Integrated structure and event-specific real-time detection of transgenic <Emphasis Type="Italic">cry1Ac/SCK</Emphasis> rice Kefeng 6

Transgenic rice Kefeng 6 is a transformation event containing two insect-resistant genes, cry1Ac and SCK (modified CpTI gene) in China. In order to monitor the probable release of Kefeng 6 in the future and execute the labeling requirements, it is necessary to develop a rapid and reliable detection method. In this study, both the 5′ and 3′-junction sequences spanning the plant DNA and the integrated gene construct of the rice event Kefeng 6 were isolated by genome walking and long-distance PCR (LD-PCR), successively. Multiple copies of truncated SCK gene and cry1Ac gene were found to integrate into the host rice genome. The event-specific real-time detection method for Kefeng 6 event based on its 5′-junction sequence was established using one plasmid molecule pMD-KF6 containing both 5′-junction sequence and rice endogenous gene gos9 sequence as the reference material (RM) with an absolute limit of quantification (LOQa) around 10 template copies. Thereafter, three different transgenic amounts of w/w mixed samples (5, 1, and 0.5%, respectively) were quantified to assess the performance characteristics of the established real-time PCR method. The accuracy expressed as bias deviated from the 4.00–26.00%, the precision expressed as standard deviation (SD) and relative standard deviation (RSD) deviated from 0.03–0.19 and 3.42–4.76%, respectively. Based on the earlier results, we concluded that the qualitative and quantitative PCR assays were reliable and accurate for Kefeng 6 measurement, and the reference plasmid pMD-KF6 could be a good substitute for the reference material for Kefeng 6 quantification.     

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.     

Selection of a Taxon-Specific Reference Gene for Qualitative and Quantitative PCR Detection of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis>

Safflower (Carthamus tinctorius) is an emerging model plant for the transgenic modification of fatty acid composition and the production of pharmaceuticals, proteins, or enzymes. Safflower is also a traditional Chinese medicine and is often used as a fake saffron product. Reliable detection of an endogenous reference gene is indispensable for the supervision of genetically modified safflower. Such an endogenous reference gene can also be used to specifically identify safflower ingredient in complex mixtures such as medicine or food. In this study, we identified and validated the CTOS gene as an endogenous reference for safflower. Conventional and real-time polymerase chain reaction (PCR) methods for detecting the CTOS gene sequence showed high interspecies specificity and intra-species stability. The lowest copy number detectable by conventional PCR was 10 haploid copies. The limit of detection and limit of quantification for the real-time PCR assay were estimated to be five and 40 haploid genome copies, respectively. Standard curves established for the real-time PCR assay exhibited good linearity (R ² > 0.99) between the cycle threshold (Ct) values and the initial template copies. The developed conventional and real-time PCR assays were validated in routine analysis of the safflower ingredient in commercial Chinese medicines. In conclusion, the developed quantitative PCR methods were sufficiently specific and sensitive to be used in safflower genomic DNA quantification and safflower ingredient identification.     

A New Multiplexed Real-Time PCR Assay to Detect <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, <Emphasis Type="Italic">C. coli</Emphasis>, <Emphasis Type="Italic">C. lari</Emphasis>, and <Emphasis Type="Italic">C. upsaliensis</Emphasis>

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 10² genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.     

Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products

A 5′ nuclease duplex real-time polymerase chain reaction (PCR) assay was developed and validated with various food products for the specific and fast detection of Salmonella spp. in food. The assay used previously published primers in combination with a newly developed probe targeting the invA gene. An internal amplification control, which is coamplified in a duplex PCR, was included in the assay. The analysis of 1,934 natural food samples with real-time PCR and the cultural method in parallel resulted in a relative accuracy of 100% and 99.84% respectively, depending on the enrichment procedure in which buffered peptone water and selective enrichment in Rappaport–Vassiliadis (RV) broth were employed. The duplex real-time PCR assay has proven to be a specific, sensitive and fast screening method for Salmonella spp. in food. The overall analysis time of the PCR method was approximately 28 h, in contrast to 4 to 5 days with conventional Salmonella diagnostics. The developed assay has been shown to be a reliable diagnostic tool for use in routine analysis. It has been validated thoroughly and has become an official method in Germany for the detection of Salmonella spp. in food.     

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.     

A real-time PCR for Detection of Pathogenic Yersinia enterocolitica in food combined with an Universal Internal Amplification Control System

A real-time PCR system with an internal amplification control was developed for detection of pathogenic Yersinia) enterocolitica in food samples. The chromosomally encoded ail gene was chosen as PCR target. Sequences of plasmid pUC19 served as target for the internal amplification control. The method was validated in combination with sample enrichment in PSB and TSB broth using different food matrices spiked with Y. enterocolitica and naturally contaminated slaughterhouse samples. The results of the real-time PCR with internal control were verified by the cultural method according to EN ISO 10273:2003. The sensitivity of the real-time PCR with internal control is about 5 genome copies per reaction. Artificial contamination of food samples resulted in a detection level of 5 cfu per 25 g Y. enterocolitica in food samples. 100% of porcine tonsils and about 22% meat from pig heads were contaminated. The screening of samples by PCR prior to cultural analysis allows focusing on positive samples in routine analysis. This could result in a higher detection rate by cultural analysis.     

A construct-specific qualitative and quantitative PCR detection method of transgenic maize BVLA430101

Transgenic phytase maize (Zea mays L.) line BVLA430101 was the first transgenic maize obtained the security certification in 2009 in China. However, the construct of the phytase gene expression cassette and the specific detection method have not been reported yet. In this study, the phytase gene expression cassette was identified, which include maize legumin promoter, signal peptide, phytase gene, and maize legumin terminator. The construct-specific qualitative and quantitative PCR methods of BVLA430101 maize were established based on the transition of signal peptide and phytase gene using a maize taxon-specific gene zSSIIb as the endogenous gene. The detection limit for the conventional qualitative PCR was 200 haploid genome copies of BVLA430101. The absolute limit of quantification of the real-time PCR was about 20 haploid genome copies. In addition, two known BVLA430101 contents (5 and 1%) of mixed genomic DNA (V/V) were quantified using the developed real-time PCR detection system, which indicated that the developed quantitative method can be employed reliably for transgenic phytase maize BVLA430101 measurement.     

An event-specific real-time PCR detection system for the transgenic rice line 114-7-2 of producing functional human serum albumin

Transgenic rice 114-7-2 is a newly developed transgenic rice line of producing human serum albumin (HSA). It has attracted much attention because of its economic potential. This paper was designated to discover the integration site of the transgenic HSA rice line 114-7-2 and to establish event-specific methods for qualitative and quantitative detection of the transgenic HSA rice based on the border junction fragment. One gene fragment of 5′ flanking region was successfully isolated using the TAIL-PCR methods. The fragment sequence showed that a 454-bp junction fragment contained 75 bp of T-DNA sequence and 379 bp of rice genome DNA, which is located in chromosome 4. Event-specific real-time PCR method for HSA rice line 114-7-2 was established with the primers (HSA-F/HSA-R) and the probe (HSA-P) targeting the 454-bp junction region. The qualitative PCR assay showed the limit of detection was 0.01 %. In the event-specific quantitative detection method, the LOQ for 114-7-2 HSA rice was estimated to be 0.025 ng or 50 copies. The method developed in this study is highly specific, sensitive, and reliable for transgenic HSA rice sample detection.     

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。     

Detection of genetically modified rice: collaborative validation study of a construct-specific real-time PCR method for detection of transgenic Bt rice

A collaborative trial study has been conducted for validation of an extraction method and a subsequent real-time PCR for detection of a transgenic Bt rice line (‘Bt63’) in rice products originating from China. A total of 17 laboratories participated in the study and each laboratory received 16 coded samples comprising of rice grain flours, rice noodle flours and plasmid DNAs. Of the accepted results all Bt63-positive rice grain samples (0.1 or 0.05% w/w) and all rice noodle samples prepared from marketed rice products were detected correctly. The result demonstrates that ‘Bt63’ rice is detectable even at low relative mass concentrations of 0.05%. The absolute LOD determined with plasmid DNA samples showed to be at least five copies of the ‘Bt63’ target sequence. The data provided in this study show that the method is fit-for-purpose to inspect Chinese rice products for the presence of EU-unauthorised rice lines carrying the ‘Bt63’ construct.     

Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction

Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the C _q (quantification cycle) from 10 ng/μl porcine DNA is (18.70 ± 0.12 to 19.08 ± 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r ² = 0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork–beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification.     

Effects of processing on detection and quantification of the parvalbumin gene in Atlantic salmon (Salmo salar)

Consumption of Atlantic salmon is a common cause of fish allergies with parvalbumin (Sal s1) being the major allergen. The presence of DNA encoding Sal s1 indicates the presence of Atlantic salmon in food. Using real-time polymerase chain reaction (PCR), the effects of food processing on the ability to detect and quantify the Sal s1 gene were determined. The method was specific for salmon and did not cross-react with 53 other species. Baking and pressure cooking caused a 5–100-fold decrease in detectable copies of the Sal s1 gene. Despite a 98% reduction in detectable copies following pressure cooking for 60 min, the relative standard deviation (RSD) between replicates was 20% and the response was 100-fold grater than the lowest copy number of Sal s1 reliably detected by the assay. Despite efforts to develop a quantitative assay, the PCR assay was qualitative. It is impossible to predict the effects of food matrices not included in this study, some of which may affect the reliability of the assay. Analyses of raw and pressure cooked salmon using a commercial PCR kit indicated comparable results to the PCR assay.     

Application of magnetic immuno-polymerase chain reaction assay for detection of <Emphasis Type="Italic">Salmonella</Emphasis> spp. in chicken meats

Since contaminated chicken meats have been the principal foodborne source of the contamination of Salmonella to human beings and cultural detection methods are labor-intensive and time-consuming, a study evaluating the performance of the combination of two techniques that are immunomagnetic separation (IMS) and polymerase chain reaction (PCR) for the detection of Salmonella in chicken meats was conducted. The IMS and PCR assay combines selective extraction of Salmonella by specific antibodies with primer-specific (primer pair based on the sequence of invA gene) PCR amplification. Initially chicken meat samples, in which no Salmonella contamination had been determined by using ISO 6579 reference method, were inoculated with Salmonella Enteritidis culture and subsequently the shortest non-selective pre-enrichment time, that had been needed for the detection of approximately 1 or 10 CFU/mL chicken meat levels of target bacteria by magnetic immuno-PCR assay, was found by using 14, 12, 10 and 8-h periods. In conclusion, it was found that magnetic immuno-PCR assay was able to detect 1–10 CFU Salmonella/25 g chicken meat, after only incorporating a non-selective pre-enrichment period of 12 h. Therefore, an overall 16-h (magnetic immuno-PCR assay in conjunction with 12-h non-selective pre-enrichment) magnetic immuno-PCR assay statistically evaluated as sufficient (p = 0.182 > 0.05) for rapid and sensitive detection of approximately 1–10 CFU Salmonella from 25 g chicken meat samples. Accordingly, 16-h magnetic immuno-PCR assay can be promising for routine use in the detection of Salmonella in chicken meat samples, and it consequently may prevent the risk of Salmonella infections in regard to chicken meats.     

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.     

Collaborative trial validation of a construct-specific real-time PCR method for detection of genetically modified linseed event ‘CDC Triffid’ FP967

A real-time PCR-based method for construct-specific detection of the genetically modified (GM) linseed event ’CDC Triffid’ FP967 originating from Canada has been validated in a collaborative trial. The construct-specific method amplifies a 105 bp long fragment of the transgenic insertion present in FP967 spanning the junction of the terminator region of the nopalin synthase gene from Agrobacterium tumefaciens (Tnos) to a sequence region coding for the dehydrofolate reductase gene (dfr) from a class I integron from Escherichia coli. This region is characteristic for the construct used to develop FP967. A total of 11 laboratories participated in the collaborative study. For PCR analysis, each laboratory received 14 DNA samples comprising 7 double-blind DNA samples. The samples consisted of two low GM-levels of FP967 DNA (10 or 50 copies per PCR), of DNA from two different GM-positive linseed products and of DNA from GM-negative linseed, potato and rapeseed materials, respectively. All but one of the FP967-positive DNA samples were detected correctly. No false-positive results were reported. The results demonstrate that the linseed event FP967 is detectable even at low copy number concentrations. The limit of detection (LOD) determined with plasmid DNA was shown to be at 5 copies of the Tnos–dfr sequence. The data provided show that the method can be applied successfully in different laboratories and is fit-for-purpose to test for the presence of the EU-unauthorised linseed event ‘CDC Triffid’ FP967.     

Determination of eight genetically modified maize events by quantitative, multiplex PCR and fluorescence capillary gel electrophoresis

Specific legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. This has necessitated the development of methods for detection and quantification of such materials. Here we present a robust, quantitative, 9-plex PCR method for event-specific detection of maize TC1507, MON863, MON810, T25, NK603, GA21, construct specific detection of BT11, BT176 and detection of the endogenous hmga maize reference gene. The method is suitable for quantification in the 0–2% range with a detection limit of approximately 0.1%. PCR is carried out in two stages. In the first stage, bipartite primers containing a universal 5′-sequence and a GMO specific 3′-sequence are used. In the second PCR stage only a universal primer is used. Trypsin digestion between the first and second PCR stages enhances signal strength and reproducibility. Probes hybridising to the PCR amplicons are then labelled by primer extension and detected by fluorescence capillary electrophoresis. Good agreement was observed in 76 of 80 determinations when 10 food and feed samples were analysed using the multiplex PCR assay and compared to results from quantitative real-time 5′-nuclease PCR. The presented method is therefore suitable for quantification purposes for food and feed containing the most common maize GMOs.     

转基因水稻潮霉素标记基因PCR检测方法的建立及其在基因水平转移研究中的应用

为建立用于基因水平转移研究, 尤其是DNA经加工和消化后稳定性研究的针对转基因水稻潮霉素标记基因hpt（hygromycin phosphotransferase）的定性和实时定量PCR体系,设计针对hpt的上游通用引物多个片段定性PCR扩增体系,以植物叶绿体基因rbcl为内对照,PCR扩增产物经测序验证.将定性PCR中最小片段（236 bp）连接到质粒载体pUC18-pMD T载体上,提取质粒经验证后做外标.应用TaqMan-MGB荧光探针和引物,建立定量的外标校正曲线法,并评价方法的精密度.建立的定性PCR体系能稳定扩增出236 bp～910 bp不同大小的5个hpt片段,并经测序验证.实时定量PCR的线性范围为105～10拷贝（R^2=0.998）,最低能检出10拷贝,重复性好.本研究已成功建立了用于转基因水稻标记基因hpt基因水平转移研究的定性和定量PCR系统。     

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。     

Real-Time Recombinase Polymerase Amplification Assay for the Detection of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> in Seafood

Vibrio cholerae, the most hazardous Vibrio species, is threatening aquacultured animals and even human beings. In recent years, a few genetic markers that target V. cholerae have been reported, but their application was limited by techniques or themselves. The outer membrane lipoprotein gene lolB has been identified as a specific marker by using PCR and quantitative PCR methods. In this study, we developed a real-time recombinase polymerase amplification (RPA) assay targeting lolB gene in an attempt to provide a sensitive, rapid, and reliable detection method of V. cholerae. The sensitivity of RPA assay was determined by amplifying the standard plasmid dilutions. The detection limit down to five copies was achieved within 10 min, which was lower than that of qPCR (ten copies after 25 cycles within 1.5 h). The reproducibility of RPA assay was verified by eight independent experiments, presenting a high R ² value of the standard regression line (0.97). In addition, the specificity was confirmed with DNA extracted from other bacteria, shrimps, clams, and fishes using both methods. Finally, V. cholerae in shrimp samples were quantified using real-time RPA and qPCR with consistent outcomes, while no amplification was obtained from clam and fish samples using both methods. The applicability in the food industry was confirmed by quantitative detection of natural seafood.