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1.
Due to the potential for intentional contamination of food with crude preparations containing ricin, a real-time PCR method was developed for the detection of castor plant material in ground beef. One primer pair was identified and confirmed to be castor-specific and efficient for amplification of ricin in DNA extracts from castor or beef matrices. Of three different DNA extraction protocols compared, the hexadecyltrimethylammonium bromide (CTAB) method yielded the highest quality of DNA for QPCR assay. The detection limit for castor contamination in ground beef samples was <0.001% (<10 microg of castor acetone powder per gram of beef, corresponding to 0.5 microg of ricin), indicating excellent sensitivity for the assay, well below the threshold for oral toxicity.  相似文献   

2.
Three methods of DNA extraction from feed products and four detection methods for the 5'-junction fragment of genetically modified (GM) Roundup Ready soybean (RRS) were compared and evaluated. The DNA extraction methods, including cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and guanidine hydrochloride (Kit), were assessed for their yields and purity of DNA, extraction time, and reagent cost. The DNA yields of CTAB, SDS, and Kit were 52-694, 164-1750 and 23-105 ng/mg sample, and their extraction time was 2.5-3, 2-2.5, and 1.5-2 h with reagent cost about US dollar 0.24, 0.13, and 1.9 per extraction, respectively. The SDS method was generally well suited to all kinds of feed matrices tested. The limits of detection for the four amplification protocols, including loop-mediated isothermal amplification (LAMP), hyperbranched rolling circle amplification (HRCA), conventional polymerase chain reaction (PCR), and real-time PCR, were 48.5, 4.85, 485, and 9 copies of the pTLH10 plasmid, respectively. The ranked results of the four detection methods were based on multiattribute utility theory as follows (from best to worse): HRCA, LAMP, PCR, and real-time PCR. This comparative evaluation was specifically useful for selection of a highly efficient DNA extraction or amplification method for detecting different GM ingredients.  相似文献   

3.
Previous research has demonstrated transmission of zearalenone and alpha- and beta-zearalenols into the milk of cows and other animals. Since human intake of zearalenone and its metabolites via milk is an unknown factor in risk assessment of zearalenone and because appropriate methodology for their determination in milk is not available, a rapid and sensitive analytical method has been developed. Essentially, the method includes extraction with basic acetonitrile, acidification, partition into methylene chloride on a hydrophilic matrix, cleanup on an aminopropyl solid phase extraction column, and reverse-phase liquid chromatography with fluorescence detection. Recoveries from milk averaged 84% for zearalenone, 93% for alpha-zearalenol, and 90% for beta-zearalenol at spiking levels of 0.5 to 20 ng/mL. As little as 0.2 ng/mL of zearalenone and alpha-zearalenol and 2 ng/mL of beta-zearalenol can be detected in milk. These 3 compounds are stable in refrigerated milk for at least 2 weeks and in milk brought to boiling. Enzymes (beta-glucuronidase and aryl sulfatase) may be added to milk prior to extraction to hydrolyze any conjugates.  相似文献   

4.
An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.  相似文献   

5.
A rapid confirmatory method for monitoring chloramphenicol (CAP) residues in honey, whole milk, and eggs is presented. This method is based on the polymer monolith microextraction (PMME) technique and high-performance liquid chromatography (HPLC)-electrospray ionization mass spectrometry (MS). A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium. To obtain optimum extraction efficiency, several parameters related to PMME were investigated. After dissolution in 20 mM phosphate solution at pH 4.0 and centrifugation, honey, eggs, or milk samples were directly passed through the extraction tube. The LC-MS instrument was equipped with an electrospray ion source and a single quadrupole. The eluates were analyzed by LC-MS in the negative-ion mode and by monitoring a pair of isotopic ions for the target compound. The in-source collision-induced dissociation process produced confirmatory ions. The recoveries of CAP from real samples spiked at 0.1-10 ng/g (honey), 0.2-10 ng/mL (milk), and 0.2-10 ng/g (egg) were in the range of 85-102%, with relative standard deviations ranging between 2.1% and 8.9%. The limits of detection (S/N = 3) were 0.02 ng/g, 0.04 ng/mL, and 0.04 ng/g in honey, milk, and eggs, respectively. The proposed method was proved to be robust in monitoring CAP residue in honey, milk, and eggs.  相似文献   

6.
Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR) amplification has easier sample preparation and detection limits are lower. Of the different methods of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green detection method showed that the method is highly sensitive to sample matrices and genomic DNA content influencing the interpretation of results. Therefore, this paper describes a real-time DNA quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with detection limits of lower than 18 copies per sample applicable and comparable to highly purified plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard deviation.  相似文献   

7.
Real-time polymerase chain reaction is currently being used for the identification and quantification of plant and animal species as well as microorganisms in food or feed samples based on the amplification of specific sequences of low copy genes. We report here the development of a new real-time PCR method for the detection and quantification of the pea (Pisum sativum) based on the amplification of a specific region of the legS gene. The specificity was evaluated in a wide range of plant species (51 varieties of Pisum sp., and 32 other plant species and varieties taxonomically related or nonrelated). The method allows the detection and quantification of as low as 21.6 pg of DNA, which corresponds to 5 haploid genome copies. The system has been shown to be sensitive, reproducible and 100% specific for the rapid detection and quantification of pea DNA in processed food and feed samples, being therefore suitable for high-throughput analysis.  相似文献   

8.
This paper is part of a set of three papers investigating metrological traceability of the quantification of DNA fragments as, for instance, used for quantification of genetic modifications. This paper evaluates the possible impact of several factors on results of real-time Polymerase Chain Reaction (PCR) measurements. It was found that the particle size of the powder samples does not have an influence, whereas the nature of the calibrant (plasmidic or genomic DNA) has a significant effect. Moreover, two real-time PCR detection methods (construct-specific and event-specific) for MON 810 corn were compared. The results obtained in a specifically designed interlaboratory study revealed a significant influence of the DNA extraction method on measurement results when the MON 810 construct-specific real-time PCR detection method was applied. Statistical analyses confirmed the importance of validating DNA extraction methods in conjunction with real-time PCR methods.  相似文献   

9.
An analytical method has been developed for the determination of total bound and extractable residues of the nitrofuran drugs furazolidone, nitrofurazone, furaltadone, and nitrofurantoin in milk of dairy cows. The method involves overnight acid hydrolysis and simultaneous derivatization of the released side chains with 2-nitrobenzaldehyde. During hydrolysis, the bound metabolites are hydrolyzed to the side chains. After pH adjustment and solid-phase extraction cleanup, the derivatives are detected and quantitated using a liquid chromatography-tandem mass spectrometry system with an atmospheric pressure chemical ionization interface. Validation of the method is accomplished by fortifying control milk with a mixture of side chains at 1, 2, and 4 ng/g. Internal standards are added at the beginning of the procedure to compensate for matrix effects and recovery losses. Method accuracies range from 83 to 104% with coefficients of variation less than 13% for all four analytes. The limits of detection are相似文献   

10.
A duplex Polymerase Chain Reaction (PCR) method able to detect bovine milk in ovine cheeses was developed. This method is based on the mitochondrial 12S and 16S rRNA genes to generate fragments of different lengths. The proposed methodology presents an alternative DNA extraction procedure faster and more economical than the kits commercially available. A linear normalized calibration curve was obtained between the log of the ratio of the bovine band intensity and the sum of bovine and ovine band intensities versus the log of cow's milk percentage. The method was applied successfully to the detection and quantification of raw, pasteurized, and powdered bovine milk in different cheeses. The proposed duplex PCR provides a simple, sensitive, and accurate approach to detect as low as 0.1% bovine milk in cheeses and to quantify bovine milk in ovine cheeses in the range of 1-50%.  相似文献   

11.
A simple, rapid, and sensitive method for the quantitative monitoring of five sulfonamide antibacterial residues in milk was developed by coupling in-tube solid-phase microextraction (SPME) to high-performance liquid chromatography with an ultraviolet detector. A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium for this on-line technique. To obtain optimum extraction efficiency, several parameters relating to in-tube SPME were investigated. By simple extraction with ethanol, dilution with phosphate buffer solution, and centrifugation, the sample solution then could be directly injected into the device for extraction. The calculated detection limits for sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium were 2.0, 2.8, 1.7, 2.5, and 22 ng/mL, respectively. The method was linear over the range of 20-5000 ng/mL (100-5000 ng/mL for sulfacetamide sodium) with a correlation coefficient R (2) value >0.9980. Excellent method reproducibility was found by intra- and interbatch precisions, yielding the relative standard deviations of <10.0 and <9.94%, respectively. The proposed method was proved to be robust in monitoring sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium residues in milk.  相似文献   

12.
The ethylenebisdithiocarbamate (EBDC) fungicide, nabam, was determined in several crop matrixes using liquid chromatography with postcolumn reaction detection. After separation by micellar liquid chromatography, nabam (EBDC sodium salt) was acid hydrolyzed to ethylenediamine and fluorigenically labeled with o-phthalaldehyde-mercaptoethanol (OPA-MERC). Standard curves were linear from the detection limit of ca 1 ng to 1000 ng. Nabam was recovered in high yield (89 plus or minus 7.7%) over a range of concentrations (0.1 to 20 ppm) from fortified samples of papaya, lettuce, cucumber, spinach, and applesauce using a simple extraction method. Efforts to convert the more popular EBDC fungicides, maneb and mancozeb, to nabam are discussed.  相似文献   

13.
中国大菱鲆虹彩病毒(turbot reddish body iridovirus, TRBIV)是一种感染养殖大菱鲆的鱼类虹彩病毒,它可以引起大菱鲆病毒性红体病并导致养殖大菱鲆大量死亡。本研究利用TRBIV主要衣壳蛋白基因序列设计的一对引物,结合内嵌式核酸染料SYBR Green І,建立了TRBIV特异的Real-time PCR检测方法。实验结果表明,该对引物具有较高的灵敏度和较强的特异性,能够检测相当于102数量级的TRBIV基因组拷贝,而不与健康大菱鲆组织DNA、淋巴囊肿病毒DNA发生交叉反应。最后,应用建立的Real-time PCR检测方法,开展了TRBIV的组织敏感性检测和病毒流行情况调查。结果发现,大菱鲆的脾脏、肾脏、脑、鳃、心脏、肝脏、消化道、血液等组织中均可检测到TRBIV的存在,其中脾和肾是TRBIV的最主要的靶器官,每毫克组织的病毒含量分别高达5.23106个和2.18106个。分子流行病学调查结果显示,在山东半岛的多个大菱鲆养殖场中均存在TRBIV的感染和流行。  相似文献   

14.
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15.
Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.  相似文献   

16.
Template DNAs were extracted from wine and purified for use as samples for PCR to differentiate grape cultivars. It has been pointed out that the authentication of grape material by PCR using wine as a material is very difficult. The problems are (1) decomposition of DNAs during fermentation; (2) contamination of DNAs from microorganisms such as yeast; (3) interference of DNA extraction by polysaccharides and polypeptides in the beverages; and (4) coexistence of PCR inhibitors, such as polyphenols. For this study was developed a novel preparation method of template DNA from wine to differentiate grape cultivars using PCR by (1) lyophilizing and pulverizing the fermented beverage to concentrate the DNAs; (2) decomposition of polysaccharides and proteins so as not to inhibit DNA extraction using heat-resistant amylase and proteinase K without DNA damage by endogenous DNase; and (3) separation of the template DNAs for PCR from PCR inhibitors, such as polyphenols, by purification using 70% EtOH extraction and isopropyl alcohol precipitation. To prevent the amplification of microorganisms' DNAs during PCR, suitable PCR primers closely related to the specific plant DNAs, such as chloroplast DNA and mitochondrial DNA, were selected. The sequences of the amplified DNAs by PCR were ascertained to be the same as those of grape materials.  相似文献   

17.
陈传君  金鹭  林华  胡滨  韩国全  陈世界  张婧  安微 《核农学报》2020,34(12):2762-2768
为了研究干制加工羊肉基因组DNA的最佳提取方法,本试验采用传统酚-氯仿法、磁珠法、改良CTAB法、离心柱法分别提取干制处理后的羊肉基因组DNA,并对4种方法提取的羊肉基因组DNA浓度、纯度、完整性以及提取所需时间、PCR扩增效果等进行比较。结果表明,采用磁珠法提取DNA的效果更好,DNA浓度为118.87 ng·μL-1,A260/A280值为1.89,而且此方法具有提取时间短、效率高、污染小等特点。本研究结果为干制加工羊肉基因组DNA的大批量提取和检测提供了参考依据。  相似文献   

18.
In this paper, the possibilities of capillary gel electrophoresis (CGE) to detect transgenic maize in flours are shown. The method is based on the extraction and amplification by the polymerase chain reaction (PCR) of a specific DNA fragment from transgenic maize and its subsequent analysis by CGE with UV detection or laser-induced fluorescence (LIF). Some useful considerations regarding the optimization of DNA extraction and amplification conditions are given. Also, a comparison is established between the two CGE protocols for DNA detection based on ultraviolet absorption (CGE-UV) and LIF (CGE-LIF). The requirements, advantages, and limitations of both CGE methods are discussed. To our knowledge, this is the first paper on the use of CGE-LIF to detect transgenic food.  相似文献   

19.
In this study, the event-specific primers for insecticide-resistant maize, MON810, and herbicide-tolerance maize, NK603, have been designed. Simplex PCR and multiplex PCR detection method have been developed. The detection limit of the multiplex PCR is 0.5% for MON810 and NK603 in 50 ng of the template for one reaction. Quantitative methods based on real-time quantitative PCR were developed for MON810 and NK603. Plasmid pMulM2 as reference molecules for the detection of MON810 and NK603 was constructed. Quantification range was from 0.5 to 100% in 100 ng of the DNA template for one reaction. The precision of real-time Q-PCR detection methods, expressed as coefficient of variation for MON810 and NK603 varied from 1.97 to 8.01% and from 3.45 to 10.94%, respectively. The range agreed with European interlaboratories test results (25%). According to the results, the methods for quantitative detection of genetically modified maize were acceptable.  相似文献   

20.
Animal feeds occasionally have some degree of contamination by Aspergillus spp. Even pasteurized milk at times contains the toxic liver carcinogen aflatoxin M1 (AFM1). Confirmation of its presence is now done with solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC)-fluorescence, using a small enough sample that SPE time is reasonable. In this study 200 mL of milk was extracted using a C18 disk at a flow rate of approximately 100 mL/min and AFM1 quantified by HPLC-tandem mass spectrometry with negative electrospray ionization. The effectiveness and cleanup efficacy of immunoaffinity columns (IAC) was compared with that of Mycosep multifunctional cleanup columns (MFC). Average recovery and detection limits of whole milk and low-fat milk cleaned up by IAC were significantly superior to those obtained with the MFC (78-87% and 0.59-0.66 ng/L, respectively). The new procedure improves extraction speed, sensitivity, and specificity.  相似文献   

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