Biomedical subjects

Hiroyuki Kikuchi

Publications and source records attributed to Hiroyuki Kikuchi.

9 recordsLinked to original sources

PubMed2006

Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard.

Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision.

Food, Genetically Modified↗

PubMed2006

A detection method of CryIAc protein for identifying genetically modified rice using the lateral flow strip assay.

We examined the lateral flow strip assay for identifying unauthorized genetically modified (GM) rice. The GM rice expresses the Bacillus thuringiensis (Bt) toxin, CryIAc protein, which confers tolerance to insects. The recombinant CryIAc protein was prepared from the inclusion bodies of an E. coli. strain into which the CryIAc gene had been inserted, using gel filtration chromatography. The lateral flow strip assay for the identification of GM cotton which also expresses CryIAc protein, was applied to unpolished rice and polished rice spiked with recombinant CryIAc protein. The spiked recombinant CryIAc protein was clearly detected at the level of 0.012 microg/g in both the unpolished and polished rice. After loading of the extract on the strip, a 60 -minute stand time is necessary to clearly detect CryIAc protein. The detection limit was approximately 12 ng CryIAc protein per gram of rice. These results suggest that the lateral flow strip assay for GM cotton can be used to detect CryIAc protein expressed in GM rice.

Bacillus thuringiensis Toxins↗

PubMed2006

[Laboratory-performance study of quantitative PCR methods to analyze an approved genetically modified maize (Mon810 Line)].

A laboratory-performance study was carried out to investigate factors affecting the reliability of the quantitative PCR method to analyze an approved genetically modified (GM) maize (Mon810 line). Test maize powdered samples were prepared as blind samples containing a high (assigned value; 5.45%) or low (assigned value; 0.35%) concentration of the Mon810 line. After confirmation of their homogeneity, they were provided to 27 laboratories participating in the collaborative study. The data were collected from all laboratories and statistically analyzed. Two laboratories, which used a Roche LightCycler (LC), reported significantly high test values. A further examination showed that the LC method is greatly affected by the equipment itself or PCR reagents, resulting in poor repeatability. On the other hand, some laboratories, which used ABI quantitative PCR equipment, reported erroneous test values. In these laboratories, the errors appeared to have been due to inadequate quality and/or yield of DNA. To identify factors affecting the test values, analysis of the measured values for the taxon-specific gene will be useful. Furthermore, the modified silica-gel membrane DNA extraction method made it possible to extract the required amounts of DNA more easily and in a shorter time than before.

DNA, Plant↗

PubMed2005

[Laboratory-performance study of the notified methods to detect genetically modified papaya (55-1)].

To investigate important factors affecting the reliability of the analytical results, proficiency tests were attempted for the histochemical method (GUS method) and the qualitative PCR method (PCR method) to detect genetically modified papaya (55-1) in the Japanease official method. The test samples were distributed to twenty-three laboratories that participated in the study and were examined according to the protocol. All the data collected from participating laboratories were statistically analyzed. In the PCR method, one negative sample was detected as positive using detection primers in one laboratory, though the sample was negative when checked using confirmation primers. Contamination might have occurred in the step of the preparation of the PCR sample solution using detection primers. In the GUS method, all the test samples were identified as expected. Thus, all the laboratories reported correct results overall.

Carica↗

PubMed2005

[Laboratory performance study of the quantitative detection method for genetically modified soybeans (roundup ready soybeans 40-3-2)].

To investigate important factors affecting the analytical results, a laboratory-performance study was attempted for the Japanese official methods to detect genetically modified (GM) soybeans (40-3-2). Test samples containing 0, 1 and 5% GM soya powder in non-GM soya powder was prepared. A set of 3 test samples was sent to the participating laboratories along with the protocol. The data were collected from all laboratories and statistically analyzed. In the real-time PCR detection method, the average values of the GM 1% and 5% samples were both much lower than the spiked value because the laboratories using a silica-membrane DNA extraction method underestimated the GM value. On the other hand, the laboratories using other extraction methods, such as the CTAB method obtained values close to the spiked value. These results suggest that use of the silica-membrane DNA extraction method may result in underestimation of the GM content in the real-time PCR method. In the ELISA method, the average value of 5% spiked samples appears to be slightly higher than the fortified value. But, overall, it was considered that reported values were close to the spiked level.

DNA, Plant↗

PubMed2004

New qualitative detection methods of genetically modified potatoes.

In Japan, 8 lines of genetically modified (GM) potato (2 lines of NewLeaf potato; NL, 3 lines of NewLeaf Plus potato; NLP, and 3 lines of NewLeaf Y potato; NLY) have already been authorized as safe for use in foods and feeds. We have developed polymerase chain reaction (PCR) methods for the qualitative detection of the GM potatoes for the screening and the identification of NL, NLP and NLY. The gene encoding uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) was used as a taxon specific gene. We designed the primer pair to detect the cryIIIA genes as a screening method for GM potatoes because the gene should be inserted in all 8 lines of the GM potatoes. For identification of NL, NLP and NLY, we further designed three specific primer pairs for the different recombinant DNAs (r-DNA) specifically introduced into NL, NLP, or NLY. In addition, to identify the 3 lines of NLY that have been introduced with the same r-DNA, the three line-specific primer pairs for the border sequence between the r-DNA and genomic DNA of NLY 3 lines were designed. Six lines of GM potato used as the test material were specifically identified using the each primer pair under the same PCR condition. The detection limits of all the GM potatoes should be approximately 0.1%. Furthermore, the specificity and reproducibility of the methods were confirmed in a six-laboratory collaborative study.

Bacillus thuringiensis Toxins↗

PubMed2002

Analysis of rubusoside and related compounds in tenryocha extract sweetener.

The main and minor constituents of tenryocha extract product, a natural sweetener, were investigated as a part of an ongoing study to evaluate its quality and safety as a food additive. Four constituents, namely rubusoside, steviolmonoside, panicloside IV, and ent-16 alpha, 17-dihydroxykauran-19-oic acid, were isolated. The concentration of rubusoside, the main sweet constituent, was 8.65% in the tenryocha extract product. In addition, it was confirmed that the origin of the extract was the leaves of Rubus suavissimus S. Lee (Rosaseae), as determined by comparing TLC and HPLC profiles of the product and hot-water extract prepared from the leaves of R. suavissimus.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Reactivation of hepatitis and lamivudine therapy in 11 HBsAg-positive renal allograft recipients: a single centre experience.

BACKGROUND: In hepatitis B virus (HBV) surface antigen (HBsAg) (+) renal allograft recipients, the mortality associated with liver disease reaches 37-78%. An antiviral agent, lamivudine, has recently been reported to be safe and effective for preventing hepatic damage in these patients, although either resurgence of HBV-DNA levels after discontinuation or emerging resistant HBV mutants caused by long-term administration are still unsettled. METHODS: Between July 1976 and December 2003, 555 renal transplantations were performed in our centre. Of these, 11 patients who were HBsAg (+) at the time of transplantation (2.0%) were selected for this study. We investigated the incidence of hepatitis reactivation for three yr after transplantation and their clinical courses, including the efficacy of lamivudine therapy in seven of the 11 patients. RESULTS: Six episodes of hepatitis reactivation developed in five of the 11 patients (45.5%) within three yr after transplantation. Five episodes of six occurred within four months after transplantation. The patient who underwent the most severe reactivation needed intensive care including lamivudine administration and plasma exchange. Lamivudine caused no severe adverse effects and HBV-DNA levels dropped to under measurable levels within four months after lamivudine administration in all patients. Resistant HBV mutant emerged in only one patient, who had the longest lamivudine administration of 49 months. CONCLUSIONS: For HBsAg (+) renal allograft recipients, careful monitoring of HBV-DNA levels and timely administration of lamivudine could prevent hepatic damage caused by reactivation of hepatitis.

Adult↗

PubMedDate not supplied

Tube cholangio-jejunal bypass in unresectable hepatic hilar carcinoma for a better quality of life.

A 76-year-old male patient with jaundice was diagnosed as having hepatic hilar cholangiocarcinoma. The patient underwent percutaneous transhepatic biliary drainage of the left and the right intrahepatic bile duct. He could not have a percutaneous transhepatic endoprosthesis placed because it was impossible to pass the guidewire through the stenotic portion of the lesion. The patient was operated, but the tumor was considered to be unresectable. Along the intrahepatic routes formed by the preexisting two percutaneous transhepatic biliary drainage tubes, silicon tubes were inserted. Through the lumen of a long jejunal limb, Roux-en-Y, the tubes with five to six side holes were passed in the distal direction and drawn out from the lumen of the jejunal limb and passed through the abdominal wall to outside. The tubes were occluded and buried in the subcutaneous space after a few clamping tests. He died of liver failure in his house four months after the operation without any symptoms of jaundice, fever or of obstruction of the tubes.

Aged↗