Development and Performance Evaluation of a New Test Kit for Quantifying the Degree of DNA Fragmentation.

BACKGROUND: PCR-based genetic testing of agricultural products and foods is widely used for detecting various analytical targets such as genetically modified organisms and food allergens. However, it is difficult to obtain accurate genetic testing results from processed foods because DNA is fragmented by heat and pressure during food processing. Thus we previously developed an analytical method to quantitatively evaluate the degree of DNA fragmentation for the purpose of quality control of genetic testing for processed foods. OBJECTIVE: Our previous analytical method requires four PCR primer sets, resulting in high reagent costs and heavy analytical workloads. Therefore, we attempted to develop an easy-to-use test kit for quantifying the degree of DNA fragmentation and to evaluate its analytical performance. METHODS: To simplify the analysis procedure, we used only two primer sets. In addition, no-fragmentation control templates were prepared to obtain stable measurement results. The precision of the simplified analysis was evaluated through blind tests among laboratories. RESULTS: It was confirmed that plant species and extracted DNA concentrations had little effect on analysis with the newly developed test kit. In addition, the analytical values indicating the degree of DNA fragmentation exhibited small variability among laboratories. CONCLUSION: We confirmed the high practicality of the developed test kit. Because DNA fragmentation in cells is a universal phenomenon, we anticipate that the test kit will be used not only for quality control of genetic testing but also for food testing, medical diagnostics and other applications in a range of fields. HIGHLIGHTS: The newly developed test kit enables quantitative evaluation of the degree of DNA fragmentation in a simple manner.

Rapid Screening Detection of Genetically Modified Papaya by Loop-Mediated Isothermal Amplification.

A loop-mediated isothermal amplification (LAMP)-mediated screening detection method for genetically modified (GM) papaya was developed targeting the 35S promoter (P35S) of the cauliflower mosaic virus. LAMP products were detected using a Genie II real-time fluorometer. The limit of detection (LOD) was evaluated and found to be ≤0.05% for papaya seeds. We also designed a primer set for the detection of the papaya endogenous reference sequence, chymopapain, and the species-specificity was confirmed. To improve cost-effectiveness, single-stranded tag hybridization (STH) on a chromatography printed-array strip (C-PAS) system, which is a lateral flow DNA chromatography technology, was applied. LAMP amplification was clearly detected by the system at the LOD level, and a duplex detection of P35S and chymopapain was successfully applied. This simple and quick method for the screening of GM papaya will be useful for the prevention of environmental contamination of unauthorized GM crops.

[Evaluation of a Species-specific, Stable, and Endogenous Sequence of Eggplant (Solanum melongena) using LAMP for the Detection of Genetically Modified Eggplants].

Species-specific endogenous reference sequences are indispensable in the development of methods to detect genetically modified (GM) crops for food and feed. We analyzed a partial sequence derived from the ß-fructosidase gene among several solanaceous species and developed a new eggplant specific detection method using loop-mediated isothermal amplification (LAMP). LAMP is a rapid, specific, and cost-effective technique. The species-specificity and stability of the developed method were evaluated using 18 eggplant cultivars and other crops including solanaceous plants. The limit of detection was also evaluated. The developed method showed high specificity for eggplants and stability among the eggplant cultivars tested. These results suggested that the developed method would be useful as a positive control for the detection of GM eggplants with LAMP.

Development of a Novel Detection Method Targeting an Ultrashort 25 bp Sequence Found in Agrobacterium-Mediated Transformed GM Plants.

Agrobacterium-mediated transformation is the most commonly used technique for plant genetic engineering. During the transformation, a T-DNA region, which is flanked by the right border (RB) and the left border, is transferred to plant nuclear chromosomes. Simultaneously, a sequence adjacent to the RB on T-DNA is frequently transferred to plant genomes together with the intentionally introduced recombinant DNA. We developed a novel polymerase chain reaction (PCR)-mediated detection method targeting this region. The conserved sequence of the region found in genetically modified (GM) crops is only 25 bp in length. To detect this ultrashort 25 bp sequence near the RB region, we designed a primer set consisting of a 12-base forward primer and a 13-base reverse primer. The predicted band was detected from GM crops by optimizing the PCR conditions. We used lateral flow DNA chromatography for rapid and inexpensive detection. The developed method would be applicable for screening the GM crops generated by Agrobacterium-mediated transformation.

Diverged and Active Partitiviruses in Lichen.

The lichen is a microbial consortium that mainly consists of fungi and either algae (Viridiplantae) or cyanobacteria. This structure also contains other bacteria, fungi, and viruses. However, RNA virus diversity associated with lichens is still unknown. Here, we analyzed RNA virus diversity in a lichen dominated by fungi and algae using dsRNA-seq technology and revealed that partitiviruses were dominant and active in the microbial consortium. The Partitiviridae sequences found in this study were classified into two genera, which have both plant- and fungi-infecting partitiviruses. This observation suggests that the lichen provides an opportunity for horizontal transfer of these partitiviruses among microbes that form the lichen consortium.

A Novel Real-Time PCR-Based Screening Test with Pooled Fecal Samples for Bovine Johne's Disease.

Johne's disease (JD) is an economically important infectious disease in livestock farming caused by Mycobacterium avium subsp. paratuberculosis As an alternative to serological tests, which are used mainly for the screening of whole herds, we developed a novel ResoLight-based real-time PCR (RL-PCR) assay with pooled fecal samples for the detection of fecal shedders in cattle herds. The RL-PCR assay included an internal amplification control (IC) which was amplified using the same primer pair as the target molecule M. avium subsp. paratuberculosis IS900 and differentiated based on melting temperatures. Individual fecal suspensions were pooled and concentrated by centrifugation to avoid a loss of sensitivity by the dilution effect. Combined with a DNA extraction kit (Johne-PureSpin; FASMAC), no inhibition of PCR amplification was observed with up to 15 fecal samples in a pool. The detection limit of RL-PCR at a pool size of 10 was 10 M. avium subsp. paratuberculosis organisms per gram of feces, which was comparable to that of individual testing. A total of 2,654 animals in 12 infected herds were screened by individual antibody-enzyme-linked immunosorbent assay (ELISA) and the RL-PCR assay using pooled feces. Fifty animals were diagnosed with JD through the screening by RL-PCR, compared with only 5 by ELISA (which were also positive in RL-PCR). In 7 JD-free herds, the results of 4 out of 327 pools (1.2%) were invalid due to the lack of IC amplification, and then animals were confirmed negative individually. Our results suggest that implementation of herd screening by pooled RL-PCR would advance the monitoring and control of JD in cattle herds.

Real-time PCR Detection Methods for Food Allergens (Wheat, Buckwheat, and Peanuts) Using Reference Plasmids.

Specific and sensitive real-time qualitative polymerase chain reaction (PCR) methods for the detection of food allergens including wheat, buckwheat, and peanuts were developed that could cancel between instrument effects and avoid risks of false-positives and false-negatives. In these real-time PCR analysis, the cutoff for determination of positive samples was set in every PCR run by using reference plasmids containing known copies of the target sequences. The copy numbers of the plasmids were used to detect the allergenic ingredients corresponding to 10 ppm (w/w) protein in highly processed foods (cooked for more than 30 min at 122 °C). Reference plasmid analysis for each real-time PCR run helped to minimize variability between runs and instruments (7900HT Real-Time PCR systems and Light Cycler Nano). It also helped to avoid false positives due to trace levels of contaminants from the laboratory environment or agricultural products. The specificity of the real-time PCR method was verified using 79 commonly used food materials and some of their relatives. The method was sensitive enough to detect those allergenic ingredients corresponding to 10 ppm (w/w) in seven types of incurred samples. The current official Japanese method was not able to detect the allergens in some of the incurred samples. The developed method can avoid false negatives due to lack of sensitivity and is useful to confirm positive ELISA screening tests.

Rapid Screening Detection of Genetically Modified Crops by Loop-Mediated Isothermal Amplification with a Lateral Flow Dipstick.

We developed a novel loop-mediated isothermal amplification (LAMP)-based detection method using lateral flow dipstick chromatography for genetically modified (GM) soybean and maize events. The single-stranded tag hybridization (STH) for the chromatography printed-array strip (C-PAS) system was used for detections targeting the cauliflower mosaic virus 35S promoter, mannose-6-phosphate isomerase gene, Pisum sativum ribulose 1, 5-bisphosphate carboxylase terminator, a common sequence between the Cry1Ab and Cry1Ac genes, and a GA21-specific sequence. The STH C-PAS system was applicable for multiplex analyses to perform simultaneous detections. The limit of detection was 0.5% or less for each target. By using the developed method, the LAMP amplification was visually detected. Moreover, the detection could be carried out without any expensive instruments, even for the DNA amplification steps, by virtue of the isothermal reaction. We demonstrated that the rapid and useful method developed here would be applicable for screening GM crops.

Development and evaluation of rapid screening detection methods for genetically modified crops using loop-mediated isothermal amplification.

We developed new loop-mediated isothermal amplification (LAMP)-based detection methods for the screening of genetically modified (GM) maize and soybean events. The LAMP methods developed targeted seven sequences: cauliflower mosaic virus 35S promoter; 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (cp4epsps); phosphinothricin acetyltransferase (pat) gene; mannose-6-phosphate isomerase gene; Pisum sativum ribulose 1, 5-bisphosphate carboxylase terminator; a common sequence between Cry1Ab and Cry1Ac genes; and a GA21 construct-specific sequence. We designed new specific primer sets for each target, and the limit of detection (LOD) was evaluated using authorized GM maize and soybean events. LODs for each target were ≤ 0.5%. To make the DNA extraction process simple and rapid, we also developed a direct LAMP detection scheme using crude cell lysates. The entire process, including pretreatments and detection, could be completed within 1 h.

Development and Evaluation of Event-Specific Quantitative PCR Method for Genetically Modified Soybean MON87701.

A real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event, MON87701. First, a standard plasmid for MON87701 quantification was constructed. The conversion factor (Cf) required to calculate the amount of genetically modified organism (GMO) was experimentally determined for a real-time PCR instrument. The determined Cf for the real-time PCR instrument was 1.24. For the evaluation of the developed method, a blind test was carried out in an inter-laboratory trial. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr), respectively. The determined biases and the RSDr values were less than 30 and 13%, respectively, at all evaluated concentrations. The limit of quantitation of the method was 0.5%, and the developed method would thus be applicable for practical analyses for the detection and quantification of MON87701.

The Development of a Novel, Validated, Rapid and Simple Method for the Detection of Sarcocystis fayeri in Horse Meat in the Sanitary Control Setting.

Sarcocystis fayeri (S. fayeri) is a newly identified causative agent of foodborne disease that is associated with the consumption of raw horse meat. The testing methods prescribed by the Ministry of Health, Labour and Welfare of Japan are time consuming and require the use of expensive equipment and a high level of technical expertise. Accordingly, these methods are not suitable for use in the routine sanitary control setting to prevent outbreaks of foodborne disease. In order to solve these problems, we have developed a new, rapid and simple testing method using LAMP, which takes only 1 hour to perform and which does not involve the use of any expensive equipment or expert techniques. For the validation of this method, an inter-laboratory study was performed among 5 institutes using 10 samples infected with various concentrations of S. fayeri. The results of the inter-laboratory study demonstrated that our LAMP method could detect S. fayeri at concentrations greater than 10(4) copies/g. Thus, this new method could be useful in screening for S. fayeri as a routine sanitary control procedure.

Inter-Laboratory Study to Validate New Rapid Screening Methods for Kudoa septempunctata.

Kudoa septempunctata is the causative agent of a food-borne disease associated with the ingestion of raw olive flounder. As the current qRT-PCR method for its detection is time-consuming, a rapid and simple method is required. Recently, a new real-time loop-mediated isothermal amplification (LAMP) method and an immunochromatography method, whose sensitivities are intended to be compatible with that designated in the official analytical method (10(5) spores/g olive flounder), have been developed. To validate these new methods, we performed an inter-laboratory study across seven laboratories. Both methods could not detect less than 10(4) spores/g; however, these methods were able to detect more than 10(5) spores/g in olive flounder samples. These results demonstrated that the sensitivities of these methods were compatible with the designated level in the official analytical method. We concluded that these new methods were acceptable as the screening methods for K. septempunctata.

Selection of Suitable DNA Extraction Methods for Genetically Modified Maize 3272, and Development and Evaluation of an Event-Specific Quantitative PCR Method for 3272.

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) maize, 3272. We first attempted to obtain genome DNA from this maize using a DNeasy Plant Maxi kit and a DNeasy Plant Mini kit, which have been widely utilized in our previous studies, but DNA extraction yields from 3272 were markedly lower than those from non-GM maize seeds. However, lowering of DNA extraction yields was not observed with GM quicker or Genomic-tip 20/G. We chose GM quicker for evaluation of the quantitative method. We prepared a standard plasmid for 3272 quantification. The conversion factor (Cf), which is required to calculate the amount of a genetically modified organism (GMO), was experimentally determined for two real-time PCR instruments, the Applied Biosystems 7900HT (the ABI 7900) and the Applied Biosystems 7500 (the ABI7500). The determined Cf values were 0.60 and 0.59 for the ABI 7900 and the ABI 7500, respectively. To evaluate the developed method, a blind test was conducted as part of an interlaboratory study. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSDr). The determined values were similar to those in our previous validation studies. The limit of quantitation for the method was estimated to be 0.5% or less, and we concluded that the developed method would be suitable and practical for detection and quantification of 3272.

Development of direct real-time PCR system applicable to a wide range of foods and agricultural products.

To improve the efficiency of DNA analysis of foods and agricultural products, we investigated a direct real-time PCR based on the real-time monitoring of DNA amplification directly from crude cell lysates of analytical samples. We established a direct real-time PCR system comprising sample pretreatment with a specified lysis buffer and real-time PCR using the developed master mix reagent. No PCR inhibition was observed in the analysis of crude cell lysates from 50 types of samples, indicating that the direct real-time PCR system is applicable to a wide range of materials. The specificity of the direct real-time PCR was evaluated by means of a model assay system for single nucleotide discrimination. Even when crude cell lysates coexisted in the reaction mixtures, the primer selectivity was not affected, suggesting that the sequence specificity of the direct real-time PCR was equivalent to that of PCR from purified DNA templates. We evaluated the sensitivity and quantitative performance of the direct real-time PCR using soybean flour samples including various amounts of genetically modified organisms. The results clearly showed that the direct real-time PCR system provides sensitive detection and precise quantitation.

Development and validation of an event-specific quantitative PCR method for genetically modified maize MIR162.

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) maize event, MIR162. We first prepared a standard plasmid for MIR162 quantification. The conversion factor (Cf) required to calculate the genetically modified organism (GMO) amount was empirically determined for two real-time PCR instruments, the Applied Biosystems 7900HT (ABI7900) and the Applied Biosystems 7500 (ABI7500) for which the determined Cf values were 0.697 and 0.635, respectively. To validate the developed method, a blind test was carried out in an interlaboratory study. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr). The determined biases were less than 25% and the RSDr values were less than 20% at all evaluated concentrations. These results suggested that the limit of quantitation of the method was 0.5%, and that the developed method would thus be suitable for practical analyses for the detection and quantification of MIR162.

Interlaboratory study of qualitative PCR methods for genetically modified maize events MON810, bt11, GA21, and CaMV P35S.

Qualitative PCR methods for the genetically modified (GM) maize events MON810, Bt11, and GA21, and the 35S promoter (P35S) region of the cauliflower mosaic virus (CaMV) were evaluated in an interlaboratory study. Real-time PCR-based quantitative methods for these GM events using the same primer pairs had already been validated in previous studies. Fifteen laboratories in Japan participated in this interlaboratory study. Each participant extracted DNA from blind samples, performed qualitative PCR assays, and then detected the PCR products with agarose gel electrophoresis. The specificity, sensitivity, and false-negative and false-positive rates of these methods were determined with different concentrations of GM mixing samples. LODs of these methods for MON810, Bt11, GA21, and the P35S segment calculated as the amount of MON810 were 0.2, 0.2, 0.1, and 0.2% or less, respectively, indicating that the LODs of MON810, Bt11, and P35S were lower than 10 copies, and the LOD of GA21 was lower than 25 copies of maize haploid genome. The current study demonstrated that the qualitative methods would be fit for the detection and identification of these GM maize events and the P35S segment.

[Development and validation of event-specific quantitative PCR method for genetically modified maize LY038].

In this article, we report a novel real-time PCR-based analytical method for quantitation of the GM maize event LY038. We designed LY038-specific and maize endogenous reference DNA-specific PCR amplifications. After confirming the specificity and linearity of the LY038-specific PCR amplification, we determined the conversion factor required to calculate the weight-based content of GM organism (GMO) in a multilaboratory evaluation. Finally, in order to validate the developed method, an interlaboratory collaborative trial according to the internationally harmonized guidelines was performed with blind DNA samples containing LY038 at the mixing levels of 0, 0.5, 1.0, 5.0 and 10.0%. The precision of the method was evaluated as the RSD of reproducibility (RSDR), and the values obtained were all less than 25%. The limit of quantitation of the method was judged to be 0.5% based on the definition of ISO 24276 guideline. The results from the collaborative trial suggested that the developed quantitative method would be suitable for practical testing of LY038 maize.

Development and interlaboratory validation of quantitative polymerase chain reaction method for screening analysis of genetically modified soybeans.

A novel real-time polymerase chain reaction (PCR)-based quantitative screening method was developed for three genetically modified soybeans: RRS, A2704-12, and MON89788. The 35S promoter (P35S) of cauliflower mosaic virus is introduced into RRS and A2704-12 but not MON89788. We then designed a screening method comprised of the combination of the quantification of P35S and the event-specific quantification of MON89788. The conversion factor (Cf) required to convert the amount of a genetically modified organism (GMO) from a copy number ratio to a weight ratio was determined experimentally. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDR), respectively. The determined RSDR values for the method were less than 25% for both targets. We consider that the developed method would be suitable for the simple detection and approximate quantification of GMO.

Quantification and identification of genetically modified maize events in non-identity preserved maize samples in 2009 using an individual kernel detection system.

We investigated the GM maize grain content of non-identity preserved (non-IP) maize samples produced in 2009 in the USA using our individual kernel detection system, involving two multiplex qualitative PCR methods coupled to microchip electrophoresis and partially real-time PCR array analysis, to clarify how many GM event maize grains were present in the samples and which GM events frequently appeared in 2009. The average percentage and standard deviation of GM maize grains on a kernel basis in five non-IP sample lots were 81.9%±2.8%, the average percentage of single GM event grains was 46.9%, and the average percentage of stacked GM event grains was 35.0%. MON88017 grains and NK603 grains were the most frequently observed as single GM event grains. The most frequent stacked GM event grains were MON88017×MON810 grains. This study shows that our method can provide information about GM maize events present in imported maize samples on a kernel basis.

Development and validation of event-specific quantitative PCR method for genetically modified maize MIR604.

A GM maize event, MIR604, has been widely distributed and an analytical method to quantify its content is required to monitor the validity of food labeling. Here we report a novel real-time PCR-based quantitation method for MIR604 maize. We developed real-time PCR assays specific for MIR604 using event-specific primers designed by the trait developer, and for maize endogenous starch synthase IIb gene (SSIIb). Then, we determined the conversion factor, which is required to calculate the weight-based GM maize content from the copy number ratio of MIR604-specific DNA to the endogenous reference DNA. Finally, to validate the developed method, an interlaboratory collaborative trial according to the internationally harmonized guidelines was performed with blind samples containing MIR604 at the mixing levels of 0, 0.5, 1.0, 5.0 and 10.0%. The reproducibility (RSDr) of the developed method was evaluated to be less than 25%. The limit of quantitation of the method was estimated to be 0.5% based on the ISO 24276 guideline. These results suggested that the developed method would be suitable for practical quantitative analyses of MIR604 maize.