Development and in‐house validation of the event‐specific qualitative and quantitative PCR detection methods for genetically modified cotton MON15985

BACKGROUND: To implement genetically modified organism (GMO) labeling regulations, an event‐specific analysis method based on the junction sequence between exogenous integration and host genomic DNA has become the preferential approach for GMO identification and quantification. RESULTS: In this study, specific primers and TaqMan probes based on the revealed 5′‐end junction sequence of GM cotton MON15985 were designed, and qualitative and quantitative polymerase chain reaction (PCR) assays were established employing the designed primers and probes. In the qualitative PCR assay, the limit of detection (LOD) was 0.5 g kg^?1 in 100 ng total cotton genomic DNA, corresponding to about 17 copies of haploid cotton genomic DNA, and the LOD and limit of quantification (LOQ) for quantitative PCR assay were 10 and 17 copies of haploid cotton genomic DNA, respectively. Furthermore, the developed quantitative PCR assays were validated in‐house by five different researchers. Also, five practical samples with known GM contents were quantified using the developed PCR assay in in‐house validation, and the bias between the true and quantification values ranged from 2.06% to 12.59%. CONCLUSION: This study shows that the developed qualitative and quantitative PCR methods are applicable for the identification and quantification of GM cotton MON15985 and its derivates. Copyright © 2009 Society of Chemical Industry     

Screening and construct‐specific detection methods of transgenic Huafan No 1 tomato by conventional and real‐time PCR

Genetically modified (GM) tomatoes have been approved for commercialization in many countries since the first GM tomato FLAVR SAVR was permitted for planting in 1994. In China, GM tomato Huafan No 1 with a character of long shelf‐life was the first GM plant which was approved for commercialization in 1996. To meet the requirement of the GM tomatoes labeling policy that has been actualized in China since 2001, screening and construct‐specific PCR detection methods for detecting the universal elements transformed into tomato, such as cauliflower mosaic virus 35S (CaMV35s) promoter and the nopaline synthase (NOS) terminator of Agrobacterium tumefaciens, and the specifically inserted heterologous DNA sequence between CaMV35s promoter and anti‐sense ethylene‐forming enzyme (EFE) gene were set up. To make the detection methods normative, a novel single copy tomato gene LAT52 was also used as an endogenous reference gene in the PCR detection systems. The limit of detection of screening and construct specific detection methods for Huafan No 1 was 68 haploid genome copies in conventional PCR detection, and three copies in TaqMan real‐time PCR detection. The limit of quantitation of screening quantitative PCR assays for Huafan No 1 was three copies and was 25 copies for construct‐specific quantitative PCR. Two samples with known Huafan No 1 tomato content were detected using the established conventional and real‐time PCR systems, and these results also indicated that the established Huafan No 1 screening and construct‐specific PCR detection systems were reliable, sensitive and accurate. Copyright © 2005 Society of Chemical Industry     

Characterization and event‐specific quantitative detection of DAS‐59122‐7 maize insert with the application of plasmidic reference material

BACKGROUND: With the development of genetically modified organisms (GMOs), event‐specific qualitative and quantitative polymerase chain reaction (PCR) detection methods have become the internationally agreed standard. RESULTS: The flanking regions of DAS‐59122‐7 maize were characterized by inverse PCR (I‐PCR). In the qualitative PCR assay, a duplex PCR was established with the event‐specific and taxon‐specific primers, and the limit of detection (LOD) was 1 g kg^?1 (approximates to 38 haploid genome copies). In the quantitative TaqMan® real‐time PCR assay, a plasmidic reference material was constructed by recombinant PCR and standard curves were set up. By using the plasmidic reference material, we obtained standard curves with good linearity and relatively high efficiency. The results indicated the usability of the plasmid as standard material. CONCLUSION: From above results, we believe that the developed event‐specific qualitative and quantitative PCR systems for DAS‐59122‐7 maize in this study are acceptable and suitable for DAS‐59122‐7 maize detection. Copyright © 2008 Society of Chemical Industry     

Event‐specific analytical methods for biotech maize MIR 604 and DAS‐59122‐7

BACKGROUND: It is very important to develop analytical methods for genetically modified organism (GMO) labelling systems and living modified organism (LMO) management. The polymerase chain reaction (PCR) is the most efficient DNA‐based analytical method for identifying and quantifying biotech crops. Qualitative PCR methods have been developed to detect the presence of biotech crops, while quantitative PCR methods have been developed to analyse the content of biotech crops. Analytical methods are now required for new biotech maize events, MIR 604 and DAS‐59122‐7. RESULTS: The event‐specific primers and probes were developed for qualitative and quantitative analysis of biotech maize MIR 604 and DAS‐59122‐7 based on the 3′ flanking regions. As a reference molecule, single standard plasmid was developed. The specificity of the qualitative primers was confirmed by the single PCR product, with limits of detection (sensitivity) of 0.01 and 0.05% respectively. In‐house validation of the quantitative methods was performed using six levels of mixing samples (0.1–10% w/w). As a result, the biases from the true value and the relative standard deviations were within the range of ± 30%. The limits of quantitation of the quantitative methods were all 0.1% for real time PCRs of MIR 604 and DAS‐59122‐7. CONCLUSION: In this study, event‐specific analytical methods were developed and applied to the qualitative and quantitative analysis of biotech maize MIR 604 and DAS‐59122‐7. Copyright © 2009 Society of Chemical Industry     

转基因棉花MON88913转化体特异性定性、定量PCR检测方法

本文以我国批准商业化的转基因耐草甘膦棉花MON88913为研究对象,建立并验证了其转化体特异性定性、定量PCR检测方法.建立的定性PCR方法的检测极限是20个拷贝棉花单倍体基因组DNA,定量PCR方法的检测和定量极限分别是10和20个拷贝棉花单倍体基因组DNA.同时,我们组织了实验室5位研究人员对建立的定量PCR检测方法进行了协同验证.对5个盲样的定量分析结果显示与真实值的偏差介于1.59% 和10.12%之间,完全满足国际标准25%偏差范围的要求,完全可用于转基因棉花MON88913的实际样品检测.     

Identification of pork genome in commercial meat extracts for Halal authentication by SYBR green I real‐time PCR

The identification of pork DNA in meat extracts is very important for Halal authentication and Muslim consumers demand protection from falsely labelled meat products. A pig‐specific SYBR green I real‐time PCR assay has been developed to address this issue. Using specific primers for pig mitochondrial DNA, successful amplification has been obtained by DNA extracted from control meat samples. With SYBR green I real‐time PCR, the specificity of the amplification was showed by Tm value. Detection limit of the real‐time PCR was down to 0.1 ng of porcine DNA. An appropriate linearity was obtained by construction of a standard curve based on Ct value and different concentrations of porcine DNA. By conventional PCR, no amplification was shown by porcine DNA less than 0.1 ng. The established method was conducted on commercially available meat extracts for detection and quantification of porcine DNA. The results showed the SYBR green I real‐time PCR could be considered a robust method for Halal authentication of meat extracts.     

Event-specific detection of genetically modified wheat B73-6-1 based on the 3′-flanking sequence

In this study, 3′-flanking sequence between the host plant DNA and the integrated gene construct of pHMW1Dx5 vector in transgenic wheat B73-6-1 was revealed by means of adaptor PCR; thus, the fragment with the length of 3.1?kb was obtained, including a 190-bp wheat genomic DNA, which demonstrates that this HMW-GS gene was located on the wheat chromosome 3B. And the event-specific PCR primers were designed based upon the revealed 3′-flanking sequence; the conventional qualitative PCR and quantitative SYBR real-time PCR detection methods employing these primers were successfully developed. In conventional qualitative PCR assay, the limit of detection was 0.1?% for B73-6-1 wheat genomic DNA for one reaction. In the quantitative SYBR real-time PCR assay, the limit of detection and limit of quantification were 10 and 100 haploid genome copies, respectively. In addition, three mixed blind wheat samples with known B73-6-1 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.     

Cooking DNA: the effect of ‘domestic’ cooking methods on detection of GM potato

The ability to detect GM material in otherwise unprocessed foods cooked using domestic methods is important should ‘ready‐to‐eat’ foods require labelling. This study addresses the issue of DNA degradation in foods as a result of cooking. A number of ‘domestic’ cooking methods were shown to affect the length of DNA sequences able to be PCR amplified from potato samples and the degree of degradation was treatment‐specific. However, a. real‐time PCR assay was developed and. GM material was positively identified in all cooked GM potato samples. This confirms that GM material should be able to be detected in otherwise unprocessed food samples cooked using domestic methods, even if the cooking process has partially degraded the DNA. Results indicate, however, that there may be implications of the cooking process on the ability to accurately quantify GM content in some cooked samples.     

A cotton‐specific gene,stearoyl‐ACP desaturase,used as a reference for qualitative and real‐time quantitative polymerase chain reaction detection of genetically modified organisms

Biotechnology has permitted the modification of agricultural materials in a very precise way to improve productivity and yields. Polymerase chain reaction (PCR)‐based methods have been the first choice of most analytical laboratories for routine use in the detection of genetically modified organisms (GMO) and their derived products. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison with an amplified reference gene. This paper describes the specific primers and probe for the cotton stearoyl‐ACP desaturase (sad1) gene, and PCR cycling conditions suitable for the use of this sequence, which acts as an endogenous reference gene in both qualitative and quantitative PCR assays. The two methods were tested with 18 cotton varieties and identical amplification products were obtained with all of them. No amplification products were detected when DNA samples from other species, including soybean, rapeseed, tobacco, maize, tomato, potato, cucumber, pea, red pepper, sunflower, sesame, rice, peach, banana, apple, pumpkin, barley and carrot, were used as templates, which demonstrates that this system is specific for cotton. In real‐time quantitative PCR analysis, the detection limit was as low as 6 pg of DNA, which indicates that this method is suitable for application to processed food samples that contain very low copies of target DNA. Southern blot analysis confirmed that the sad1 gene was a single copy in the tested cotton varieties. Copyright © 2006 Society of Chemical Industry     

转基因"华番一号"番茄的筛选和特异性的定性、定量PCR检测方法

为给转基因植物监测提供技术支持，建立了转基因“华番一号”番茄筛选和特异性的定性、定量PCR检测方法。转基因“华番一号”的筛选PCR检测主要以转基因通用元件CaMV35S启动子和NOS终止子为目的基因片段，特异性PCR检测以转基因外源重组子的CaMV35S启动子和反义EFE基因的相邻序列为目的片段；实验同时设立番茄的LAT52基因为转基因番茄定性、定量PCR检测的内对照基因。在所建立的PCR检测体系中，定性PCR筛选和特异性检测的检测极限为68个拷贝，实时定量PCR方法的检测极限为3个拷贝；筛选定量．PCR检测的定量极限为3个拷贝，特异性定量PCR检测的定量极限为25个拷贝。最后通过对2个已知含量的转基因番茄“华番一号”混合试样的检测，证明了该体系可以有效地用于转基因番茄“华番一号”的筛选和特异性的定性、定量PCR检测。     

转基因大米TT51-1品系精准定量检测方法的比较研究

基于大米蔗糖磷酸合成酶(SPS)基因和TT51-1品系特异性基因序列筛选适用于数字PCR的内、外源基因特异性引物探针并建立转基因大米TT51-1品系的双重数字PCR定量方法。其定量的绝对灵敏度和相对灵敏度分别达2 copies/μL和0.1%。当样品中TT51-1转基因大米成分含量低至0.1%时,6次定量值的相对标准偏差在7.30%~18.63%之间,偏差在-8.77%~9.62%之间,精密度和稳定性均较为理想,而同样的引物探针所建立的实时荧光PCR方法定量的相对灵敏度仅达到1%。为促进该方法的标准化应用,将微滴式数字PCR平台上建立的定量方法在芯片式数字PCR平台上进行室内验证,结果表明该方法的定量精密度和准确性符合要求。该方法可应用于大米、稻谷及其初加工产品中TT51-1转基因大米成分的精准定量检测。     

基于微滴式和芯片式数字PCR技术的转基因克螟稻成分的定量检测

以转基因克螟稻品系为研究对象,通过大米内源蔗糖磷酸合成酶基因和转基因克螟稻品系特异性序列的绝对拷贝数分析,建立转基因克螟稻成分的双重数字聚合酶链式反应（polymerase chain reaction,PCR）定量分析方法。本研究中转基因克螟稻定量体系中最适DNA添加量在10 pg～13 ng之间,模板断裂程度、PCR扩增退火温度等因素对定量结果的影响不大。其定量的绝对灵敏度达1 copies/μL,可在微滴式和芯片式数字PCR平台上准确检测质量分数在0.1%～100%之间的转基因克螟稻成分,尤其是对低于1%的样品,其定量准确性高于传统的实时荧光PCR方法。本研究建立的转基因克螟稻成分定量分析方法适用性较好,可用于转基因水稻规范化与标准化的定量分析。     

Development of two screening duplex PCR assays for genetically modified organism quantification using multiplex real-time PCR master mixes

Since 2001, the traceability and labelling of genetically modified organism (GMO) food and feed derived products are obligatory in the European Union. Genetically modified organisms (GMO) are commonly detected via PCR tests. These tests typically involve several steps: (1) screening (2) construct specific (3) event specific and (4) reference gene. Screening tests are based on sequences frequently used for GM development, allowing for the detection of a large number of GMOs. To improve GMO detection efficiency, using specific multiplex master mixes, we developed two real-time PCR screening duplex PCR assays for the detection of P35S/Tnos and Pnos/T35S sequences. By combining these tests, we were able to reduce the time and cost of analysis. For the Pnos/T35S duplex, good sensitivity was obtained using one of the mixes compared to the others. Both duplexes had 100% specificity when tested on DNA from GM maize, rapeseed and soybean. When the duplexes were tested on DNA containing various amounts of GM maize and soybean, the corresponding targets were detected. The detection limit of our methods was found to be between 2 and 8 haploid genome copies for both P35S/Tnos and Pnos/T35S tests. In summary, with high efficiency and good linearity, the proposed two screening duplexes allow for more efficient GMO detection.     

Quantitative detection method for Roundup Ready soybean in food using duplex real‐time PCR MGB chemistry

BACKGROUND: Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non‐authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost‐efficient solution. RESULTS: Construct‐specific primers and probe were developed for quantitative analysis of Roundup Ready ^® soybean (RRS) event glyphosate‐tolerant soybean (GTS) 40‐3‐2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS‐ and Le1‐specific quantitative real‐time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. CONCLUSION: In this study, a duplex qRTPCR using TaqMan minor groove binder‐non‐fluorescent quencher (MGB‐NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40‐3‐2 that can be used for practical monitoring in processed food products. Copyright © 2010 Society of Chemical Industry     

Multiplex Real‐Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera

Abstract: Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real‐time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene‐producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON‐ and OTA‐producing fungi can be detected and quantified in a single reaction tube using this multiplex real‐time PCR method. Practical Application: This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal‐based foods.     

副溶血弧菌的SYBR Green Ⅰ实时定量PCR检测方法建立

基于副溶血弧菌gyrB基因保守序列设计1对特异性引物,建立SYBR GreenⅠ实时定量聚合酶链式反应(polymerase chain reaction,PCR)检测副溶血弧菌的方法。SYBR GreenⅠ实时定量PCR的Tm为90℃,扩增产物的熔解曲线只出现1个单特异峰,无引物二聚体,表明该引物具有较好的特异性;所制作的实时定量PCR扩增标准曲线在2.06×108～2.06×103拷贝数之间有较好的线性关系,相关系数为0.992,能对副溶血弧进行准确的定量分析。该方法检测时间从核酸抽提到结果分析仅需4～5h,且较传统方法敏感、操作简单,可用于针对副溶血弧菌的进出口检验检疫、食品安全检测及该菌引起的水产动物疾病的诊断与分子流行病学调查。     

Amplification Facilitators and Pre‐Processing Methods for PCR Detection of Strictly Anaerobic Beer‐Spoilage Bacteria of the Class Clostridia in Brewery Samples

The aim of this study was to evaluate easy pre‐PCR processing procedures to allow rapid and reliable detection of strictly anaerobic beer‐spoilage bacteria throughout the brewing process by end‐point and real‐time PCR techniques. The efficiencies of the new procedures were evaluated using spiked brewery samples and specific PCRs for the target group bacteria. We found for the first time that the inclusion of 0.25% (w/v) bovine serum albumin (BSA) or 0.5% (w/v) polyvinyl pyrrolidone (PVP) in the end‐point PCR mixture reduces the inhibiting effect of brewery sample extracts (3–10%, v/v) on PCR. Membrane filtration with a PVP or a sodium tri‐polyphosphate‐EDTA wash, and cross‐flow filtration were the most promising new methods to reduce inhibitors from beer samples before cell lysis. Together with BSA, they allowed the analysis of 10% (v/v) of crude extracts instead of <3% (v/v). Moreover, we developed a one‐hour procedure to prepare target DNA from process samples containing brewer's yeast. It involved removal of inhibitors by a two‐step centrifugation followed by physical disruption of cells. The detection limit of the procedure was 10¹‐10³ CFU/mL. The developed procedures help to reduce the risk of partial or complete PCR failure due to inhibition and target DNA losses, with minimal sample handling.     

The 50:50 method for PCR‐based seamless genome editing in yeast

The ability to edit the yeast genome with relative ease has contributed to the organism being a model eukaryote for decades. Most methods for deleting, inserting or altering genomic sequences require transformation with DNA that carries the desired change and a selectable marker. One‐step genome editing methods retain the selectable marker. Seamless genome editing methods require more steps and a marker that can be used for both positive and negative selection, such as URA3. Here we describe the PCR‐based 50:50 method for seamless genome editing, which requires only two primers, one PCR with a URA3 cassette, and a single yeast transformation. Our method is based on pop‐in/pop‐out gene replacement and is amenable to the facile creation of genomic deletions and short insertions or substitutions. We used the 50:50 method to make two conservative loss‐of‐function mutations in MATα1, with results suggesting that the wild‐type gene has a new function outside of that presently known. Copyright © 2013 John Wiley & Sons, Ltd.     

Biodegradation of genetically modified seeds and plant tissues during composting

BACKGROUND: The increasing global market of genetically modified (GM) crops amplifies the potential for unintentional contamination of food and feed with GM plants. Methods proposed for disposal of crop residues should be assessed to prevent unintended distribution of GM materials. Composting of organic material is inexpensive and location‐independent. The objective of this study was to determine the effectiveness of composting for disposal of GM plants in terms of reducing seed viability and promoting the degradation of endogenous as well as transgenic DNA. RESULTS: Duplicate samples of corn kernels, alfalfa leaves, and GM canola seeds, meal and pellets were sealed in porous nylon bags and implanted in duplicate 85 000 kg (initial weight) feedlot manure compost piles. Samples were collected at intervals over 230 days of composing. Canola seeds and corn kernels were not viable after 14 days of composting with temperatures in the piles exceeding 50 °C. In all samples, PCR analyses revealed that plant endogenous and transgenic fragments were substantially degraded after 230 days of composting. Southern blotting of genomic DNA isolated from canola seeds identified differences in the persistence of endogenous, transgenic, and bacterial DNA. CONCLUSION: Composting GM and non‐GM plant materials with manure rendered seeds non‐viable, and resulted in substantial, although not complete, degradation of endogenous and transgenic plant DNA. This study demonstrates that composting could be effective for disposing of GM crops in the event of their inadvertent entry into the food or feed chain. Copyright © 2010 Crown in the right of Canada. Published by John Wiley & Sons, Ltd     

微滴式数字PCR定量检测转基因玉米品系VCO-01981-5

建立基于QX100微滴式数字聚合酶链式反应(polymerase chain reaction,PCR)平台的我国未批准转基因玉米品系VCO-01981-5的二重微滴式数字PCR定量检测方法。该方法选择基因组中单拷贝的玉米内源基因hmg和VCO-01981-5品系边界序列为定量靶序列,分别设计不同的PCR扩增引物和TaqMan探针,并对两种探针用不同的荧光进行标记,然后将上述探针和引物置于同一个PCR反应体系中以同时定量两个靶标序列。特异性实验结果显示该法只有VCO-01981-5品系的两个靶序列才都有扩增信号。灵敏度、线性和准确性实验结果显示在定量结果相对标准偏差不大于25%时,最低可稳定定量5个拷贝的VCO-01981-5品系特异性序列分子和4个拷贝的内源基因hmg分子;而在高达50 ng模板DNA以下范围内,PCR反应模板量与测定样品拷贝数之间呈高度正相关,相关系数达0.99以上;平均误差小于10%。结果表明本研究建立的该玉米品系定量方法特异性强,稳定性好,精确性、准确性以及灵敏度高,定量范围广,可用于进、出口农产品和食品中该转基因玉米品系成分的定量检测。此外,该法还可为其他转基因玉米品系及其他转基因作物品系建立类似定量检测方法提供参考。