Interlaboratory validation study of an event-specific real-time polymerase chain reaction detection method for genetically modified 55-1 papaya.

Genetically modified (GM) papaya line 55-1 (55-1) is resistant to papaya ringspot virus infection, and is commercially available in several countries. A specific detection method for 55-1 is required for mandatory labeling regulations. An event-specific real-time PCR method was developed by our laboratory. To validate the method, interlaboratory validation of event-specific qualitative real-time PCR analysis for 55-1 was performed in collaboration with 12 laboratories. DNA extraction and real-time PCR reaction methods were evaluated using 12 blind samples: six non-GM papayas and six GM papayas in each laboratory. Genomic DNA was highly purified from all papayas using an ion-exchange column, and the resulting DNA sample was analyzed using real-time PCR. Papaya endogenous reference gene chymopapain (CHY) and the event-specific 55-1 targeted sequence were detected in GM papayas whereas CHYalone was detected in non-GM papayas in all laboratories. The cycle threshold values of CHYand the 55-1 targeted sequence showed high repeatability (RSD, 0.6-0.8%) and reproducibility (RSDR 2.2-3.6%). This study demonstrates that the 55-1 real-time PCR detection method is a useful and reliable method to monitor 55-1 papaya in foods.

Application of a qualitative and quantitative real-time polymerase chain reaction method for detecting genetically modified papaya line 55-1 in papaya products.

Genetically modified (GM) papaya (Carica papaya L.) line 55-1 (55-1), which is resistant to papaya ringspot virus infection, has been marketed internationally. Many countries have mandatory labeling regulations for GM foods, and there is a need for specific methods for detecting 55-1. Here, an event- and construct-specific real-time polymerase chain reaction (PCR) method was developed for detecting 55-1 in papaya products. Quantitative detection was possible for fresh papaya fruit up to dilutions of 0.001% and 0.01% (weight per weight [w/w]) for homozygous SunUp and heterozygous Rainbow cultivars, respectively, in non-GM papaya. The limit of detection and quantification was as low as 250 copies of the haploid genome according to a standard reference plasmid. The method was applicable to qualitative detection of 55-1 in eight types of processed products (canned papaya, pickled papaya, dried fruit, papaya-leaf tea, jam, puree, juice, and frozen dessert) containing papaya as a main ingredient.

Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.

[Selection of suitable polypropylene tubes for DNA testing using real-time PCR].

Polypropylene microtubes (tubes) are generally used for bio-material tests in addition to PCR tests such as genetically modified organism (GMO) testings. However, the choice of suitable tubes is quite important, because it might influence the results: DNA binding and/or elution of chemical substances sometimes occurs. In this study, we established methods to select tubes with the most suitable characteristics for DNA testing.

Development of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstuffs and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this.

Investigation of residual DNAs in sugar from sugar beet (Beta vulgaris L.).

Genetically modified (GM) sugar beets have been bred for use as food and animal feed. To evaluate the applicability of GMO analyses to beet sugar products, we investigated residual DNA in eight sorts of in-process beet sugar samples and commercial beet sugar products. Polymerase chain reaction (PCR) analyses with taxon-specific primers indicated that sugar beet DNA was degraded at an early stage of sugar processing, and no PCR amplification was detected from the investigated sugar products because of low DNA recovery and/or PCR inhibition.

Impaired function of dendritic cells in alymphoplasia (aly/aly) mice for expansion of CD25+CD4+ regulatory T cells.

Alymphoplasia (aly/aly) mice are from a naturally occurring strain with a mutation in nuclear factor-kappa B inducing kinase (NIK). The NIK mutation causes disruption of the architecture of the thymus and spleen and aly/aly mice show decreased numbers of CD25+CD4+T cells in the spleen. For the expansion of CD25+CD4+T cells, interactions between dendritic cells (DCs) and CD25+CD4+ regulatory T cells are necessary. We investigated the ability of DCs to induce expansion of CD25+CD4+T cells. We found that DCs are reduced in the spleen of aly/aly mice, and showed low expressions of CD80, CD86 and MHC class II molecules on the surface. DCs from aly/aly mice showed decreased ability to present ovalbumin (OVA) to T cells from OVA specific TCR transgenic mice, and a decreased ability for alloantigen presentation. Further, DCs showed a decreased ability to induce expansion of CD25+CD4+T cells in vitro. Our results suggested that the impairment of DCs in aly/aly mice is responsible, at least in part, for the decreased numbers of CD25+CD4+T cells in the periphery of aly/aly mice.