The montmorillonite-DNA complex required at least 100-fold dilution before amplification could be detected. DNA bound on goethite was amplified irrespective of whether the complex was used directly, or diluted 10- and 20-fold. The amplification of mineral-bound plasmid DNA by PCR is, therefore, markedly influenced by the type and concentration of minerals used. This information is of fundamental importance to soil molecular microbial ecology with particular reference to monitoring the fate of genetically engineered microorganisms and their recombinant DNA in soil environments.展开更多
The fungus Magnaporthe oryzae is the causal agent of a wide range of cereal diseases. For long-term preservation, the fungus is grown and stored desiccated on filter papers at –20 °C.Inoculated filter papers are...The fungus Magnaporthe oryzae is the causal agent of a wide range of cereal diseases. For long-term preservation, the fungus is grown and stored desiccated on filter papers at –20 °C.Inoculated filter papers are cut into pieces of 0.5–1.0 cm diameter prior to storage. In the present study, a fast(11 min) and simple method of preparing DNA suitable for amplifying avirulence genes of M. oryzae by polymerase chain reaction(PCR) was developed. A piece of filter paper containing the fungus was removed from a glass bottle and placed in a 0.2 mL Eppendorf tube containing 100 μL 10 × TE. The suspension was heated for 10 min at 95 °C in a PCR machine. The tube was then centrifuged for 1 min at 3000 r min-1. One μL of 10 × TE solution containing DNA was used for PCR. A total of 28 samples were PCR tested. As a positive control, fungal DNA was extracted using a conventional DNA preparation method. DNA samples obtained from both methods were stored at 4 °C. PCR was performed with DNA on the preparation day and after 4, 8,10, and 18 days of refrigerated storage. In four samples, samples 12, 13, 14, and 28, AVR-Pi9 failed to be amplified. These four samples were tested with a different set of primers for AVR-Pi9, and for AVR-Pita1, confirming that the quality of the samples was insufficient for PCR. Overall, for nearly 90%(24/28) of the samples, the quality of the DNA prepared directly from the fungus on filter paper appeared suitable for a rapid survey of genetic identity of the rice blast fungus by PCR.This method will be useful and effective for reducing cost and time and could readily be adopted worldwide for analysis of M. oryzae and possibly other fungi.展开更多
基金Supported by the Jilin Science & Technology Development Plan,China(No.201201060)the Scientific Research Foundation of Jilin Province,China(No.20100942)+1 种基金the Fund of Developing and Reforming Community of Jilin Province,China(No.2010-1928)the Health Scientific Research Foundation of Jilin Province,China(Nos.2009z081,2010Z083)
文摘For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor（TKI） therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded（FFPE） tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction（PCR） are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100（pH=10.0） was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs（bp）.However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.
文摘[Objectives]This study was conducted for the purpose of widely using molecular biology techniques in the detection of seafood species.[Methods]In this study,such four kinds of seafood as fish,shrimps,crabs and shellfish were used as samples to compare three DNA extraction methods,including Tiangen marine animal tissue genome extraction kit method(DP324)(a),SN/T 3589.6-2013/8.3(b)method and modified SN/T 3730.8-2013/8.1(c)method.The purity and concentrations of DNA and the amplification effects of real-time fluorescene PCR were compared.[Results]The three methods had different extraction effects while satisfying the real-time fluorescent PCR detection.The DNA templates extracted by the SN/T 3730.8-2013/8.1 standard method had higher purity and concentrations,and showed clearer bands in gel electrophoresis,indicating that the method has less effect on real-time fluorescent PCR and less inhibition,and can meet the detection needs of the four types of seafood,including fish,shrimps,crabs and shellfish.[Conclusions]This study provides a theoretical basis for the detection of seafood animal-derived components.
基金Supported by Most-USDA Cooperation Program in Agriculture Science and Technology(10-8100-1452-CA)National Natural Science Foundation of China(31071712)
文摘[ Objective] This study aimed to develop a simple and effective DNA extraction method for citrus Huanglongbing pathogen detection. [ Method] From aspects of preparing procedure, prepare time and the quality of DNA, advantages and disadvantages of three sample preparation methods were compared, include two Direet-PCR extraction methods and one universal genomic DNA extraction kit method. In addition, PCR amplification effect on specific primers for 16S rDNA of ＂Candidatua Libefibacter asiaticus＂ （CLas） had also been evaluated. [ Result] The results showed that RT-qPCR detected CLas by using DNA obtained from one of Direct-PCR extraction method as templates. Under improved Direct-PCR extraction method, 16S rDNA of CLas could also be amplified by routine PCR. [ Conclusion] A simple and effective DNA extraction method of citrus Huanglongbing pathogen have been established, which provides technical supports for prepa- ration of large number of samples for detection of CLas.
基金Supported by Dean Youth Innovation Fund of Anhui Academy of Agricultural Sciences"ITS Sequence and Evolutionary Analysis of Local Mulberry Germplasm Resources in Anhui Province"(15B0634)Hefei Comprehensive Test Station of National Sericulture Technology System"Special Project for Construction of China Agricultural Industry Research System"(CARS-22-SYZ09)+1 种基金Special Talent Development Fund of Anhui Academy of Agricultural Sciences"Breeding of High Quality Ecological Mulberry Varieties and Integration and Demonstration of Matching Cultivation Technology"(17F0610)Discipline Construction of Anhui Academy of Agricultural Sciences"Research of Silkworm Gender Tag Technology"(16A0618)
文摘[Objective] The paper aimed to get a efficient, stable and low-priced DNA extraction method of mulberry leaves. [Method] With tender leaves of mulberry varieties in Anhui Province as materials, genomic DNA was extracted with CTAB method, and further identified by ultraviolet spectrophotometer, agarose gel electrophoresis and PCR methods. [ Result] The A260/A280 ratio of extracted DNA was 1.80 - 1.90, and the yield was 0. 187 -0.275μg/mg. With extracted DATA as the template, clear target bands were obtained by PCR. [Conclusion] Extracted DNA was good in quality, which could satisfy the needs of follow-up mo-lecular biological operation.
基金supported by grants from the National Major Special Project for the Development of Transgenic Organisms (grant No. 2016ZX08012-005, 2016ZX08012-003)the National Natural Science Foundation of China (31601581)
文摘To ensure food safety, it's vital to accurately detect genetically modified(GM)ingredient adulteration in food products and effectively detect the adulteration of vegetable oils from GM organisms(GMO). Therefore, it's essential to establish efficient DNA isolation method from vegetable oil. Here, we evaluate 5 DNA isolation methods using 25 commercial vegetable oils produced from soybean, peanut, corn, sunflower, rapeseed as well as blended oils. Quality of isolated DNA was determined by Nanodrop 2000 spectrophotometry. Real-time PCR and universal gene tRNA-Leu was used to assess resolution of methods. Our results showed that only DNA samples isolated by modified emulsification method based on cetyl trimethylammonium bromide(CTAB) were able to amplify t RNA-Leu gene.Moreover, Ct values of species specific endogenous reference genes were greater than 36 in these samples. In summary, CTAB method showed the best resolution on GMO adulteration detection for commercial vegetable oils, especially in fully refined oils.
基金Supported by the National Natural Science Foundation of China(No. 30371328), the Key Project of Natural Science Foundationof Shandong Province (No. Z2002C01), and the Key Project ofShandong Academy of Medical Sciences (No. 2005007)
文摘AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 104/mL, 6.3 × 102/mL and 1.6 × 103/ mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 109/mL, 2.08 × 106/mL and 4.40 × 107/mL respectively, the relative Ct valuewas 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 105/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.
文摘利用磁珠法对甘蓝、白菜、芥菜、洋葱、黄瓜、番茄、辣椒等7种形状、大小不同的蔬菜单粒种子和幼苗进行DNA提取,采用内参基因Actin进行实时荧光定量PCR评价DNA质量,利用KASP(kompetitive allele specific PCR)标记检验DNA质量是否达到KASP检测要求。结果显示,单粒种子磁珠法提取的各蔬菜DNA平均Ct值均处于19~24范围内。KASP检测进一步证实种子提取DNA的质量达到KASP标记分型的要求。综上所述,本研究建立了一种简单、经济、实用性强的单粒种子DNA提取方法,该方法对不同大小的蔬菜种子具有较好的稳定性,且大幅缩短了获得基因型数据的时间。
文摘探究了不同DNA提取方法对人体肠道微生物菌群研究的影响,为今后研究人体肠道微生物时选择提取方法提供参考依据。选择市面上3种商业化肠道微生物DNA提取试剂盒和1种实验室改良方法对20份人类粪便样本中微生物DNA进行提取,根据DNA浓度、纯度和微生物多样性比较4种DNA提取方法差异。结果表明:Q-试剂盒法(QIAamp PowerFecal Pro DNA Kit,Qiagen)、ET-试剂盒法(ET粪便DNA快速提取试剂盒,IDMBIO)、P-实验室改良法3种方法提取的DNA得率较高;方法 P获得的OTU数量最多;α多样性分析结果显示4种提取方法差异不显著;方法 P、方法 Q得到肠道微生物组成结构相似度较高,方法 ET、方法 Y(MolPure Stool DNA Kit,Yeasen)得到肠道微生物组成相似度较高;4种DNA提取方法所得微生物群落构成在门水平和属水平差异明显。每种DNA提取方法都有各自提取偏好性菌属,经过综合比较,方法 Q和方法 ET提取DNA的得率更高、速度更快。
基金Supported by National Natural Science Foundation of China(Nos.21077321,21105028,21075128)K.C.Wong Education Foundation,Hong Kong,China
文摘Deoxyribonucleic acid (DNA) damage arising from radiations widely occurred along with the development of nuclear weapons and clinically wide application of computed tomography (CT) scan and nuclear medicine. All ionizing radiations (X-rays, γ-rays, alpha particles, etc.) and ultraviolet (UV) radiation lead to the DNA damage. Polymerase chain reaction (PCR) is one of the most wildly used techniques for detecting DNA damage as the amplification stops at the site of the damage. Improvements to enhance the efficiency of PCR are always required and remain a great challenge. Here we establish a multiplex PCR assay system (MPAS) that is served as a robust and efficient method for direct detection of target DNA sequences in genomic DNA. The establishment of the system is performed by adding a combination of PCR enhancers to standard PCR buffer. The performance of MPAS was demonstrated by carrying out the direct PCR amplification on 1.2 mm human blood punch using commercially available primer sets which include multiple primer pairs. The optimized PCR system resulted in high quality genotyping results without any inhibitory effect indicated and led to a full-profile success rate of 98.13%. Our studies demonstrate that the MPAS provides an efficient and robust method for obtaining sensitive, reliable and reproducible PCR results from human blood samples.
文摘We examined 50 Escherichia coli (E. coli) strains isolated from broiler chickens between January 2013 to March 2014 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Jordan by multiplex PCR and random amplification of polymorphic DNA (RAPD) tests. The multiplex polymerase chain reaction (PCR) which was used as tentative criteria of APEC targets 8 virulence associated genes;enteroaggregative toxin (astA), Type 1 fimbria adhesion (fimH), iron-repressible protein (irp2), P fimbriae (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon (cva/cvi) genes. The number of detected genes could be used as a reliable index of their virulence. E. coli strains already typed as an APEC always harbor 5 to 8 genes, but non-APEC strains harbor less than 4 genes. Assuming the criteria of an APEC is possession of 5 or more virulence associated genes;we found that all 50 E. coli strains were classified as APEC strains. The RAPD analysis showed that the E. coli strains could be grouped into 35 of RAPD types by using these two different RAPD primer sets, RAPD analysis primer 4 5'AAGAGCCCGT5', and RAPD analysis primer 6 5'CCCGTCAGCA3'. The current study confirmed the endemic nature of APEC in broiler flocks in Jordan. It is essential that the biosecurity on poultry farms should be improved to prevent the introduction and dissemination of APEC and other agents. Furthermore, farmers need to be educated about the signs, lesions, and the importance of this agent.
文摘[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses （WSSV, IHHNV, TSV and YHV ）. [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies （E = 1.06, 1.07, 0.92 and 0.92, respectively）, good hnear relationship （ r = 1 ）, uniform repeatability （ standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ） and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR （average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ）. The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.
基金Project supported by the National Natural Science Foundation of China(No.40271064)
文摘Polymerase chain reaction （PCR） was used to amplify a 600-base pair （bp） sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil （Alfisol） and Red soil （Ultisol）, and three different minerals （goethite, kaolinite, montmorillonite）. DNA bound on soil colloids, kaolinite, and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted, 10- and 20-fold. The montmorillonite-DNA complex required at least 100-fold dilution before amplification could be detected. DNA bound on goethite was amplified irrespective of whether the complex was used directly, or diluted 10- and 20-fold. The amplification of mineral-bound plasmid DNA by PCR is, therefore, markedly influenced by the type and concentration of minerals used. This information is of fundamental importance to soil molecular microbial ecology with particular reference to monitoring the fate of genetically engineered microorganisms and their recombinant DNA in soil environments.
基金supported in part by Agriculture and Food Research Initiative Competitive Grant 2013-68004-20378 from the USDA National Institute of Food and Agriculture
文摘The fungus Magnaporthe oryzae is the causal agent of a wide range of cereal diseases. For long-term preservation, the fungus is grown and stored desiccated on filter papers at –20 °C.Inoculated filter papers are cut into pieces of 0.5–1.0 cm diameter prior to storage. In the present study, a fast(11 min) and simple method of preparing DNA suitable for amplifying avirulence genes of M. oryzae by polymerase chain reaction(PCR) was developed. A piece of filter paper containing the fungus was removed from a glass bottle and placed in a 0.2 mL Eppendorf tube containing 100 μL 10 × TE. The suspension was heated for 10 min at 95 °C in a PCR machine. The tube was then centrifuged for 1 min at 3000 r min-1. One μL of 10 × TE solution containing DNA was used for PCR. A total of 28 samples were PCR tested. As a positive control, fungal DNA was extracted using a conventional DNA preparation method. DNA samples obtained from both methods were stored at 4 °C. PCR was performed with DNA on the preparation day and after 4, 8,10, and 18 days of refrigerated storage. In four samples, samples 12, 13, 14, and 28, AVR-Pi9 failed to be amplified. These four samples were tested with a different set of primers for AVR-Pi9, and for AVR-Pita1, confirming that the quality of the samples was insufficient for PCR. Overall, for nearly 90%(24/28) of the samples, the quality of the DNA prepared directly from the fungus on filter paper appeared suitable for a rapid survey of genetic identity of the rice blast fungus by PCR.This method will be useful and effective for reducing cost and time and could readily be adopted worldwide for analysis of M. oryzae and possibly other fungi.
