Impact of national pneumococcal vaccination program on invasive pneumococcal diseases in South Korea.

Following the introduction of pneumococcal conjugate vaccines (PCVs), the rate of invasive pneumococcal disease (IPD) declined, however, IPDs replaced by serotypes that are not included in the vaccine have emerged. We describe the epidemiology of IPD in South Korea over a 4.5-year period, encompassing the impact following introduction of PCV10/13 and PPSV23 into the public immunization program, and assess serotype dynamics in pediatric and adult population. This was a nationwide, retrospective review of surveillance of all IPD cases in Korea between September 2014 to December 2019. We analyzed VT13 (serotypes included in 13-valent conjugate vaccine) and NVT (nonvaccine type) cases by age, sex, IPD type, vaccination status, and deaths. A total of 893 cases with serotype data were included; 306 (34%) VT13 cases and 587 (66%) NVT cases. Serotype 3 (n = 155) was the most common VT13 serotype, followed by serotypes 19A (n = 70) and 14 (n = 28). Among the NVTs, serotype 10A (n = 74) was the most common serotype, followed by serotypes 23A (n = 60) and 34 (n = 58). Persons who had PCV13 vaccination were at lower risk (aOR = 0.11, 95% CI 0.02-0.73, P = 0.022) of death compared to unvaccinated persons. Introduction of PCV10/13 and PPSV23 vaccination program has had different impacts on the serotype-specific IPD across age groups. The most common serotypes included serotypes 3 and 19A (VT13), and 10A, 23A, and 34 (NVT). Our findings suggest continued monitoring in the midst of new vaccine development, and a need to develop novel strategies to mitigate the IPDs from emerging pneumococcal serotypes.

Analysis of national surveillance of respiratory pathogens for community-acquired pneumonia in children and adolescents.

BACKGROUND: Respiratory infections among children, particularly community-acquired pneumonia (CAP), is a major disease with a high frequency among outpatient and inpatient visits. The causes of CAP vary depending on individual susceptibility, the epidemiological characteristics of the community, and the season. We performed this study to establish a nationwide surveillance network system and identify the causative agents for CAP and antibiotic resistance in Korean children with CAP. METHODS: The monitoring network was composed of 28 secondary and tertiary medical institutions. Upper and lower respiratory samples were assayed using a culture or polymerase chain reaction (PCR) from August 2018 to May 2020. RESULTS: A total of 1023 cases were registered in patients with CAP, and PCR of atypical pneumonia pathogens revealed 422 cases of M. pneumoniae (41.3%). Respiratory viruses showed a positivity rate of 65.7% by multiplex PCR test, and human rhinovirus was the most common virus, with 312 cases (30.5%). Two hundred sixty four cases (25.8%) were isolated by culture, including 131 cases of S. aureus (12.8%), 92 cases of S. pneumoniae (9%), and 20 cases of H. influenzae (2%). The cultured, isolated bacteria may be colonized pathogen. The proportion of co-detection was 49.2%. The rate of antibiotic resistance showed similar results as previous reports. CONCLUSIONS: This study will identify the pathogens that cause respiratory infections and analyze the current status of antibiotic resistance to provide scientific evidence for management policies of domestic respiratory infections. Additionally, in preparation for new epidemics, including COVID-19, monitoring respiratory infections in children and adolescents has become more important, and research on this topic should be continuously conducted in the future.

Early Laboratory Preparedness of the Korea Disease Control and Prevention Agency and Response to Unknown Pneumonia Outbreak from Wuhan, China, in January 2020.

We report the response process of the Laboratory Analysis Task Force (LATF) for Unknown Disease Outbreaks (UDOs) at the Korea Disease Control and Prevention Agency (KDCA) during January 2020 to coronavirus disease 2019 (COVID-19), which developed as a UDO in Korea. The advanced preparedness offered by the laboratory diagnostic algorithm for UDOs related to respiratory syndromes was critical for the rapid identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enabled us to establish and expand the diagnostic capacity for COVID-19 on a national scale in a timely manner.

2019 Tabletop Exercise for Laboratory Diagnosis and Analyses of Unknown Disease Outbreaks by the Korea Centers for Disease Control and Prevention.

OBJECTIVES: The Korea Centers for Disease Control and Prevention has published "A Guideline for Unknown Disease Outbreaks (UDO)." The aim of this report was to introduce tabletop exercises (TTX) to prepare for UDO in the future. METHODS: The UDO Laboratory Analyses Task Force in Korea Centers for Disease Control and Prevention in April 2018, assigned unknown diseases into 5 syndromes, designed an algorithm for diagnosis, and made a panel list for diagnosis by exclusion. Using the guidelines and laboratory analyses for UDO, TTX were introduced. RESULTS: Since September 9th, 2018, the UDO Laboratory Analyses Task Force has been preparing TTX based on a scenario of an outbreak caused by a novel coronavirus. In December 2019, through TTX, individual missions, epidemiological investigations, sample treatments, diagnosis by exclusions, and next generation sequencing analysis were discussed, and a novel coronavirus was identified as the causal pathogen. CONCLUSION: Guideline and laboratory analyses for UDO successfully applied in TTX. Conclusions drawn from TTX could be applied effectively in the analyses for the initial response to COVID-19, an ongoing epidemic of 2019 - 2020. Therefore, TTX should continuously be conducted for the response and preparation against UDO.

Recent trends of antigenic variation in Bordetella pertussis isolates in Korea.

Pertussis is a representative vaccine-preventable disease. However, there have been recent outbreaks in countries where even higher vaccination against the disease. One reason is the emergence of antigenic variants, which are different to vaccine type. In Korea, reported cases have rapidly increased since 2009. Therefore, we analyzed genotype of strains isolated in 2011-2012 by multilocus sequence typing method. As expected, the genotype profiles of tested genes dramatically changed. The major sequence type changed from ST1 to ST2, and new sequence type (ST8) appeared. In the minimum spanning tree, recent isolates belonging to the ACC-I-ST3 subgroup were detected that were composed of ST2, ST3, and ST6. In particular, the ST2 frequency increased to 81%. The novel ST8 was linked to the increased frequency of ST2. In addition, toxic strains carrying the ptxP3 promoter type were confirmed. This ptxP3 type emerged from 2009 and its frequency had increased to 100% in 2012. Based on these results, it can be inferred that the genotypic changes in the currently circulating strains are strongly associated with the recent increasing of pertussis in Korea. Therefore, the surveillance system should be strengthened, and genetic characterization of the isolates should be expanded to the whole genome sequence level.

A Multicenter Study of Pertussis Infection in Adults with Coughing in Korea: PCR-Based Study.

BACKGROUND: Limited data on the incidence and clinical characteristics of adult pertussis infections are available in Korea. METHODS: Thirty-one hospitals and the Korean Centers for Disease Control and Prevention collaborated to investigate the incidence and clinical characteristics of pertussis infections among adults with a bothersome cough in non-outbreak, ordinary outpatient settings. Nasopharyngeal aspirates or nasopharyngeal swabs were collected for polymerase chain reaction (PCR) and culture tests. RESULTS: The study enrolled 934 patients between September 2009 and April 2011. Five patients were diagnosed as confirmed cases, satisfying both clinical and laboratory criteria (five positive PCR and one concurrent positive culture). Among 607 patients with cough duration of at least 2 weeks, 504 satisfied the clinical criteria of the US Centers for Disease Control and Prevention (i.e., probable case). The clinical pertussis cases (i.e., both probable and confirmed cases) had a wide age distribution (45.7±15.5 years) and cough duration (median, 30 days; interquartile range, 18.0~50.0 days). In addition, sputum, rhinorrhea, and myalgia were less common and dyspnea was more common in the clinical cases, compared to the others (p=0.037, p=0.006, p=0.005, and p=0.030, respectively). CONCLUSION: The positive rate of pertussis infection may be low in non-outbreak, ordinary clinical settings if a PCR-based method is used. However, further prospective, well-designed, multicenter studies are needed.

Yeast ribosomal protein S3 possesses a ß-lyase activity on damaged DNA.

Yeast ribosomal protein S3 has multifunctional activities that are involved in both protein translation and DNA repair. Here, we report that yeast Rps3p cleaves variously damaged DNA that contains not only AP sites and pyrimidine dimers but also 7,8-hydro-8-oxoguanine. This study also revealed that Rps3p has a ß-lyase activity with a broad range of substrate specificity which cleaves phosphodiester bonds of UV or oxidatively damaged DNA substrates. Mutation analysis of the yeast Rps3 protein including introduction of domain deletions and residue replacements identified the residues Asp154 and Lys200 are important for the catalytic activity. In addition, the repair enzyme activity of yeast Rps3p was confirmed by complementation in xth, nfo Escherichia coli cells in which the DNA repair process is defective.

Multilocus Sequence Analysis of Housekeeping Genes and Antigenic Determinant Genes in Bordetella pertussis Strains Isolated in Korea.

OBJECTIVES: To confirm genotype diversities of clinical isolates of Bordetella pertussis and to evaluate the risk of pertussis outbreak in Korea. METHODS: Seven housekeeping genes and 10 antigenic determinant genes from clinical B. pertussis isolates were analyzed by Multilocus sequence typing (MLST). RESULTS: More variant pattern was observed in antigenic determinant genes. Especially, PtxS1 gene was the most variant gene; five genotypes were observed from eight global genotypes. In the bacterial type, the number of observed sequence types in the isolates was seven and the most frequent form was type 1 (79.6%). This major sequence type also showed a time-dependent transition pattern. Older isolates (1968 and 1975) showed type 1 and 6 in housekeeping genes and antigenic determinant genes, respectively. However, these were changed to type 2 and 1 in isolates 1999-2008. This transition was mainly attributed to genotype change of PtxS1 and Fim3 gene; the tendency of genotype change was to avoid vaccine-derived genotype. In addition, there was second transition in 2009. In this period, only the sequence type of antigenic determinant genes was changed to type 2. Based Upon Related Sequence Types (BURST) analysis confirmed that there were two clonal complexes (ACCI and ACCII) in the Korean isolates. Moreover, the recently increased sequence type was revealed as AST2 derived from AST 3 in ACCI. CONCLUSIONS: Genotype changes in Korean distributing strains are still progressing and there was a specific driving force in antigenic determinant genes. Therefore continuous surveillance of genotype change of the distributing strains should be performed to confirm interrelationship of genotype change with vaccine immunity.

Mitochondrial genome of Thais clavigera (Mollusca: Gastropoda): affirmation of the conserved, ancestral gene pattern within the mollusks.

Class Gastropoda includes a large number of described species, many with extensively rearranged mitochondrial genomes. We sequenced the mitogenome of the rock shell, Thais clavigera (Gastropoda: Muricidae), an intertidal snail, using long PCR with primers designed on the basis of expressed sequence tags. The mitogenome of T. clavigera consists of 2 rRNAs, 22 tRNAs, and 13 protein-coding genes, but no control region. Structural comparisons revealed that the order Sorbeoconcha, including T. clavigera, have nearly identical mitochondrial gene patterns. However, they have an inversion between a tRNA(Phe)-tRNA(Glu) cluster that comprises 21 genes, but most of the remaining structure is similar to the putative mollusk ground pattern. These findings will provide a better insight into mitochondrial gene rearrangement over the course of gastropod evolution.

Complete mitochondrial genome of the bullhead torrent catfish, Liobagrus obesus (Siluriformes, Amblycipididae): Genome description and phylogenetic considerations inferred from the Cyt b and 16S rRNA genes.

Mitochondrial DNA (mtDNA) from the bullhead torrent catfish, Liobagrus obesus, was isolated by long-polymerase chain reaction (Long-PCR) with universal primers and was fully sequenced by primer working using flanking sequences. The complete mtDNA from L. obesus was 16,531 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region, demonstrating a structure very similar to that of other bony fish. An analysis of the protein-coding genes revealed a statistically substantiated bias in (T+C): (A+G) content, supporting earlier findings regarding this peculiarity. As indicated by a chi-square test, the observed scores for pyrimidine and purine content were different from those expected assuming a 50:50 ratio: chi(2)=41.63, d.f.=5, p<0.000001 for three categories, including the 1st, 2nd, and 3rd codon positions. Further, there was a difference in nucleotide content between ND6 and the other 12 protein-coding genes in L. obesus. The values of p-distances, as summarized for different scales of evolutionary history at the Cyt b gene, revealed a clear pattern of increased nucleotide diversity at four levels: (1) intraspecies, (2) intragenus, (3) intrafamily, and (4) intraorder. Scores of average p-distances of the four categories in catfish were (1) 1.59+/-0.54%, (2) 5.28+/-1.72% (3) 16.37+/-1.26%, and (4) 19.81+/-0.14%, respectively. These data support the hypothesis that speciation in the order Siluriformes, in most cases, follows a geographic mode through the accumulation of a numerous small genetic changes over a long time period. A phylogenetic tree for the bullhead torrent catfish and several other fish species belonging to the order Siluriformes was developed on the basis of respective Cyt b sequences (1138 bp); the analysis revealed a monophyletic origin for the five examined families. A species-specific clustering of sequences from single species was obtained, supporting additionally basic phylogenetic information for the catfish and the barcoding suitability of Cyt b sequence data. Lastly, one of the well-supported properties of our phylogenetic tree (99% repetition level in our analysis) was the monophyletic placement of all catfish (order Siluriformes) among other ray-finned fish of the class Actinopterigii. Also discussed herein are the aspects of phylogeny based on the 16S rRNA gene.

Molecular cloning and characterization of theta-class glutathione S-transferase (GST-T) from the hermaphroditic fish Rivulus marmoratus and biochemical comparisons with alpha-class glutathione S-transferase (GST-A).

We cloned and sequenced full-length cDNA of a theta-class-like glutathione S-transferase (GST-T) from liver tissue of the self-fertilizing fish Rivulus marmoratus. The full-length cDNA of rm-GST-T was 907 bp in length containing an open reading frame of 666 bp that encoded a 221-amino acid putative protein. Its derived amino acid sequence was clustered with other vertebrate theta-class GSTs in a phylogenetic tree. The deduced amino acid sequence of theta-like rm-GST (rm-GST-T) was compared with both classes (alpha and theta) of GST and alpha-class rm-GST (rm-GST-A). Tissue-specific expression of two rm-GST mRNAs was investigated using real-time RT-PCR. To further characterize the catalytic properties of this enzyme along with rm-GST-A, we constructed the recombinant theta-like rm-GST plasmid with a 6 x His-Tag at the N-terminal of rm-GST-T cDNA. Recombinant rm-GST-T was highly expressed in transformed Escherichia coli, and its soluble fraction was purified by His-Tag affinity column chromatography. The kinetic properties and effects of pH and temperature on rm-GST-T were further studied, along with enzyme activity and inhibition effects, and compared with recombinant rm-GST-A. These results suggest that recombinant rm-GSTs such as rm-GST-A and rm-GST-T play a conserved functional role in R. marmoratus.

Nonylphenol modulates expression of androgen receptor and estrogen receptor genes differently in gender types of the hermaphroditic fish Rivulus marmoratus.

To uncover the effect of estrogenic chemicals [4-nonylphenol (NP) and bisphenol A (BisA)] on the expression of androgen receptor (AR) and estrogen receptors (ERalpha and ERbeta) in the hermaphroditic fish Rivulus marmoratus, we cloned the full length of the cDNAs encoding AR, ERalpha, and ERbeta from gonadal tissue of R. marmoratus and analyzed the modulation of expression of these genes following exposure to estrogenic chemicals using real-time RT-PCR. R. marmoratus AR, ERalpha, and ERbeta genes showed a high similarity to the relevant fish species on amino acid residues, respectively. Rm-ERalpha and Rm-ERbeta cDNAs included a serine-rich region when compared to other teleost fish ER genes. Tissue-specific expression of Rm-AR and Rm-ERbeta mRNAs in adult hermaphrodite R. marmoratus was high in the gonad, while Rm-ERalpha mRNA was high in the liver based on real-time RT-PCR. In addition, Rm-AR and Rm-ERalpha mRNAs increased along with developmental stage from stage 3 (5 dpf) to hatching, while Rm-ERbeta mRNA increased from stage 2 (2 dpf). To uncover the effect of estrogenic chemicals on R. marmoratus, we exposed the fish to NP (300 microg/l) and BisA (600 microg/l) for 96 h. Significant down-regulation of Rm-AR, Rm-ERalpha, and Rm-ERbeta mRNA was observed in gonadal tissue after exposure to NP but not BisA. In the liver, there were gender differences in gene expression after EDC exposure. These results demonstrate that expression patterns of the Rm-AR, Rm-ERalpha, and Rm-ERbeta genes in the hermaphroditic fish, R. marmoratus, vary according to tissue and developmental stage as well as the specificity of environmental estrogenic chemicals. These genes can be useful as molecular biomarkers in assessing the potential impact of estrogenic compounds using this species as a model system.

Cloning and characterization of glutathione S-transferase gene in the intertidal copepod Tigriopus japonicus and its expression after exposure to endocrine-disrupting chemicals.

To investigate the impacts of marine pollution on aquatic organisms, we tested the intertidal copepod Tigriopus japonicus as a model species. To analyze the copepods' responses to endocrine-disrupting chemicals (EDCs), we exposed them to two different chemicals: 4,4'-octylphenol (4,4'-OP, 12.5-100 microg/L for 2 h) and polychlorinated biphenyl (PCB, 6.25-25 microg/L for two days). 4,4'-OP was toxic, although exposure time was limited to 2h. After extracting total RNA from the exposed T. japonicus, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) to determine gene expression patterns following chemical exposure. To analyze the gene expression of T. japonicus, we used glutathione S-transferase with GAPDH as an internal control. Of the genes tested using EDC-exposed samples, 4,4'-OP induced upregulation of the glutathione S-transferase (GST) gene, while PCB caused downregulation of the GST gene. These results suggest that the two EDCs act in different manners in T. japonicus.

Cloning of Ki-ras and Ha-ras cDNAs from the hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and its expression after exposure to 4-nonylphenol.

Previous studies on ras proto-oncogene genes in fish have been focused on chemical-associated carcinogenesis, and the expression of fish ras genes was not well-characterized. We investigated Ki- and Ha-ras genes from the hermaphroditic fish Rivulus marmoratus to understand better their expression patterns in specific tissues, as well as their responses to endocrine-disrupting chemicals such as 4-nonylphenol (4-NP). By investigating expression patterns, we found that the R. marmoratus Ki-ras (Rm Ki-ras) gene showed an alternative splicing event between exons 4A and 4B according to tissue types, which is different from the expression pattern of mammalian Ki-ras genes. In the Rm Ki-ras gene, there were two different expressed types, with exons 1-2-3-4A-4B (long form) and with exons 1-2-3-4B (short form). In the Rm Ki-ras gene, the long form was expressed strongly in the gonad and intestine, and the short form was expressed ubiquitously, except for a low level of expression in the liver. Following 4-NP exposure (300 microg/L), the Rm Ki-ras long form in the liver was significantly expressed, while it was expressed moderately in the ovaries. However, the Rm Ha-ras gene was significantly over-expressed in the brain, while its expression in the gonad was down-regulated. In relation to these modulations after 4-NP exposure, we searched the Rm Ha- and Ki-ras promoter regions and found several ERE-half sites, that may be involved in the modulation of ras gene expression following 4-NP exposure. These genes could be applicable as new biomarker genes for assessing exposure to endocrine-disrupting chemicals (EDCs). Further, this implies the disturbance of ras-dependent signal transduction following EDC exposure.

cDNA cloning and expression of ferritin heavy chain 1, ferritin heavy chain 2 and hemoglobin genes from the fire-bellied frog Bombina orientalis.

We cloned two Bombina orientalis ferritin heavy chains (ferritin heavy chains 1 and 2) and one hemoglobin beta-chain gene from a B. orientalis oviduct cDNA library, and the length of transcripts was 882, 858 and 611 bp encoding 177, 177 and 148 aa, respectively. B. orientalis ferritin heavy chain genes showed high similarity to those of amphibia (88-93%), mammals (70-71%), and fishes (70-72%), and the hemoglobin beta-chain gene showed moderate similarity to amphibian (65-68%) and mammalian (54-57%) hemoglobin beta-chain genes, respectively. Based on phylogenetic analysis, the genes were clustered to the same clade in amphibia. The two B. orientalis ferritin heavy chain genes showed different tissue-specific gene expression patterns. Thus, ferritin heavy chain 1 gene was highly expressed in intestine and oviduct but ferritin heavy chain 2 gene was ubiquitously expressed in most of the examined tissues. The hemoglobin beta-chain gene was more highly expressed in liver than in oviduct. These findings indicate that the genes may play different roles in different tissues. In this paper, we discuss the basic characteristics of B. orientalis ferritin heavy chain genes and hemoglobin beta-chain gene.

Analysis of 686 expressed sequence tags (ESTs) from the intertidal harpacticoid copepod Tigriopus japonicus (Crustacea, Copepoda).

The intertidal harpacticoid copepod Tigriopus japonicus is an important species in the study of marine pollution. To facilitate molecular biomonitoring using T. japonicus, we constructed a T. japonicus unidirectional cDNA library using lambdaZAP expression vector, excised to pBluescript vector with the aid of helper phage, and analyzed 686 randomly picked expressed sequence tags (ESTs) from this species. From the 686 ESTs sequenced, we found several functional genes such as vitellin, kinases and potential detoxification-related genes. We are now preparing a T. japonicus cDNA chip for molecular ecotoxicological studies. In this paper, we discuss the potential use of T. japonicus ESTs and their importance in ecotoxicology.

The complete mitochondrial genome of the rayfish Raja porosa (Chondrichthyes, Rajidae).

We isolated mitochondrial DNA from the rayfish Raja porosa by long-polymerase chain reaction (Long-PCR) with conserved primers, and sequenced it by primer walking method using flanking sequences as sequencing primers. R. porosa mitochondrial DNA consists of 16,972 bp and its structural organization is conserved in comparison with other fishes and mammals. Based on the mitochondrial cytochrome b (cyt b) sequence, the phylogenetic position of R. porosa among cartilaginous fishes was inferred using different phylogenetic methods (ML-based quartet puzzling, Neighbor-joining (NJ) and Bayesian approaches). In this paper, we report the characteristics of the R. porosa mitochondrial genome including structural organization, base composition of rRNAs, tRNAs and protein-encoding genes and characteristics of mitochondrial tRNAs. These findings are applicable to comparative mitogenomics of R. porosa with other related taxa.

Cloning and expression of alpha class glutathione S-transferase gene from the small hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae).

In order to assess its potential as a biomarker of aquatic pollution, an alpha class glutathione S-transferase gene (GSTalpha gene) was cloned from the small hermaphroditic fish Rivulus marmoratus. The R. marmoratus GSTalpha gene spanned 1.3 kb, consisting of 6 exons encoding 221 amino acid residues. It showed high similarity to zebrafish GST. We named this R. marmoratus GSTalpha gene as rm-GSTalpha. The cDNA of the rm-GSTalpha gene was also investigated for its phylogeny, tissue-specific and chemical-induced expression. Rm-GSTalpha was subcloned into a 6 x His-tagged pCRT7 TOPO TA expression vector to produce the recombinant 6 x His-tagged rm-GST protein. This will be used in future to raise an rm-GSTalpha antibody for use in the study of phase II metabolism involved in detoxification. We also exposed R. marmoratus to 300 microg/l of 4-nonylphenol in water, and found approximately 4-fold induction of R. marmoratus GSTalpha mRNA in the treated animals. In this paper, we discuss the characteristics of the R. marmoratus GSTalpha gene as well as its potential use in relation to environmental pollution.

cDNA cloning and expression of a cytochrome P450 1A (CYP1A) gene from the hermaphroditic fish Rivulus marmoratus.

We previously described the genomic structure of the cytochrome P450 1A (CYP1A) gene from the hermaphroditic fish Rivulus marmoratus [Kim, I.-C., Kim, Y.J., Yoon, Y.-D, Kawamura, S., Lee, Y.-S., Lee, J.-S., 2004a. Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphroditic fish Rivulus marmoratus and the Japanese medaka Oryzias latipes. Mar. Environ. Res. 58, 125-129]. To further characterize R. marmoratus CYP1A, we cloned the cDNA sequence of a CYP1A gene from this species and also expressed its recombinant protein in an E. coli system. We exposed R. marmoratus to 4-nonylphenol, and found a small induction of CYP1A mRNA in the treated animals. In this paper, we discuss the characteristics of R. marmoratus CYP1A gene as well as its potential use in a biomonitoring assay.

New preparation of PM2 phage DNA and an endonuclease assay for a single-strand break.

PM2 is a bacteriophage which has closed circular double-stranded DNA as a genome, which is the sole source for endonuclease assay for a single strand break in the fmol range. Therefore, it is important to isolate PM2 DNA with low control nicks for the endonuclease assay. Usually, the isolation method of phage DNA is to use ultracentrifugation which takes at least 4 days. In this report, a fast and effective method which takes only 2 days was developed to purify DNA using polyethylene glycol (PEG) 8000 and the yields of phage DNA isolated by these two methods were compared. The method using PEG 8000 increased the yield of PM2 DNA from 31.2% to 45.2%, and decreased the nick from 17.1% to 13.1%. Recently, the complete PM2 DNA genome sequence of 10,079 bp was published. The exact number of nucleotides of PM2 DNA is important for the correct enzyme assay which measures nicks generated by an endonuclease. The correct calculation of endonuclease activity of rpS3 for nick-circle assay was performed to measure single-strand breaks in this report.