Pyrophosphate detection method using 5-Br-PAPS to detect nucleic acid amplification - Application to LAMP method.

Genetic testing has been increasingly used in several fields. In many applications, nucleic acid amplification technology is required. However, current methods to detect nucleic acid amplification require expensive reagents and special equipment or exhibit limited sensitivity, which hinders their use. To address this issue, this study reports an assay method for detecting occurrence of acid amplification in post-amplification samples using pyrophosphate, a highly sensitive byproduct of nucleic acid amplification. The method proposed requires two reagents and an automated analyzer. First, hydrogen peroxide is derived from pyrophosphate, an indicator of nucleic acid amplification, and the oxidizing power of hydrogen peroxide is used to produce Fe (III) from Fe (II). The specific metal chelator 5-Br-PAPS forms a complex with the trivalent iron produced, resulting in a highly sensitive coloration. The within-run reproducibility of our method (n = 20) was less than 3.67% at each concentration tested, and the detection limit was 0.075 µmol/L, sufficient for quantitative analysis. The technique described could detect pyrophosphate in a sample that was amplified using the loop-mediated isothermal amplification method after only 10 min. Therefore, the proposed method has the potential to be a new, rapid, and simple detection technique for amplified nucleic acids.

Development of a plasma maltose assay method as a screening test for upper gastrointestinal disorders.

BACKGROUND AND OBJECTIVE: The disaccharide loading test is a method to assess gastric mucosal damage. Since Trelan-G75, which is used for the sugar tolerance test, contains disaccharide maltose, if maltose is detected at a high sensitivity in the sample blood used in the sugar tolerance test, screening for upper gastrointestinal mucosal damage can be made simultaneously with the sugar tolerance test for the diagnosis of diabetes. METHODS: Glucose-6-phosphate is generated by treating maltose with maltose phosphorylase, ß-phosphoglucomutase, and glucose-1,6-bisphosphate. Then, change in the absorbance at 405 nm is measured by the enzymatic cycling method using Thio-NADP, ß-NADPH, and Glucose-6-phosphate dehydrogenase. After evaluating the optimal condition for this method, it is mounted on an automatic biochemical analyzer, and samples after the sugar tolerance test were assayed. RESULTS: Regarding the performance of this method, the repeatability was 10-50 µmol/L with a CV of ≤1.1%. Concerning the assay range, a curve passing the origin with a range of linearity up to 120 µmol/L was obtained. No effect of dyes or sugars in the blood was noted. As a result of application to patients with gastric mucosal disorders (those who had a health checkup), significant differences were observed depending on the stage of atrophic gastritis. DISCUSSION: This method has a high sensitivity and a high precision and can be used for high-speed analysis on an automatic analyzer. It has the potential to be used as a screening test for gastric mucosal damage.

Development of a protein assay with copper chelator chromeazurol B, based on the biuret reaction.

This study aimed to provide a novel and highly sensitive protein assay based on the biuret reaction and using chromeazurol B, a metal chelate compound. The method consists of two reagents and an automated analyzer. First, a complex of copper and protein (biuret reaction) is formed. Second, a chelating reagent containing chromeazurol B forms a three-dimensional complex of protein, copper, and chromeazurol B at neutral pH, resulting in highly sensitive coloration. The intra-assay (n = 20) variation for the three levels was 3.54 % or lower at each concentration. Each response with α, ß-, and γ-globulin was 103.8 % and 104.3 %, respectively, against albumin. The molar absorption coefficient (ε) of the present method was 2.5 × 105 m2/mol against human albumin, higher than that of the commercially available Lowry method (ε = 8.7 × 104 m2/mol), which is based on the same principle. The correlation test for the pyrogallol method with 30 urine samples showed good performance (r = 0.961). The method described here (the Biuret-based CAB method) is a more sensitive and rapid assay than the Lowry method, and it may also be applied to biological samples because of its similar reactivity towards various proteins.

A survey of the reactivity of in vitro diagnostic bilirubin reagents developed in Japan using artificially prepared bilirubin materials: A comparison of synthetic delta, unconjugated, and taurine-conjugated bilirubin.

BACKGROUND: In vitro diagnostic bilirubin reagents based on oxidation with bilirubin oxidase or vanadic acid for total and direct-reacting bilirubin are widely used in Japan; however, their reactivity to unconjugated and conjugated bilirubin and delta bilirubin has not been completely disclosed by manufacturers. We used artificially prepared bilirubin materials to investigate the reactivity with four in vitro diagnostic bilirubin reagents. METHODS: Porcine unconjugated bilirubin solution, chemically synthesized ditaurobilirubin solution, and chemically synthesized delta bilirubin solution were used as surrogates of naturally occurring unconjugated bilirubin, conjugated bilirubin, and delta bilirubin, respectively. The total bilirubin and direct-reacting bilirubin concentrations were measured by three bilirubin oxidase methods and one vanadic acid method, and the observed concentrations were compared with those obtained by the diazo-based reference measurement procedure. RESULTS: The unconjugated bilirubin and delta bilirubin concentrations were similar when any of the four in vitro diagnostic bilirubin reagents were used during total bilirubin measurement. This was consistent with reference measurement procedure and exhibited a converged inter-method variation. Compared with reference measurement procedure, significantly low ditaurobilirubin concentrations were observed by the in vitro diagnostic bilirubin reagents despite the converged inter-method variation. In delta bilirubin measurement, some reagents reacted doubtfully with unconjugated bilirubin, while showed lower ditaurobilirubin concentrations than its corresponding total bilirubin concentration. Reactivity with delta bilirubin was different for each method including reference measurement procedure. Some reagents were developed to react less with delta bilirubin and others to strongly react with delta bilirubin. CONCLUSIONS: We revealed the reactivity of IVD-TB and IVD-DB reagents to artificially prepared bilirubin materials, and their consistency with reference measurement procedure. The delta bilirubin data results vary depending on the reagents used.

A highly sensitive and rapid enzymatic method using a biochemical automated analyzer to detect inorganic pyrophosphate generated by nucleic acid sequence-based amplification.

BACKGROUND AND AIMS: Polymerase chain reaction-based techniques require expensive equipment for fluorescence detection of the products. However, the measurement of inorganic pyrophosphate (PPi) released during DNA synthesis can be used to quantify target genes without such equipment. Here, we devised a high-sensitivity enzymatic assay for detection of PPi. MATERIALS AND METHODS: In our assay method, PPi was converted to hypoxanthine by hypoxanthine phosphoribosyl transferase. Xanthine dehydrogenase converted the hypoxanthine to uric acid and yielded two molecules of NADH, which in turn reduced Fe3+ to Fe2+ (mediated by 1-methoxy-5-ethylphenazinium ethylsulfate). 2-Nitroso-5-(N-propyl-N-sulfopropylamino) phenol chelated the Fe2+, which resulted in an intensely colored product that could be measured using a biochemical automated analyzer. RESULTS: The assay was able to detect PPi within 10 min. It was linear between 0 and 10 µmol/L PPi, and intra-run and inter-run coefficients of variation were 1%-2%. Other validation tests with a biochemical automated analyzer were satisfactory. The assay could potentially be used to directly quantify samples after isothermal nucleic acid sequence-based amplification of a target gene. CONCLUSION: The method developed here for detection of PPi can be used to measure nucleic acid biomarkers in biological samples in clinical practice using a high-throughput biochemical automated analyzer.

Characterization and application of a novel nicotinamide mononucleotide adenylyltransferase from Thermus thermophilus HB8.

Herein, we describe a novel enzymatic cycling method to measure nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN), which are precursors of NAD biosynthesis. A gene encoding an NMN adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified in Thermus thermophilus HB8. The gene from T. thermophilus (TtNMNAT) was engineered for expression in Escherichia coli and the recombinant enzyme found to be stable, retaining full activity after incubation for 45 min at 70°C. The Km values for NMN and ATP were calculated to be 0.263 and 1.27 mM, respectively, with a Vmax value of 60.3 µmoL/min/mg. TtNMNAT was successfully applied to the colorimetric NMN or NaMN assays, which employed (i) adenylation of NMN to NAD by TtNMNAT or adenylation of NaMN to deamido-NAD (NaAD) by TtNMNAT followed by amidation of NaAD to NAD by NAD synthetase (NADS, EC 6.3.1.5) and (ii) an NAD cycling reaction using 12α-hydroxysteroid dehydrogenase (12α-HSD, EC 1.1.1.176) and diaphorase (DI, EC 1.6.99.3) to accumulate reduced WST-8. This enzymatic cycling method enabled detection of 0.5 µM (12.2 nM in the reaction mixture) NMN or NaMN in an automatic clinical analyzer.

Development of a simple indocyanine green measurement method using an automated biochemical analyser.

Background The indocyanine green retention rate is important for assessing the severity of liver disorders. In the conventional method, blood needs to be collected twice. In the present study, we developed an automated indocyanine green method that does not require blood sampling before intravenous indocyanine green injections and is applicable to an automated biochemical analyser. Methods The serum samples of 471 patients collected before and after intravenous indocyanine green injections and submitted to the clinical laboratory of our hospital were used as samples. The standard procedure established by the Japan Society of Hepatology was used as the standard method. In the automated indocyanine green method, serum collected after an intravenous indocyanine green injection was mixed with the saline reagent containing a surfactant, and the indocyanine green concentration was measured at a dominant wavelength of 805 nm and a complementary wavelength of 884 nm. Results The coefficient of variations of the within- and between-run reproducibilities of this method were 2% or lower, and dilution linearity passing the origin was noted up to 10 mg/L indocyanine green. The reagent was stable for four weeks or longer. Haemoglobin, bilirubin and chyle had no impact on the results obtained. The correlation coefficient between the standard method (x) and this method (y) was r=0.995; however, slight divergence was noted in turbid samples. Conclusion Divergence in turbid samples may have corresponded to false negativity with the standard procedure. Our method may be highly practical because blood sampling before indocyanine green loading is unnecessary and measurements are simple.

Multianalyte Conventional Reference Material (MacRM): A Useful Tool for Nationwide Standardization of Laboratory Measurements for Medical Care-A Model Study in Japan.

BACKGROUND: The Japanese Committee for Clinical Laboratory Standards (JCCLS) has developed a multianalyte conventional reference material (MacRM) for nationwide standardization of laboratory measurements. METHODS: To prepare the MacRM, pooled sera were obtained from healthy Japanese individuals. Target values of the pooled sera for 30 analytes were assigned on the basis of the measurement results of 45 certified clinical laboratories whose calibration was verified by measuring certified reference materials (CRMs) provided by the National Institute of Standards and Technology, the Institute for Reference Materials and Measurements, and JCCLS. Commutability of MacRM was assessed by comparison with results for 150 individual inpatients at Fukuoka University Chikushi Hospital. Survey samples were prepared by essentially the same method for MacRM but without target values. The survey samples were used to assess agreement among 165 laboratories that used various assay kits and platforms calibrated with the MacRM. RESULTS: The commutability of MacRM was confirmed for 30 analytes with sera from 150 individual patients. The imprecision (CV) of measurements of survey samples (high and low concentrations) among the 165 laboratories was 0.4%-10.0%. Twenty-six of 30 analytes were within the goals for interinstitutional allowable bias. An aliquot of MacRM stored frozen at -80 °C remained stable for ≥4 years. CONCLUSIONS: The MacRM was successfully applied as a calibrator to achieve nationwide standardization for 30 analytes measured by 165 laboratories that used various methods from different manufacturers.

[Role of Medical Technologists as Certified Clinical Chemistry and Inununochemistry Test Quality Assurance Managers in International Standardization].

In Japan, biochemical testing has been standardized by establishing secondary reference measurement procedures for each method under the leadership of the Japan Society of Clinical Chemistry (JSCC). This has led to the assignment of values to secondary calibration materials, enabling laboratories to manage the accuracy of measurement values using them. To promote the sharing of test data by standardizing base facilities on a nationwide basis, the Japanese As- sociation of Medical Technologists (JAMT) launched the Laboratory Test Data Standardization Project. The range of reference for biochemical testing was calculated, and the absence of regional differences in such a range was confirmed by standardized base facilities throughout Japan. Through collaboration between the JSCC and JAMT, the Clinical Chemistry and Immunochemistry Test Quality Assurance Manager Certification System was established in 2014, with a view to promoting quality assurance in laboratories. It is expected that medical technologists who are certified as such managers will contribute to the nationwide provision of high-quality medical services. [Review].

[Strategy Development for International Cooperation in the Clinical Laboratory Field].

The strategy of international cooperation in the clinical laboratory field was analyzed to improve the quality of intervention by reviewing documents from international organizations and the Japanese government. Based on the world development agenda, the target of action for health has shifted from communicable diseases to non-communicable diseases (NCD). This emphasizes the importance of comprehensive clinical laboratories instead of disease-specific examinations in developing countries. To achieve this goal, the World Health Organization (WHO) has disseminated to the African and Asian regions the Laboratory Quality Management System (LQMS), which is based on the same principles of the International Organization of Standardization (ISO) 15189. To execute this strategy, international experts must have competence in project management, analyze information regarding the target country, and develop a strategy for management of the LQMS with an understanding of the technical aspects of laboratory work. However, there is no appropriate pre- and post-educational system of international health for Japanese international workers. Universities and academic organizations should cooperate with the government to establish a system of education for international workers. Objectives of this education system must include: (1) training for the organization and understanding of global health issues, (2) education of the principles regarding comprehensive management of clinical laboratories, and (3) understanding the LQMS which was employed based on WHO's initiative. Achievement of these objectives will help improve the quality of international cooperation in the clinical laboratory field.

[Development of the fingertip blood dilution method for blood cell counting].

For simple examination of blood disorders such as anemia, the results of blood cell counting using a small volume of blood sampled from a finger were evaluated. Because the sampling volume varies, tyramine was added to the blood dilution buffer as an internal standard. When blood is added to a tyramine-containing buffer, tyramine is diluted according to the blood volume regardless of the hematocrit. The degree of blood dilution can be calculated from the difference in the tyramine concentration. The diluted blood was measured for blood cell count items using XE-2100. After cell counting, the diluted blood was centrifuged, and the concentration of the internal standard in the supernatant was measured using the automated biochemical analyzer JCA-BM2250. Correlation coefficients of 0.824-0.995 were obtained in the blood cell counts between fingertip and venous blood samples from 48 volunteers. However, as platelet aggregation was observed by fingertip blood sampling, the correlation coefficient for the platelet count was 0.450. This method is considered to be useful for screening of blood disorders. It may also be applicable to medical care in remote areas and during disasters.

Nationwide multicenter study aimed at the establishment of common reference intervals for standardized clinical laboratory tests in Japan.

BACKGROUND: The Japanese Association of Medical Technologists (JAMT) sought to establish common reference intervals (RIs) applicable nationwide in Japan for 27 serum constituent analytes for which certified reference materials are available and nine analytes frequently measured in routine tests. More than 100 laboratories certified for metrological traceability collaborated in the recruitment, sampling, and measurement of analytes for the establishment of RIs. No previous attempt has been made to establish RIs by such a large number of laboratories. The allowable limits of trueness and intermediate precision based on the JAMT criteria were applied to the reference values measured by these laboratories, and measured values within the allowance limits were used to establish RIs. METHODS: Reference individuals included 5748 healthy volunteers aged 18-65 years who were engaged in medical care-related work based on the CLSI guidelines. After secondary exclusion of individuals in whom abnormal values were detected in basic routine test items and adjustment for the distribution of age and gender, 3371 reference individuals were chosen in the parametric determination of RIs. Employing the three-level nested ANOVA, between-laboratory, -region, -sex, and -age variations were evaluated. RESULTS: No significant difference was noted in between-region variations in any item. Results of ANOVA revealed between-sex and -age variations in 14 and 15 analytes, respectively. Based on these results of variation, RIs were established with and without partition by sex. CONCLUSIONS: Since no between-region variation was detected in reference values among accuracy-certified core laboratories, RIs applicable nationwide were established.

Enzymatic assay of phosphatidylethanolamine in serum using amine oxidase from Arthrobacter sp.

BACKGROUND: In human serum, as for phospholipids not containing choline, phosphatidylethanolamine (PE) exists approximately 5% in a whole phospholipid. PE is well known as one of the main components of biological membranes, and also plays important roles that contribute to apoptosis and cell signaling. However, it could not measure PE with other phospholipids due to a lack of choline in them. METHODS: Using an amine oxidase (EC 1.4.3.6), from Arthrobacter species, a simple and rapid enzymatic assay for measurements of PE in serum was established. That assay used the Hitachi 7170 analyzer to evaluate the analytical performance. RESULTS: The average within-run CVs were 0.38-1.27% (n=20) at 69-160 µmol/l. The correlation between values obtained with the present method (y) and the high-performance liquid chromatography (HPLC) method (x) was: y=0.944x+9.441 (r=0.977, S(y|x)=5.82, n=34). In addition, the reference interval of healthy subjects was 115±45 µmol/l. CONCLUSIONS: This new enzymatic method shows a high specificity for serum PE and can be easily applied to an automated analyzer. The present method is available as a novel marker of changes in the clinical condition of serum phospholipids.

Development of a new measurement method for serum calcium with chlorophosphonazo-III.

BACKGROUND: Although serum calcium has been measured using the o-cresolphthalein complexone (oCPC) method in the clinical laboratory, this method still has some problems regarding linearity and reagent stability. We developed a new measurement procedure using chlorophosphonazo-III (CPZ-III: 2,7-bis (4-chloro-2-phosphonophenylazo) -1,8- dihydroxy-3, 6-naphthalenedisulphonic acid, disodium salt) as a chelator with an acid medium for serum calcium measurement. The present method showed better linearity and reagent stability compared with the oCPC method. METHODS: Characteristics were studied in optimized conditions measuring wavelength by absorption spectra analysis, and interference of protein and metals with Mg(2+), Fe(2+), Cu(2+) and Zn(2+). The method was applied to an automated analyser (7170; Hitachi High Technologies Corp). The measurement performance was evaluated for accuracy, precision, recovery rate, linearity and reagent stability with a comparison study against atomic absorption spectrophotometry (AAS). RESULTS: The within-run and between-run variations (coefficient of variation [CV]) were 0.92-1.01% and 0.75-1.43%, respectively. The linearity was 0-7.0 mmol/L. The comparison study obtained y = 1.002x (AAS) - 0.10, Sy/x = 0.18 mmol/L, n = 50. Reagent stability was at least 20 d at 4 degrees C without daily calibration. CONCLUSION: The new calcium measurement method in serum was demonstrated to have reliable and acceptable performances as a routine test in clinical laboratory.

Enzymatic characterization of an amine oxidase from Arthrobacter sp. used to measure phosphatidylethanolamine.

Ethanolamine oxidase was screened with the aim of using it to establish a novel enzymatic phosphatidylethanolamine assay. Ethanolamine oxidase activity was detected in the crude extract of Arthrobacter sp., and the enzyme was purified more than 15-fold in three steps with a 54% yield. SDS-PAGE revealed the presence of only one band, which migrated, with an apparent molecular mass of 70 kDa. Biochemical characterization of the enzyme showed phenylethylamine to be the preferred substrate, with the highest kcat/Km value. The primary structure, determined by sequencing the cloned gene, showed a high degree of identity to Cu-containing phenylethylamine oxidase (64%). When heterologously overexpressed in Escherichia coli, the enzyme exhibited only a trace of amine oxidase activity, but high levels of activity emerged after exposure to Cu2+, as is typical of recombinant copper amine oxidases. Preliminary application of this enzyme coupled with phospholipase D for determination of phosphatidylethanolamine is also described. This is the first enzymatic method for the measurement of phosphatidylethanolamine.

Mapping of rRNA gene loci in the mice, Mus musculus molossinus (Japan) and Mus musculus musculus (Russia) by double color FISH.

Ribosomal RNA gene (rDNA) loci of Russian Mus musculus musculus and of Japanese Mus musuculus molossinus were mapped by double color FISH. The total number of rDNA loci was varied from 5 to 12, although the loci on chromosomes 12, 15, 16, 18, and 19 were common to all mice examined. Instead, polymorphisms of the rDNA loci were found on chromosomes 1, 5, 8, 9, 10, 11, 13 and 17. The novel rDNA loci of M. m. musculus were found in Nov/TUA strain on chromosomes 8 and 17. These observations, together with those of previous reports, suggest that the rDNA loci of Mus musculus species are in the evolutionary process of further translocation to other chromosomes.

[Establishment of mail medical examination system using immediate plasma separating device by the self-collection blood--the method of dilution ratio calculation by using Internal standard for the sample with different amount of collecting blood].

A method of calibrating unquantified samples was developed in order to conduct tests from approximately 65 microl or less of self-collected whole blood from the finger. In this method, the dilution ratio is estimated by absorption analysis of an internal standard substance in the blood dilution buffer. Good results for replication of the dilution ratio were obtained, with a CV value of 0.99-2.78% and a rate of concordance with the theoretical dilution ratio of 99.4-99.8%. Evaluations of the utility of this system (Demecal System) were performed using total cholesterol measurement, and excellent correlation with the measurement value in plasma was achieved, with a correlation coefficient of r=0.975 at n=40 and a regression equation of y=1.02x-2.83. Dilution of 7-fold or less is required to assure accuracy with this system.

Identification of rRNA gene loci in the wild mouse (Mus musculus molossinus) captured at Hachioji, Tokyo.

Mus musculus (M. m.) molossinus has been considered an independent subspecies of Mus musculus. To elucidate the evolutional origin of this subspecies, we carried out double-color FISH using 18s-28s ribosomal DNA and mouse chromosome paint probes. Among eleven rDNA loci detected, five loci on chromosomes 12, 15, 16, 18 and 19 were common to both Mus musculus (M. m.) musculus and M. m. molossinus and the other six loci, on chromosomes 1, 5, 10, 11, 13 and 17, were characteristic in M. m. molossinus. As M. m. molossinus is thought to originate from a hybrid between ancestral colonies of M. m. musculus and Mus musculus castaneus, we supposed that these six rDNA loci might have evolved after geographical isolation of the ancestral hybrid animals from M. m. musculus and M. m. castaneus.