Detection of genetically modified rice: a construct-specific real-time PCR method based on DNA sequences from transgenic Bt rice

Genetically modified rice varieties developed in China are close to approval for agricultural cultivation and production. However, so far no method has been reported for specific detection of transgenic varieties of this crop. In the present study, rice seeds assumed to consist of field-tested Bt rice (‘Anti-pest Shanyou 63’ and ‘Anti-pest Jinyou 63’) were used as reference material to determine transgenic DNA sequences. The transition between the cryIA(b) and cryIA(c) fusion gene and the nopaline synthase terminator (nos) sequence was used to develop a construct-specific real-time PCR based detection method. This Bt rice specific detection system was combined with a recently published quantitative real-time PCR method for the rice-specific (Oryza sativa L.) reference gene gos9. The complete PCR assay for detection of transgenic Bt rice was in-house validated and the limit of quantification was found to be below 0.1% Bt rice relative to the rice content. Application of the PCR assay should allow more precise detection of transgenic rice varieties in imported food products which are so far not approved in the EU.     

基于上转换荧光标记和磁分离技术的沙门氏菌DNA检测新方法

利用水热法制备NaYF4:Yb3+Er3+荧光纳米颗粒,表面氨基化修饰后与探针核酸单链共价偶联,形成荧光标记显示探针。再将氨基化的Fe3O4磁性纳米颗粒与捕获核酸单链进行共价偶联,制备磁分离捕获探针。基于DNA杂交互补反应,加入体系中的目标DNA链分别与两端互补的荧光显示探针和磁分离捕获探针形成三明治夹心结构,通过外加磁场收集分离。加入的目标DNA链浓度越大,体系荧光强度越大。结果表明,复合结构的荧光强度与目标DNA链浓度成正比,在0.01~10 pmol/L范围内呈现良好的线性关系,最低检测限达3 fmol/L。     

Validation and collaborative study of a P35S and T-nos duplex real-time PCR screening method to detect genetically modified organisms in food products

In this work the intra and inter-laboratory validation of a duplex real-time PCR screening method for the detection of genetically modified (gm) plants is described. Target DNA sequences from Cauliflower Mosaic Virus 35S promoter (P35S) and nos-terminator from Agrobacterium tumefaciens (T-nos) are amplified. The duplex real-time PCR method is using primer and probe sequences that have already been published for the individual (“single”) detection of both target sequences. The validation showed sensitivity comparable to the single PCR standard methods. In addition, combined with a reference gene and using reference standard material, the method can be used to semiquantitatively estimate the amount of gm plants in an unknown sample.     

Development of three real-time PCR assays to detect peanut allergen residue in processed food products

Hypersensitivity to peanut is a public health problem, since the ingestion of even low amounts of peanut can trigger severe allergic reactions. Allergic consumers rely on the information provided on the label of foodstuffs to identify products that might endanger their health. In order to protect the allergic consumer methods are required for the detection of allergenic ingredients. For this purpose we have developed three real-time polymerase chain reaction (PCR) assays, based on TaqMan chemistry, that are capable of detecting peanut specific DNA sequences in food products. The peanut specific sequence targeted for detection is located within the gene family of the allergen Ara h 3. The occurrence of multiple Ara h 3 sequences in the peanut genome increases the chance to achieve a good sensitivity. DNA extraction is also known to affect detection by PCR, therefore the efficiency of several different DNA extraction methods was compared. The methods reported here are capable of detecting 2.5 pg peanut DNA (less than one copy of peanut genome equivalent) and all three assays were successfully applied to detect peanut traces in a model food product where they could detect 10 mg kg⁻¹ peanut.     

基于dsDNA-CuNCs的荧光ELISA检测牛奶中的E.coli O157:H7

目的:建立了一种灵敏检测牛奶中大肠杆菌O157:H7的新型荧光酶联免疫吸附方法。方法:以碱性磷酸酶水解焦磷酸盐-Cu²⁺配位复合物,释放出Cu²⁺形成铜纳米簇作为信号报告探针,通过对关键因素Cu²⁺浓度、焦磷酸盐浓度、DNA模板的浓度和长度进行优化。结果:在最优条件下,大肠杆菌O157:H7的菌数为5×10⁴～1×10⁸ CFU/mL时,与荧光信号值有较好的线性关系(R²=0.980 8),最低检出限为2.4×10⁴ CFU/mL。结论:该方法成功地应用于低脂牛奶和脱脂牛奶样本中大肠杆菌O157:H7的测定,平均回收率为92.2%～108.5%,相对标准偏差为4.9%～10.4%。     

A sensitive immunochromatographic assay using colloidal gold–antibody probe for the rapid detection of semicarbazide in meat specimens

A rapid immunochromatographic assay (ICA) was developed for the detection of semicarbazide (SEM), a nitrofurazone metabolite in meat specimens. Colloidal gold–labeled monoclonal antibody specific to a SEM derivative, 4[(4-carboxyphenyl)methylene]-hydrazinecarboxamide (CPSEM), was used as a probe for the presence of SEM. The assay is based upon the competitive reactivity theory that SEM derivatized with 4-carboxybenzaldehyde (4-CBA) competes with CPSEM-conjugated bovine serum albumin for binding the colloidal gold–labeled monoclonal antibody, the result of which could be read directly by naked eyes. Under optimal conditions, the visual detection limit is 0.72 ng mL⁻¹, which demonstrated the high sensitivity of this assay. The stability test indicated that the immunochromatographic strips could be used within 7 weeks at room temperature without significant loss of activity. Spiked pork samples were detected by ICA and confirmed by enzyme-linked immunosorbent assay (ELISA). The assay time for SEM detection was within 5 min, suitable for a rapid on-site testing for meat samples.     

速冻食品中沙门氏菌和金黄色葡萄球菌多重PCR检测方法的建立与应用

为实现速冻食品中沙门氏菌(Salmonella spp.)和金黄色葡萄球菌(Staphylococcus aureus)的多重聚合酶链式反应(PCR)检测,首先优化多重PCR扩增的反应条件,比较基因组DNA提取方法,结果表明：退火温度采用60℃、各引物浓度200nmol/L及扩增循环35次,本多重PCR检测技术可以有效地将沙门氏菌和金黄色葡萄球菌同时检出,检测特异性为100%。3种DNA提取方法中试剂盒法纯度最高,检出限分别是31、26DNA copies/reaction。经过在人工污染致病菌的速冻水饺中应用试验后,该多重PCR方法经过4h的增菌培养即可从速冻水饺中同时检测出起始菌落数低至100CFU/g的沙门氏菌和金黄色葡萄球菌。     

转基因玉米品系及转化体成分四重实时荧光PCR快速鉴定

Bt11转基因玉米品系具有抗草铵膦除草剂,鳞翅目昆虫抗性的耐除草剂抗虫玉米,MIR162和Mon89034是鳞翅目昆虫抗性的单抗虫玉米,均是国内外出入境监管主要关注的转基因玉米品系。本研究通过靶标基因筛选,转基因阳性样品采集,核酸样本制备,多重引物和荧光探针组合筛选,反应体系优化以及方法学验证等过程开发建立了四重荧光定量PCR检测技术。结果表明该技术的使用可实现一个反应管中同时检测MIR162、Bt11、Mon89034三个玉米品系的特异性基因序列和一个编码玉米淀粉合成酶异构体zSTSII-2 (zSSIIb) 玉米内源基因。通过阳性对照,阴性对照和空白对照特异性S曲线与对应的阈值大小分析可判定样品中是否含有这三个转基因玉米品系及其转化体成分。经方法学特异性检测,结果与转基因检测金标准的单实时荧光PCR结果一致;经平行样和灵敏度测试,最低检测限为18个拷贝;经标准曲线扩增分析,四个基因的扩增效率均在90%~110%范围内,扩增效果良好。该方法从DNA提取到报告结果不足3小时,可缩短检测时限,节约试剂耗材成本,操作简单易行,满足高通量特征,可为市场流通产品品系和转基因成分的实时监管和快速鉴定提供技术支撑。     

Detection of Pathogenic Yersinia enterocolitica in Meat using Real-Time PCR

Yersinia enterocolitica is an important zoonosis, which can cause disease in humans and animals. The culture methods available for detection of Y. enterocolitica in food samples are time-consuming and seldom successful. Using DNA-based methods, like PCR, this pathogen can be detected more rapidly and with greater sensitivity. The aim of this study was to establish a rapid and accurate real-time PCR method to detect pathogenic Y. enterocolitica in pork. The chromosomal ail−gene, which is only present in pathogenic Y. enterocolitica strains, was used as DNA target for the 5' nuclease PCR assay. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). A 2-step protocol with 45 cycles was used in a multicolour real-time PCR detection system. A Ct value over 40 indicated a negative result. The DNA extraction procedure for the natural samples was rapid and simply. This qualitative real-time PCR method was shown to be specific and sensitive. Detection rate of ail-positive Y. enterocolitica in 200 pig tonsils was 88 % and 35 % with PCR and culture methods, respectively. When 100 raw pork samples were studied, 7 were positive with PCR and all were culture negative. Received: January 30, 2007; Received in revised form: February 27, 2007; Accepted: February 28, 2007.     

Development of an event-specific detection method for genetically modified rice Kefeng?6 by quantitative real-time PCR

The genetically modified (GM) rice Kefeng?6 has gained resistance against several rice pests by inserting the cpti and cry1Ac genes. As this transgenic line is not approved for import, processing and cultivation in the European Union (EU), sensitive and specific detection methods need to be available to monitor any illegal presence of Kefeng?6 in food products within the EU. The aim of this study was to develop and validate an event-specific detection method by means of quantitative real-time PCR (qPCR) for the detection of Kefeng?6 in foodstuff. A primer pair and hydrolysis probe were designed according to the right border junction sequence of the transgene. The qPCR assay was validated according to the ENGL/EURL-GMFF guidelines for GMO testing and is presented according to the MIQE guidelines. The in-house validation process resulted in a limit of detection of 5 DNA copies of the transgene with confidence intervals (95?%) between 0.07 and 0.52, a PCR efficiency of 105?% and a correlation coefficient (R ²) value of 0.9997. The specificity of the assay was tested by end-point PCR, gel electrophoresis and subsequent sequencing of the PCR products. By testing DNA of several GM and non-GM crops, cross reactivity of the assay was not observed. Further, 35 food products were analyzed for the presence of Kefeng?6 by means of the event-specific detection method. For 9 out of 35 samples, PCR products for Kefeng?6 DNA were observed.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

A sandwich-type DNA array platform for detection of GM targets in multiplex assay

Over the last decade, array detection has been developed to qualitatively assess the presence of genetically modified organisms (GMOs). To date, DNA array systems have the highest capabilities as a result of GMOs analysis. We describe the construction of an array platform in the sandwich hybridization format for the detection of transgenic promoter of Cauliflower mosaic virus (CaMV; p35S). Sequence-specific signal development has been achieved by a sandwich complex composed of a surface immobilized capture probe and a fluorescein-tagged signal probe, which are partially complementary to the p35S oligonucleotide. We used poly-l-lysine-coated glass slides as support material, on which capture probes were immobilized by a heterobifunctional cross-linker. The comparative results of optimization studies including cross-linker types probe concentrations and hybridization conditions (sequence, temperature and duration) were reported. An optimum hybridization signal was obtained with a 32.5 ? cross-linker, 10???M capture and 20???M signal probe concentrations, respectively. A relatively short hybridization time (2.5?h) provided reproducible array signals. No significant effect of hybridization sequence on the fluorescence intensity was observed. The described platform can specifically detect label-free transgenic sequences with a target of 0.01???M concentration, while the optimized system exhibits great potential for the application of different GMO target sequences (p35S, tNOS, bar and cry) to multiplex array formats.     

Detection of Clostridium botulinum neurotoxin coding genes: analysis of PCR products by real time versus capillary gel electrophoresis methods

In this work, two PCR-based methods have been developed for the detection of Clostridium botulinum strains carrying the gene coding for C. botulinum neurotoxin C (BoNTC) responsible for avian botulism. Both methods are based on the same amplification primers designed using multiple sequence alignments between toxin C coding sequences from DNA sequence databases. The first is a real-time PCR method, using a Taqman-MGB probe. The second uses conventional end-point PCR, followed by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). A comparison between both methods has been established for the individual and simultaneous detection of toxin C (BONTC) or bacterial 16S (BACT) sequences from C. botulinum. The results indicate that, in general, the same sensitivity was achieved by using RT-PCR and PCR-CGE-LIF allowing the detection of both C. botulinum amplicons from concentrations as low as 7 × 10⁻⁵ μg/ml of total genomic DNA. Some other features from RT-PCR and CGE-LIF are also critically discussed in this work, including quantification capability, size determination, analysis speed and identification strategies, to provide enough information to adequately select the best analytical technique in each case. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of an event-specific detection method for genetically modified rice Kefeng?6 by quantitative real-time PCR

The genetically modified (GM) rice Kefeng 6 has gained resistance against several rice pests by inserting the cpti and cry1Ac genes. As this transgenic line is not approved for import, processing and cultivation in the European Union (EU), sensitive and specific detection methods need to be available to monitor any illegal presence of Kefeng 6 in food products within the EU. The aim of this study was to develop and validate an event-specific detection method by means of quantitative real-time PCR (qPCR) for the detection of Kefeng 6 in foodstuff. A primer pair and hydrolysis probe were designed according to the right border junction sequence of the transgene. The qPCR assay was validated according to the ENGL/EURL-GMFF guidelines for GMO testing and is presented according to the MIQE guidelines. The in-house validation process resulted in a limit of detection of 5 DNA copies of the transgene with confidence intervals (95 %) between 0.07 and 0.52, a PCR efficiency of 105 % and a correlation coefficient (R ²) value of 0.9997. The specificity of the assay was tested by end-point PCR, gel electrophoresis and subsequent sequencing of the PCR products. By testing DNA of several GM and non-GM crops, cross reactivity of the assay was not observed. Further, 35 food products were analyzed for the presence of Kefeng 6 by means of the event-specific detection method. For 9 out of 35 samples, PCR products for Kefeng 6 DNA were observed.     

Improved Sample Preparation for Real-Time PCR Detection of Listeria monocytogenes in Hot-Smoked Salmon using Filtering and Immunomagnetic Separation Techniques

This work evaluated the application of filtration and immunomagnetic separation (IMS) as sample pretreatments for use in combination with real-time polymerase chain reaction (PCR) to detect and quantify Listeria monocytogenes in hot-smoked salmon. Salmon was artificially inoculated with L. monocytogenes at levels ranging from 8 × 10⁰ to 8 × 10⁵ cfu/g of sample, and homogenates obtained from these samples were filtered to recover bacterial cells without a pre-enrichment step. High recovery of bacterial cells was achieved using standard coffee filters. IMS significantly reduced the co-extraction of PCR inhibitors present in the samples to increase the assay sensitivity with regression line parameters applicable for quantification. The limit of detection and quantification were equal to 2 × 10¹–4 × 10¹ and 2 × 10² cfu/g of sample, respectively. The entire detection procedure could be completed within 3.5 h. This study demonstrated that coupling filtration and IMS with real-time PCR has contributed to improve the sensitivity of L. monocytogenes detection from hot-smoked salmon.     

转基因大豆的PCR-免疫层析筛查方法研究初探

目的建立转基因大豆的PCR-免疫层析（PCR—ICT）快速筛查新技术。方法利用转基因植物通常含有的CaMV35S启动子作为转基因成分的筛查标记，根据其序列设计特异性引物和探针，分剐用生物素和地高辛标记，用胶体金免疫层析技术检测并鉴定PCR产物。结果用新建立的PCR—ICT方法可以检出含0．5％转基因大豆的标准品，对大豆样品的检测结果与琼脂糖凝胶电泳检测结果一致。结论PCR—ICT方法通过DNA杂交和金标显色来同时检测、鉴定PCR产物，可以简便、快速地筛查转基因产品。     

Development of a rapid and sensitive immunomagnetic-bead based assay for detecting <Emphasis Type="Italic">Bacillus cereus</Emphasis> in milk

Bacillus cereus is a major food-born pathogen in Taiwan and its major syndromes include vomiting, fever and diarrhea. To minimize the possibility of exposing consumers to pathogenic B. cereus, this study develops a rapid and sensitive assay that utilizes immunoliposomal nanovesicles (IMLNs) and immunomagnetic beads (IMBs). In this work, fluorescent dyes (sulforhodamine B)-loaded IMLNs were employed to increase the detection signal; anti-B. cereus antibody-conjugated IMBs were applied to capture B. cereus in samples. Hence in this assay, a sandwich complex was formed as “IMBs-B. cereus-IMLNs”. The optimal IMLNs had a diameter of 300 nm with a conjugated antibody molar percentage (mol%) of 0.25 mol%. The limit of detection (LOD) of this developed assay reaches 10 CFU/mL of B. cereus with the false negative value as zero in 20 parallel assays in milk samples. To evaluate the specificity of this assay, nine Gram positive and negative bacteria were tested and found to cause no significant interference problems. In conclusion, this study elucidates the feasibility of using a novel IMB/IMLN assay for detecting B. cereus and its LOD without pre-enrichment could amount to 10 CFU/mL within 4 h.     

基于内参系统的阪崎肠杆菌实时荧光定量PCR检测方法的建立

根据阪崎肠杆菌ompA靶基因设计特异性引物和探针,并加入内参(IAC),建立能够实时监控反应过程的荧光定量PCR检测方法。结果表明,该方法对阪崎肠杆菌基因组DNA的最低检测限为1pg;对细菌的最低检测限为1×10~4 CFU;对含有靶基因质粒的最低检测限为10~3拷贝;Ct值与模板拷贝数均呈良好的线性关系(R~2=0.999)。人工污染试验结果表明,在初始菌量为10CFU/25g奶粉样品时,采用水洗加试剂盒法和水煮法提取DNA,阪崎肠杆菌均在增菌10h时检出。研究结果为进一步优化和完善阪崎肠杆菌分子生物学方法的标准化提供了参考。     

Catalytic hairpin assembly combined with graphene oxide for the detection of emetic Bacillus cereus in milk

A fluorescence assay combined with PCR, catalytic hairpin assembly (CHA), and graphene oxide (GO) was established to detect emetic Bacillus cereus in milk samples. The processes of the assay are not new, but components of the processes make the assay useful. Two partially complementary hairpin probes (H1 and FAM-H2) were designed according to the target single-strand DNA (ssDNA). The CHA reaction could be initiated only by the target ssDNA, which was generated by the denaturation of PCR amplicons. In the absence of the target ssDNA, CHA reaction could not be triggered, which caused the H1 and FAM-H2 adsorbing on the surface of GO and exhibiting a low fluorescence intensity. Addition of the target ssDNA resulted in opening of the hairpin H1 that subsequently hybridized with H2. Then, target ssDNA would be replaced from the H1 and recycled to promote another CHA reaction. Through the CHA reaction, multiple H1-H2 duplexes were generated that could not adsorb on the surface of GO. Thus, a strong fluorescence signal would be obtained. The assay showed a limit of detection for emetic B. cereus of 6.2 × 10¹ cfu/mL in pure culture and 5.9 × 10² cfu/mL in spiked milk without enrichment. By changing the PCR primer, the assay developed in this study had potential to detect other bacteria.     

Real-Time PCR Quantification of Protease-Producing Bacteria in Traditional Chinese Fish Sauce

This study aims to isolate, identify, and quantify protease-producing bacteria in traditional Chinese fish sauce. Fifty-five protease-producing bacteria were isolated from 10 fermented fish sauce samples in five growth media based on their morphology and caseinolytic activity. These isolates were identified through their 16S rDNA gene sequences. BLAST analysis of 16S rDNA sequences revealed that 46 and 9 strains belonged to Bacillus sp. and Virgibacillus halodenitrificans, respectively. We used EvaGreen dye in real-time PCR assay to target the partial bacterial 16S rDNA gene sequences of the 55 strains from fish sauce. Two primer pairs (A and B) were designed. Primer pair B was more suitable than primer pair A for real-time PCR assay, in which the optimum annealing temperature was 60 °C. The significance of the developed method was proven by the highly linear characteristic of the standard curve that relates lower threshold cycle (Ct) values and 16S rDNA copy numbers of the standard DNA sample. This method was used to calculate the number of protease-producing bacteria in fish sauce. The minimum level of detection (1.44?×?10³ copies/μL) was also verified. The concentration of protease-producing bacteria in fish sauce was estimated to be (1.47?±?0.73)?×?10³ colony-forming units (cfu)/mL by real-time PCR assay and showed a percentage of positive results correctly assigned of 91.8 % when compared to the plate culture method used as reference. The results may serve as a foundation for future studies on the microbial succession and variation of microorganisms during fish sauce fermentation.