An empirical approach to target DNA quantification in environmental samples using real-time polymerase chain reactions

Recent advances in molecular techniques have allowed for the routine examination of nucleic acids in environmental samples. Although current methodologies are very sensitive, accurate target DNA quantification from environmental samples remains challenging. To facilitate high-throughput DNA quantification from environmental samples, we developed a novel DNA quantification method based on a non-linear curve-fitting approach to extract additional information from quantitative PCR amplification curves and used the fitted parameters to develop multiple regression standard equations for target DNA quantification. A 3-parameter sigmoidal function performed superior to a 4-parameter Weibull function for generating the multiple regression standard equations. In a verification experiment, target DNA was quantified in a series of ‘unknown’ samples in three soils using this approach and the results were compared to target DNA values determined using corrected and uncorrected Ct-based (threshold cycle) methods. For each method, the deviations from the expected target DNA content were determined. Results clearly showed that over all DNA concentrations, target DNA content determined by the non-linear curve-fitting method was more accurate and more precise than values predicted by all other methods. Analysis of variance conducted on the predicted DNA contents also revealed fewer statistical artifacts with the non-linear curve fitting method compared to the conventional Ct-based methods. The novel approach described here is accurate, inexpensive, and very amenable for automation and high-throughput applications.     

Application of a real time polymerase chain reaction method to detect castor toxin contamination in fluid milk and eggs

The castor seed contains ricin, which is one of the most potent biological toxins and is widely considered to be a threat agent for bioterrorism. In this study, a rapid and sensitive PCR method was applied to the detection of castor contamination in milk and liquid egg samples. The targeting gene sequence of the primer set, Ricin-F4/R4, was not found in either the bovine or chicken genome. Primers against a highly conserved sequence from the 18S ribosomal RNA gene were used as a positive control for DNA extraction and PCR reaction efficiency. The quantity and quality of DNA prepared from castor spiked or nonspiked milk and egg samples obtained from three different DNA extraction methods were compared. The cetyl trimethylammonium bromide (CTAB) method yielded the highest quality of DNA and is most suitable for the sensitive detection of castor DNA by real-time PCR in both milk and liquid egg matrixes. However, taking time and cost into consideration, a commercial kit designed for extraction of DNA from stool samples could be used as an alternative method for the routine extraction of DNA from milk for real-time PCR assays. The egg matrix was found to inhibit PCR amplification and interfere with two of the three methods tested for DNA extraction. Egg yolk had a greater negative effect on PCR amplification than the egg white matrix. Our results affirm the necessity of performing individual validations for each food matrix. Both real-time PCR systems used in this study, TaqMan and SYBR Green I dye, were capable of detecting 100 ng of castor acetone powder, corresponding to 5 ng of ricin, in 1 mL of milk or liquid egg, well below the toxic dose for humans. On the basis of these results, the real-time PCR method for detection of intentional castor contamination is applicable to milk and egg matrixes.     

Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction

Transgenic soybean line GTS-40-3-2, marketed under the trade name Roundup Ready (RR) soy, was developed by Monsanto (USA) to allow for the use of glyphosate, the active ingredient of the herbicide Roundup, as a weed control agent. RR soy was first approved in Canada for environmental release and for feed products in 1995 and later for food products in 1996 and is widely grown in Canada. Consumer concern issues have resulted in proposed labeling regulations in Canada for foods derived from genetically engineered crops. One requirement for labeling is the ability to detect and accurately quantify the amount of transgenic material present in foods. Two assays were evaluated. A conventional qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of soy and RR soy and a real-time PCR to quantify the amount of RR soy present in samples that tested positive in the first assay. PCR controls consisted of certified RR soy reference material, single transgenic soybeans, and a processed food sample containing a known amount of RR soy. To test real-world applicability, a number of common grocery store food items that contain soy-based products were tested. For some samples, significant differences in amplification efficiencies during the quantitative PCR assays were observed compared to the controls, resulting in potentially large errors in quantification. A correction factor was used to try to compensate for these differences.     

Application of relative quantification TaqMan real-time polymerase chain reaction technology for the identification and quantification of Thunnus alalunga and Thunnus albacares

A novel one-step methodology based on real-time Polymerase Chain Reaction (PCR) technology has been developed for the identification of two of the most valuable tuna species. Nowadays, species identification of seafood products has a major concern due to the importing to Europe of new species from other countries. To achieve this aim, two specific TaqMan systems were devised to identify Thunnus alalunga and Thunnus albacares. Another system specific to Scombroidei species was devised as a consensus system. In addition, a relative quantification methodology was carried out to quantify T. alalunga and T. albacares in mixtures after the relative amount of the target was compared with the consensus. This relative quantification methodology does not require a known amount of standard, allowing the analysis of many more samples together and saving costs and time. The utilization of real-time PCR does not require sample handling, preventing contamination and resulting in much faster and higher throughput results.     

Specific detection of potentially allergenic kiwifruit in foods using polymerase chain reaction

Kiwifruit (Actinidia deliciosa and Actinidia chinensis) is allergenic to sensitive patients, and, under Japanese regulations, it is one of the food items that are recommended to be declared on food labeling as much as possible. To develop PCR-based methods for the detection of trace amounts of kiwifruit in foods, two primer pairs targeting the ITS-1 region of the Actinidia spp. were designed using PCR simulation software. On the basis of the known distribution of a major kiwifruit allergen (actinidin) within the Actinidia spp., as well as of reports on clinical and immunological cross-reactivities, one of the primer pairs was designed to detect all Actinidia spp. and the other to detect commercially grown Actinidia spp. (i.e., kiwifruit, Actinidia arguta, and their interspecific hybrids) except for Actinidia polygama. The specificity of the developed methods using the designed primer pairs was verified by performing PCR experiments on 8 Actinidia spp. and 26 other plants including fruits. The methods were considered to be specific enough to yield target-size products only from the target Actinidia spp. and to detect no target-size products from nontarget species. The methods were sensitive enough to detect 5-50 fg of Actinidia spp. DNA spiked in 50 ng of salmon testis DNA used as a carrier (1-10 ppm of kiwifruit DNA) and 1700 ppm (w/w) of fresh kiwifruit puree spiked in a commercial plain yogurt (corresponding to ca. 10 ppm of kiwifruit protein). These methods would be expected to be useful in the detection of hidden kiwifruit and its related species in processed foods.     

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.     

Quantification of bovine milk oligosaccharides using liquid chromatography-selected reaction monitoring-mass spectrometry

Oligosaccharides are important components of milk with bioefficacy as prebiotics, anti-infectives, and immune system modulators and as a possible source of sialic acid for neural function. Bovine milk oligosaccharides are lower in concentration and lack the diversity of human milk oligosaccharides but could be a commercial source of milk oligosaccharides for pediatric foods. For this development, an ability to quantify the oligosaccharides is required. This study validated a hydrophilic interaction chromatography high-performance liquid chromatography-high-resolution selected reaction monitoring-mass spectrometry (HILIC HPLC-HRSRM-MS) method for measuring six different oligosaccharides in bovine milk, bovine colostrum, and infant formulas. The extraction resulted in a high recovery (90-103%) with a repeatability coefficient of variation ranging from 2 to 9% for the two dominant oligosaccharides, 3'-sialyllactose and 6'-sialyllactose, and ranging from 1 to 17% for the much lower concentration oligosaccharides, 6'-sialyllactosamine, disialyllactose, and N-acetylgalactosaminyllactose. The sixth oligosaccharide, 3'-sialyllactosamine, was not detected in any of the samples.     

Real-time polymerase chain reaction quantification of the transgenes for roundup ready corn and roundup ready soybean in soil samples

A method for quantification of recombinant DNA for Roundup Ready (RR) corn and RR soybean in soil samples is described. Soil DNA from experimental field samples was extracted using a soil DNA extraction kit with a modified protocol. For the detection and quantification of recombinant DNA of RR corn and RR soybean, a molecular beacon and two pairs of specific primers were designed to differentially target recombinant DNA in these two genetically modified crops. Soil DNA extracts were spiked with RR corn or RR soybean DNA, and recombinant DNA was quantified using real-time PCR with a molecular beacon. As few as one copy of RR corn genome or one copy of RR soybean genome was detected in the soil DNA extract.     

Development of a screening method for genetically modified soybean by plasmid-based quantitative competitive polymerase chain reaction

A novel type of quantitative competitive polymerase chain reaction (QC-PCR) system for the detection and quantification of the Roundup Ready soybean (RRS) was developed. This system was designed based on the advantage of a fully validated real-time PCR method used for the quantification of RRS in Japan. A plasmid was constructed as a competitor plasmid for the detection and quantification of genetically modified soy, RRS. The plasmid contained the construct-specific sequence of RRS and the taxon-specific sequence of lectin1 (Le1), and both had 21 bp oligonucleotide insertion in the sequences. The plasmid DNA was used as a reference molecule instead of ground seeds, which enabled us to precisely and stably adjust the copy number of targets. The present study demonstrated that the novel plasmid-based QC-PCR method could be a simple and feasible alternative to the real-time PCR method used for the quantification of genetically modified organism contents.     

Specific detection of potentially allergenic peach and apple in foods using polymerase chain reaction

Two PCR methods were developed for specific detection of the trnS-trnG intergenic spacer region of Prunus persica (peach) and the internal transcribed spacer region of Malus domestica (apple). The peach PCR amplified a target-size product from the DNA of 6 P. persica cultivars including 2 nectarine and 1 flat peach cultivar, but not from those of 36 nontarget species including 6 Prunus and 5 other Rosaceae species. The apple PCR amplified a target-size product from the DNA of 5 M. domestica cultivars, but not from those of 41 nontarget species including 7 Maloideae and 9 other Rosaceae species. Both methods detected the target DNA from strawberry jam and cookies spiked with peach and apple at a level equivalent to about 10 μg of total soluble proteins of peach or apple per gram of incurred food. The specificity and sensitivity were considered to be sufficient for the detection of trace amounts of peach or apple contamination in processed foods.     

Differential detection of shrimp and crab for food labeling using polymerase chain reaction

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.     

Novel reference gene, PKABA1, used in a duplex real-time polymerase chain reaction for detection and quantitation of wheat- and barley-derived DNA

We report the development of a duplex real-time Polymerase Chain Reaction (PCR) for the simultaneous detection and quantification of wheat- and barley-derived DNA. We used a single primer pair to amplify the single-copy gene PKABA1 from wheat and barley, using minor-groove-binding probes to distinguish between the two cereals. The assay was fully specific, and different wheat and barley cultivars exhibited similar Ct values, indicating stability across cultivars with respect to allelic and copy number composition. The limits of detection were 5 and 10 PCR-forming units for wheat and barley, respectively, making the duplex assay as sensitive as other singleplex reference gene systems published. We were able to detect both wheat and barley simultaneously in real food samples, and the duplex assay is considered to be suitable as an endogenous reference gene system for the detection and quantification of wheat and barley in genetically modified organisms (GMO) and other food and feed analyses.     

Development and comparison of four real-time polymerase chain reaction systems for specific detection and quantification of Zea mays L

Four real-time polymerase chain reaction systems aiming at the specific detection and quantification of maize DNA are described. They have been developed in four independent laboratories targeting different maize sequences, i.e., alcohol dehydrogenase (Adh1), high mobility group protein (hmga), invertase A (ivr1), and zein, respectively. They were all fully specific, showing a very similar quantification accuracy along a number of distantly related maize cultivars and being either single or low copy number genes. They were highly sensitive and exhibited limits of quantification below 100 maize genomic copies. In consequence, they are considered suitable for use as maize specific endogenous reference genes in DNA analyses, including GMO quantitative tests.     

Detection of genetically modified coho salmon using polymerase chain reaction (PCR) amplification

A PCR-based protocol for the identification of genetically modified salmon carrying a growth hormone transgene was developed. Several primer pairs were examined, and the primers that gave consistent results were selected to conduct routine testing. Comparison among several DNA extraction procedures, as well as different buffer compositions, led to the adoption of TriZol as the method of choice. Low potassium and high magnesium chloride concentrations were very important in the overall success of the PCR reaction, whereas buffer pH, ranging from 8.3 to 9.2, had little impact on the amplification reaction. The optimal primer annealing temperature was 52 degrees C. Although fish muscle tissues were the primary source for DNA samples, detection of the transgene was also possible in bones, skin, fins, and other organs. No benefits were achieved by the addition of additives such as dimethyl sulfoxide and betaine to the PCR reaction. This optimized PCR method was used to identify all samples tested (61 samples and 17 controls) with 100% accuracy.     

RT-PCR检测猪瘟病毒初探

本研究建立了反转录聚合酶链反应 ( RT PCR)技术检测猪瘟病毒 ( Hog Cholera Virus,HCV)方法。合成的二对引物 HCV 1 / HCV 2和 HCV A1 / HCV A2成功地扩增兔化弱毒株、法国株和石门株的核酸 ,并对采自博白县 3个、贵港市 1个和武宣县 1个猪场共 1 3份病料进行检测。结果从博白县和贵港市的 1 1份材料中检测到 HCV的核酸。     

Development of a multiplex polymerase chain reaction method for simultaneous detection of eight events of genetically modified maize

In this study, we developed a novel multiplex polymerase chain reaction (PCR) method for simultaneous detection of up to eight events of genetically modified (GM) maize within a single reaction. The eight detection primer pairs designed to be construct specific for eight respective GM events (i.e., Bt11, Event176, GA21, MON810, MON863, NK603, T25, and TC1507) and a primer pair for an endogenous reference gene, ssIIb, were included in the nonaplex(9plex) PCR system, and its amplified products could be distinguished by agarose gel and capillary electrophoreses based on their different lengths. The optimal condition enabled us to reliably amplify two fragments corresponding to a construct specific sequence and a taxon specific ssIIb in each of the eight events of GM maize and all of nine fragments in a simulated GM mixture containing as little as 0.25% (w/w) each of eight events of GM maize. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM maize.     

Real-time TaqMan polymerase chain reaction detection and quantification of cow DNA in pure water buffalo mozzarella cheese: method validation and its application on commercial samples

Mozzarella cheese obtained from buffalo (Bubalus bubalis) milk is a typical Italian product certificated by means of the European Protected Designation of Origin (PDO). Mozzarella cheese can also be obtained from bovine milk or bovine/buffalo milk mixtures, but in this case, it cannot be sold as PDO product, and its label must report the actual ingredients. However, bovine milk in PDO products was frequently detected in the past, suggesting fraudulent addition or accidental contamination. Several methods based on end-point polymerase chain reaction (PCR) have been profitably applied in a large number of tests to detect the presence of undeclared ingredients, also in dairy products. In the present study we report a real-time PCR method able to quantify bovine milk addition to pure buffalo cheese products. We validated a normalized procedure based on two targets: bovine mitochondrial cytochrome b (cyt b) to detect and quantify the bovine DNA and nuclear growth hormone (GH) gene used as a universal reference marker. With the use of this real-time PCR assay, 64 commercial mozzarella di bufala cheese samples purchased at local supermarkets, dairy shops, or directly from cheese manufacturers were analyzed. The results obtained demonstrate that most of the commercial samples were contaminated with bovine milk. Therefore, this assay could be conveniently employed to carry out routine and accurate controls aimed not only to discourage any fraudulent behavior but also to reduce risks for consumer health.     

Interlaboratory evaluation of a real-time multiplex polymerase chain reaction method for identification of salmon and trout species in commercial products

This interlaboratory study evaluated a real-time multiplex polymerase chain reaction (PCR) method for identification of salmon and trout species in a range of commercial products in North America. Eighty salmon and trout products were tested with this method by three independent laboratories. Samples were collected in the United States and Canada, and only the collecting institution was aware of the species declaration. Following analysis with real-time PCR, all three laboratories were able to identify species in 79 of the 80 products, with 100% agreement on species assignment. A low level of fraud was detected, with only four products (5%) found to be substituted or mixtures of two species. The results for two of the fraudulent products were confirmed with alternate methods, but the other two products were heavily processed and could not be verified with methods other than real-time PCR. Overall, the results of this study show the usefulness and versatility of this real-time PCR method for the identification of commercial salmon and trout species.     

New method of DNA isolation from two food additives suitable for authentication in polymerase chain reaction assays

Locust bean gum and guar gum are galactomannans used as additives (E 410 and E 412, respectively) in the food industry as stabilizing agents. Analytical discrimination between the two additives in gums and foods is now feasible by molecular techniques. However, only complex and time-consuming DNA isolation protocols are available to date. We have developed simple improved protocols to obtain enough DNA suitable for PCR amplification from a few milligrams of commercial E 410 and E 412 additives (containing more than 75% polysaccharides). The suspension of additives in water or 10 mM Tris-HCl, pH 8.5, efficiently recovers DNA suitable for authentication in PCR assays. However, the Tris method was much more efficient for the extraction of DNA from E 410 than for E 412 additives. Conversely, the water method was the most suitable for detecting DNA extracted from E 412 or from E 410/E 412 mixtures. Combined with the use of the two specific ribosomal primer pairs previously designed, our methods are well-suited for a fast and simple high-throughput sample treatment of commercial gums for molecular certification.