Novel method for determination of phenol degradation kinetics

In this study, we proposed a new method for estimating biokinetic parameters in phenol degradation kinetics. The new method relies on the new formulation of q–S relation where degradation rate q is calculated from the changes of substrate concentration S for each time segment during the course of entire degradation, while in the conventional method q is obtained from the slope of the straight line that is given as substrate concentration changes with time in a semi-logarithmic scale. Thus, this new method provided more data points than the conventional method. The q–S relations obtained from the new method and the conventional method were fitted with three inhibitory kinetic models of Haldane, Yano and Edwards. Simulation of degradation profile with each kinetic model and comparison with the observed profile revealed that the new method offered a better prediction with Edwards model as the best inhibitory model.     

Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.     

Biotin assay using lyophilized and glycerol-suspended cultures

An improved instant and convenient biotin bioassay method using lyophilized cells of Lactobacillus plantarum and glycerol-suspended cells of Saccharomyces cerevisiae were established. In addition, a new biotin bioassay method with a bioB⁻ mutant strain (C 162) of Escherichia coli was established. Polyvinyl pyrrolidone solution was effective as the suspending medium for lyophilization or glycerol suspension of the cells. Satisfactory standard curves were obtained by the paper disk method and turbidimetric method with lyophilized cells of L. plantarum and E. coli C 162 and glycerol-suspended cells of S. cerevisiae. These lyophilized or glycerol-suspended cells, which were preserved at −20°C, could be used for the assay for more than one year.     

Acanthamoeba-Campylobacter Coculture as a Novel Method for Enrichment of Campylobacter Species

In this study, we present a novel method to isolate and enrich low concentrations of Campylobacter pathogens. This method, Acanthamoeba-Campylobacter coculture (ACC), is based on the intracellular survival and multiplication of Campylobacter species in the free-living protozoan Acanthamoeba polyphaga. Four of the Campylobacter species relevant to humans and livestock, Campylobacter jejuni, C. coli, C. lari, and C. hyointestinalis, were effectively enriched by the coculture method, with growth rates comparable to those observed in other Campylobacter enrichment media. Studying six strains of C. jejuni isolated from different sources, we found that all of the strains could be enriched from an inoculum of fewer than 10 bacteria. The sensitivity of the ACC method was not negatively affected by the use of Campylobacter-selective antibiotics in the culture medium, but these were effective in suppressing the growth of seven different bacterial species added at a concentration of 10⁴ CFU/ml of each species as deliberate contamination. The ACC method has advantages over other enrichment methods as it is not dependent on a microaerobic milieu and does not require the use of blood or other oxygen-quenching agents. Our study found the ACC method to be a promising tool for the enrichment of Campylobacter species, particularly from water samples with low bacterial concentrations.     

Quantification of toxigenic Microcystis spp. in freshwaters by quantitative real-time PCR based on the microcystin synthetase A gene

A method to estimate the abundance of toxigenic Microcystis in environmental samples by using quantitative real-time PCR was developed and optimized. The basis of this method is the amplification of a highly conserved region of the mcyA gene within the microcystin synthetase gene cluster. Using this method, the average copy number of mcyA gene per cell in toxigenic Microcystis strains was estimated. The molecular markers and method developed in this study can be used to monitor toxigenic strains of Microcystis in Korean freshwaters, in which harmful cyanobacterial blooms are routinely found.     

Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.     

Cryptosporidium hominis Infection Diagnosed by Real-Time PCR-RFLP

There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections.     

Amino-terminal analysis of polypeptide using dimethylaminoazobenzene isothiocyanate

A new method for qualitative and quantitative N-terminal analysis of polypeptide using dimethylaminoazobenzene-isothiocyanate is presented. The method can recover all naturally occurring N-terminal amino acids, including asparagine, glutamine, and tryptophane in a nearly quantitative yield. Less than 1 nmol of polypeptide is required for qualitative N-terminal analysis and 5 to 10 nmol of polypeptide is used for quantitative N-terminal analysis. Applications and expected limitations of this new N-terminal method are described.     

Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies

A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 °C without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use.     

Modified method for determination of hippuric acid and methylhippuric acid in urine by gas chromatography

A modified method for the simultaneous determination of hippuric acid (HA) and o-, m- and p-methylhippuric acids (o-, m- and p-MHAs) in urine is described. These metabolites were extracted, derivatized into their methyl ester derivatives and analyzed using a gas chromatograph equipped with flame ionization detector and a DB-1 capillary column. The derivatives of HA, o-, m- and p-MHAs were well separated within 11 min. The accuracy and precision in the present method were sufficient for quantitative analysis, and the results obtained by the GC method were highly correlated with those by the HPLC method (NIOSH 8301).     

High-performance liquid chromatographic analysis of nitrite and nitrate in human plasma as S-nitroso-N-acetylcysteine with ultraviolet absorbance detection

A rapid HPLC method with UV absorbance detection at 333 nm for the measurement of nitrite and nitrate in ultrafiltrate samples of human plasma is described. The method is based on hydrochloric acid-catalyzed conversion of nitrite by N-acetyl-l-cysteine to S-nitroso-N-acetyl-l-cysteine and isocratic elution using 10 mM NaH₂PO₄ in acetonitrile–water, pH 2.0 (15:85, v/v). The limit of detection of the method is 50 nM nitrite. The method was validated by gas chromatography–mass spectrometry.     

Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.     

A Comparison of Culture- and PCR-Based Methods to Detect Six Major Non-O157 Serogroups of Shiga Toxin-Producing Escherichia coli in Cattle Feces

Culture-based methods to detect the six major non-O157 (O26, O45, O103, O111, O121 and O145) Shiga toxin-producing E. coli (STEC) are not well established. Our objectives of this study were to develop a culture-based method to detect the six non-O157 serogroups in cattle feces and compare the detection with a PCR method. Fecal samples (n = 576) were collected in a feedlot from 24 pens during a 12-week period and enriched in E. coli broth at 40° C for 6 h. Enriched samples were subjected to immunomagnetic separation, spread-plated onto a selective chromogenic medium, and initially pooled colonies, and subsequently, single colonies were tested by a multiplex PCR targeting six serogroups and four virulence genes, stx1, stx2, eae, and ehxA (culture method). Fecal suspensions, before and after enrichment, were also tested by a multiplex PCR targeting six serogroups and four virulence genes (PCR method). There was no difference in the proportions of fecal samples that tested positive (74.3 vs. 77.4%) for one or more of the six serogroups by either culture or the PCR method. However, each method detected one or more of the six serogroups in samples that were negative by the other method. Both culture method and PCR indicated that O26, O45, and O103 were the dominant serogroups. Higher proportions (P < 0.05) of fecal samples were positive for O26 (44.4 vs. 22.7%) and O121 (22.9 vs. 2.3%) serogroups by PCR than by the culture method. None of the fecal samples contained more than four serogroups. Only a small proportion of the six serogroups (23/640; 3.6%) isolated carried Shiga toxin genes. The culture method and the PCR method detected all six serogroups in samples negative by the other method, highlighting the importance of subjecting fecal samples to both methods for accurate detection of the six non-O157 STEC in cattle feces.     

Evaluation of immunomagnetic separation in combination with ALOA Listeria chromogenic agar for the isolation and identification of Listeria monocytogenes in ready-to-eat foods

A study was conducted to evaluate the performance of the ALOA (chromogenic media) in combination with immunomagnetic separation (IMS) for the detection of Listeria monocytogenes in ready-to-eat food products. IMS-ALOA method was found to be equivalent to Health Canada's reference culture method as well as comparable to BAX-PCR method in terms of the sensitivity of the methods for the detection of L. monocytogenes in ready-to-eat foods such as turkey roast, beef roast, mixed vegetable salads, potato and egg salad, soft cheese and smoked salmon. The IMS-ALOA method gave 100% sensitivity in the inclusivity tests with 42 pure L. monocytogenes strains. Exclusivity testing with five other species of Listeria genus and 29 pure non-L. monocytogenes strains from 21 different genera showed 97% specificity. The method was able to detect L. monocytogenes at levels near or below 1 cfu/25 g regulatory limit in ready-to-eat food matrices after 24 h enrichment, with a turnaround time of 3 days compared to 7-8 days for culture method. IMS-ALOA method is a valuable alternate test method for the screening of L. monocytogenes in a variety of foods especially ready-to-eat foods.     

Development and evaluation of a novel loop-mediated isothermal amplification (LAMP) method targeting Theileria parasites infecting Yezo sika deer

The increasing Yezo sika deer (Cervus nippon yesoensis) population is creating a large problem. Yezo sika deer are an important blood meal source, and these deer contribute to the maintenance of tick populations. Theileria spp. infections in Yezo sika deer and T. orientalis infections in cows occur at high frequencies, and the same tick species infests both deer and cows. Therefore, a specific detection method to identify deer Theileria spp. is important. In this study, we establish a novel molecular detection method for identifying Theileria spp. from deer and tick samples using loop-mediated isothermal amplification (LAMP). This method targets a metalloprotease/cell division cycle protein gene homologue. Our LAMP protocol was able to detect deer Theileria and did not show cross reactivity with other closely related protozoan parasites, including T. orientalis. The LAMP method showed sensitivity and specificity equivalent to those of nested PCR performed on the same field samples from deer and ticks. These results demonstrate the applicability of LAMP to field surveys in which the detection of deer Theileria spp. is required. In conclusion, due to its simplicity, specificity, and reliability, we suggest our LAMP protocol as an appropriate method for routine surveys to detect Yezo sika deer and ticks infected with deer Theileria spp. parasites. Additionally, this LAMP method offers great promise as a useful tool to distinguish Yezo sika deer Theileria from related Theileria parasites present in livestock.     

Estimation of coalescence probabilities and population divergence times from SNP data

We present a method called the G(A|B) method for estimating coalescence probabilities within population lineages from genome sequences when one individual is sampled from each population. Population divergence times can be estimated from these coalescence probabilities if additional assumptions about the history of population sizes are made. Our method is based on a method presented by Rasmussen et al. (2014) to test whether an archaic genome is from a population directly ancestral to a present-day population. The G(A|B) method does not require distinguishing ancestral from derived alleles or assumptions about demographic history before population divergence. We discuss the relationship of our method to two similar methods, one introduced by Green et al. (2010) and called the F(A|B) method and the other introduced by Schlebusch et al. (2017) and called the TT method. When our method is applied to individuals from three or more populations, it provides a test of whether the population history is treelike because coalescence probabilities are additive on a tree. We illustrate the use of our method by applying it to three high-coverage archaic genomes, two Neanderthals (Vindija and Altai) and a Denisovan.Subject terms: Rare variants, Evolutionary genetics

One of the goals of population genetics is to estimate the divergence time of isolated populations. We will review several methods that have been proposed and present a new method that is closely related to two existing methods. We will emphasize the assumptions made when using different methods. It will be useful to make the distinction between estimating coalescence probabilities within populations and estimating population divergence times. We will also introduce a test for a treelike population history based on our method.For distantly related populations, the numbers of mutational differences between sequences indicate relative times of divergence. Relative times are converted to absolute times by assuming a mutation rate. This method traces to Zuckerkandl and Pauling (1962, 1965) and has been used and refined extensively. This class of methods estimates genomic divergence times. Using it to estimate population or species divergence times assumes that those times are so large that the difference between them can be ignored.For recently diverged populations, the numbers of mutational differences probably do not provide a reliable estimate of population divergence times both because there may be too few mutations that differentiate populations and because the difference between the genomic and population divergence times may be substantial. To overcome this problem, Green et al. (2010) (in Supplement 14) introduced a method that accounts for the difference between genomic and population divergence. This method was used in later papers from the same group (Meyer et al. 2012; Prüfer et al. 2014, 2017).The Green et al. (2010) method is applicable when one genome is sampled from each of two populations. It depends on the statistic F(A|B), which is the fraction of sites in population A that carry the derived allele when that site is heterozygous in population B. Green et al. (2010) showed by simulation that the expectation of F(A|B) decreases roughly exponentially with the separation time of A and B. The rate of decrease depends on the history of population sizes both in B and in the population ancestral to A and B. Green et al. (2010) estimated population divergence times by interpolating their simulation results.More recently, Schlebusch et al. (2017), in Section 9.1 of their supplementary materials, introduced a similar method, called the TT method. Their method is based on analytic expressions for the configuration probabilities of SNPs that are polymorphic in the two populations. The TT method assumes that ancestral and derived alleles can be distinguished and the population before divergence was of constant size. The TT method is developed and elaborated on by Sjödin et al. (2020).In the present paper, we present a new method that is closely related to the F(A|B) and TT methods. We call it the G(A|B) method to emphasize its similarity to F(A|B). Our method is based on a method presented by Rasmussen et al. (2014) to test whether an ancient DNA sequence is from a population directly ancestral to a present-day population. We will show that our method provides a way to test whether the history of three or more populations is accurately represented by a population tree even if the demographic histories of those populations are not known.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

陆桥岛屿环境下社鼠种群数量的估算方法

根据2010年3月—11月在千岛湖地区2个岛屿上社鼠(Niviventer confucianus)的标志重捕数据,分别用Jolly-Seber法、修正Lincoln指数法、Schnabel法和MNA法计算两个岛屿上社鼠种群数量,并深入探讨在陆桥岛屿环境下估算社鼠种群数量的适用方法。研究结果显示,在满足Jolly-Seber法的条件下,通过该方法计算的结果与修正Lincoln指数法无显著差异。但在野外实验中,并不是所有的重捕数据都满足Jolly-Seber法的条件,而且该方法不能估算头尾两月的数量。因此,修正Lincoln指数法更适于估算陆桥岛屿环境下社鼠的种群数量。可为今后开展陆桥岛屿环境下鼠类种群生态学研究奠定基础。     

A sensitive,fluorometric method for the assay of microsomal hydroxylase: N-demethylation of p-chloro-N-methylaniline

A fluorometric method for the assay of microsomal hydroxylase activity is described. N-Demethylation of p-chloro-N-methylaniline yields p-chloroaniline, which is coupled with fluorescamine, extracted with ethylacetate, and measured fluorometrically. This method can determine low levels of N-demethylase activity.     

A novel method employing polymerase chain reaction to disrupt genes lacking convenient restriction enzyme sites in yeast

A novel method employing polymerase chain reaction was developed for the disruption of yeast genes lacking convenient restriction enzyme sites. The method was found to be easy and effective. Using this method, a yeastYKE2 gene (a yeast homolog of murinek-region expressed genes) was successfully disrupted by replacement ofHIS3 marker gene.