DNA Probe Method Research Articles

PCA3 and TMPRSS2: ERG Urine Level as Diagnostic Biomarker of Prostate Cancer.

Prostate cancer is a highly prevalent urological carcinoma with an increasing incidence in Indonesia and all around the world. Early diagnosis can greatly affect treatment outcomes and increase life expectancy. Several biomarkers for detecting prostate cancer have been studied and showed great promise. This study aims to analyze prostate cancer antigen 3 (PCA3) as well as transmembrane serine protease 2:ERG (TMPRSS2:ERG) for diagnosing and serving as urine biomarkers in predicting prostate cancer incidences. We conducted an analytical study to assess the utility of PCA3 and TMPRSS2:ERG for detecting prostate cancer. Thirty samples were included in this study to see the utilization of PCA3 and TMPRSS2:ERG as diagnostic biomarkers of prostate cancer. A urine sample was taken and the PCA3 test was performed using the PCA3 PROGENSA test, while the TMPRSS2:ERG was performed using the chemiluminescent DNA probe method with a hybridization protection test. The average age of the subject was 61.07±8.3 years. Based on calculations using the Mann-Whitney test, there was a significant relationship between prostate-Specific Antigen (PSA) overexpression (p<0.001), TMPRSS2:ERG (p=0.001), and PCA3 (p=0.003) with prostate cancer incidence. The sensitivity of PCA3 and TMPRSS2:ERG in detecting prostate cancer was 76.9% and 92.3%, respectively. Hence, TMPRSS2:ERG and PCA3 can be used as biomarkers for the occurrence of prostate cancer. We also performed a Kruskal-Wallis test; however, there was no significant relationship between PSA (p=0.236), TMPRSS2:ERG (p=0.801), and PCA3 (p=0.091) with the Gleason score. There is a significant correlation between overexpression of PSA, TMPRSS2:ERG and PCA3 with the incidence of prostate cancer, and TMPRSS2:ERG and PCA3 can be used as biomarkers of prostate cancer.

Thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection

Detection of point mutations in driver genes is of great significance for the early diagnosis, treatment, and prognostic evaluation of cancer. However, current detection methods do not offer versatility, specificity, and rapid performance simultaneously. Thus, multiple mutation detection processes are necessary, which results in long processing times and high costs. In this study, we developed a thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection (TTI-CS). This strategy is based on the DNA probe, which changes the thermodynamic balance of the DNA cascade by the designed bubble structure, thereby achieving a good distinction between mutant and wild-type DNA. The designed method greatly shortens the detection time through two-way intrusion. In addition, this method only changes two inexpensive trigger and bridge sequences, which replace the specific and expensive nucleic acid probes used in analyses based on traditional DNA probe methods, thereby enabling multiple detections. We performed the detection of synthetic single-stranded DNA for the five mutation points and successfully detected in endometrial cancer specimens. The detection limit of this method is 0.1%, which better meets the needs of clinical low-abundance multiple mutation detection. Overall, TTI-CS is currently one of the best methods for detecting multiple mutation detections.

Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs.

The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed. From each column, tRNAPhe, tmRNA and 16S rRNA could be purified in a single step. This is the first report of purification of native tmRNA from T. thermophilus and the purification demonstrates that the solid-phase DNA probe method is applicable to non-coding RNA, which is present in lower amounts than tRNA. Furthermore, if a long non-coding RNA is cleaved site-specifically and the fragment can be purified by the solid-phase DNA probe method, modified nucleosides in the long non-coding RNA can be analysed. Therefore, we designed a deoxyribozyme (DNAzyme) to perform site-specific cleavage of 16S rRNA, examined optimum conditions and purified the resulting RNA fragment. Sequencing of complimentary DNA and mass spectrometric analysis revealed that the purified RNA corresponded to the targeted fragment of 16S rRNA. Thus, the combination of DNAzyme cleavage and purification using solid-phase DNA probe methodology can be a useful technique for analysis of modified nucleosides in long non-coding RNAs.

Immunohistochemical detection of V600E BRAF mutation is a useful primary screening tool for malignant melanoma

ABSTRACTBackground: We compared the use of an immunohistochemical (IHC) method using a monoclonal antibody to BRAF V600E (which detects the main BRAF mutation) with existing DNA probe screening in tissue samples from 71 patients with malignant melanoma.Materials and methods: Paraffin blocks were cut to provide consecutive slides for haematoxylin and eosin staining, and for known positive micro-array DNA control material. IHC was performed by the Optiview detection system. All slides were scored independently by the clinical lead and the laboratory lead using a positive/negative system.Results: The DNA method found 26 samples to be positive, the IHC found 21 to be positive, giving a sensitivity value for IHC of 80.8%. However, all of the 45 samples found to be negative by DNA were also negative by IHC, giving a specificity of 100%. There were 66 instances of full agreement, giving a concordance of 93%. Together, these data give a kappa statistic of 0.843, indicating very good agreement.Conclusion: The data reveal a very close link between the two methods, supporting the use of the V600E as a primary screen for BRAF mutations in malignant melanoma. Samples found to be negative by this method may be retested by the DNA probe method. IHC detection conserves patient DNA from tumour blocks as only one section is required to perform the assay. The V600E antibody method is considerably cheaper and faster than the DNA probe assay, with a turn-around time of 24-48 hours, enabling more rapid clinical management.

The Caries Microbiome: Implications for Reversing Dysbiosis.

The oral microbiome plays a critical role in maintaining oral health. Frequent dietary carbohydrate intake can lead to dysbiosis of the microbial community from overproduction of acid with selection for increases in acidogenic, acid-tolerant bacteria. Knowledge of the caries-associated microbiome is key in planning approaches to reverse the dysbiosis to achieve health. For risk assessment and treatment studies, it would be valuable to establish whether microbial monitoring requires assay of multiple species or whether selected key species would suffice. Early investigations of the oral microbiota relied on culture-based methods to determine the major bacteria in health and disease. Microbial monitoring using gene probes facilitated study of larger populations. DNA probe methods confirmed and expanded the importance of transmission of bacteria from mother to infant and association of preselected species, including mutans streptococci and lactobacilli with caries in larger populations. 16S ribosomal RNA (rRNA) probes confirmed the wide diversity of species in oral and caries microbiomes. Open-ended techniques provide tools for discovery of new species, particularly when strain/clone identification includes gene sequence data. Anaerobic culture highlighted the caries association of Actinomyces and related species. Scardovia wiggsiae, in the Actinomyces/Bifidobacterium family, and several Actinomyces species have the cariogenic traits of acid production and acid tolerance. Next-generation sequencing combined with polymerase chain reaction methods revealed a strong association with mutans streptococci in a high caries population with poor oral hygiene and limited access to care. A population with a lower caries experience generally had lower or no Streptococcus mutans detection but harbored other acidogenic taxa in the microbiome. Study of the microbiome suggests a role for the assay of selected putative cariogenic species in more aggressive diseases. For many populations with caries progression, however, assay of multiple species will likely be warranted to determine the caries profile of the population and/or individuals under study.

Improved solid-phase DNA probe method for tRNA purification: large-scale preparation and alteration of DNA fixation.

The solid-phase DNA probe method, in which a target transfer RNA (tRNA) is hybridized with a complementary DNA oligomer, is generally used for tRNA purification. However, purification of tRNAs from thermophiles by this method is not easy because of their high melting temperatures. To overcome this problem, the use of tetraalkylammonium salts was previously reported [Yokogawa, T., Kitamura, Y., Nakamura, D., Ohno, S., and Nishikawa, K. (2010) Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts. Nucleic Acids Res. 38, e89]. In this study, we initially devised a large-scale purification system using tetraalkylammonium salts. The yield of tRNA was increased more than 10-fold and the manual steps were decreased as compared with the previous procedure. However, deterioration of column was very rapid owing to shedding of the biotinylated DNA probe. We therefore devised a method of covalent DNA fixation, in which a 5'-aminohexyl (dT)8 oligomer was fixed onto the N-hydroxysuccinimide-activated agarose, and then a DNA oligomer containing the tRNA and repeated A8 sequences was annealed. The probe sequence for tRNA purification was synthesized in column with Klenow enzyme. This DNA fixation method enabled us to use the column repeatedly and to wash the column with warmed buffers. Thus, this DNA fixation method is economical as compared with the previous method using the biotinylated DNA probe.

Distinct tRNA modifications in the thermo-acidophilic archaeon, Thermoplasma acidophilum

Thermoplasma acidophilum is a thermo-acidophilic archaeon. We purified tRNALeu (UAG) from T. acidophilum using a solid-phase DNA probe method and determined the RNA sequence after determining via nucleoside analysis and m7G-specific aniline cleavage because it has been reported that T. acidophilum tRNA contains m7G, which is generally not found in archaeal tRNAs. RNA sequencing and liquid chromatography–mass spectrometry revealed that the m7G modification exists at a novel position 49. Furthermore, we found several distinct modifications, which have not previously been found in archaeal tRNA, such as 4-thiouridine9, archaeosine13 and 5-carbamoylmethyuridine34. The related tRNA modification enzymes and their genes are discussed.

Standardization of laboratory tests for tuberculosis and their proficiency testing

Explanations of proper collection procedures are imperative for accurate laboratory analysis. The quality of specimens collected and the proper transport of those specimens to the laboratory are critical to successful isolation of etiological agents. Most mycobacteria grow at a relatively slow rate. Therefore, the acid-fast smear plays an important role in the early diagnosis of mycobacterial infection. There are several methods of determining the acid-fast nature of an organism. In the carbolfuchsin procedures (Ziehl-Neelsen, Kinyoun), acid-fast organisms appear red, and in the fluorochrome procedures (auramine O, auramine-rhodamine, acridine orange), the acid-fast organisms fluoresce yellow to orange. Fluorochrome-stained slides may be directly restained with any of the carbolfuchsin staining procedures. This may be done to confirm a positive fluorochrome slide and to study organism morphology. In the last 10 years, there were many advances in the culture examinations of mycobacteria. The newly developed Mycobacteria Growth Indicator Tube (MGIT), BacT/Alert, ESP Myco, MB Redox and KRD "Nichi B", biphasic Septi-Chek AFB and Myco-Acid, and radiometric BACTEC 460TB systems based on liquid media, proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens. In addition to liquid-based medium, agar (Middlebrook 7H10 or 7H11)- or egg (Ogawa or Löwenstein-Jensen)-based media should be used in the primary isolation of mycobacteria. To identify mycobacteria, conventional biochemical tests are traditionally used. Key test can be used to identify species, or further preliminary grouping may be used. Other approaches to identifying some species of mycobacteria are available. They include niacin accumulation, p-nitrobenzoic acid and p-nitro-alpha-acetylamino-beta-hydroxypropiophenone tests for discrimination of the Mycobacterium tuberculosis complex from mycobacteria other than M. tuberculosis (MOTT); DNA probe methods for identification or confirmation of the M. tuberculosis complex, M. avium complex, M. kansasii, and M. gordonae; DNA-DNA hybridization method for identification of 22 Mycobacterium species; and gas-liquid chromatography or high performance liquid chromatography analyses for recognizing the patterns of the mycobacterial cell wall fatty acids or mycolic acids. The advantages of the last four methods are that they are capable of providing definitive identification within 2 to 5 h after adequate growth. Capilia TB is the newly developed immunochromatographic assay for rapid discrimination between the M. tuberculosis complex and MOTT bacilli. The kit can be easily used for rapid identification of the M. tuberculosis complex in combination with the culture systems based on liquid media. In addition, Capilia TB could correctly detect the M. tuberculosis complex from mixed cultures with the M. tuberculosis complex and MOTT bacilli. The WHO/IUATLD supranational reference laboratory (SRL) network was created in 1994, to ascertain the accuracy of the susceptibility test methods used in different laboratories across the world, and to allow comparability of the surveillance data gathered in countries participating in the Global Project on Anti-tuberculosis Drug Resistance Surveillance. Today, the network has evolved and 23 SRLs actively participate. Results of five rounds of proficiency testing in the SRL network suggest that performance of the network has improved substantially through the years. This progress has been particularly evident for streptomycin and ethambutol sensitivity, which was very low in the first rounds of proficiency testing. In 1998 sensitivity for these two drugs was higher than 95%. For isoniazid and rifampin, sensitivity has been consistently high since the beginning of the Global Project. This indeed reflects the enhanced efforts made by the SRLs to improve their individual performance.

In vitro and in vivo production and purification of circular RNA aptamer

RNA aptamers are potential candidates for RNA therapeutics. They must be clinically modified for medical applications because they are vulnerable to indigenous ribonucleases. Since circular RNA molecules without any chemical modification are much more stable than linear ones in a cell extract, we report the production of a circular form of streptavidin RNA aptamer both in vitro and in vivo. Circularization was accomplished by self-splicing permuted intron–exon sequences derived from T4 bacteriophage gene td. This sequence was producible in both Escherichia coli cells and in vitro. The circularized streptavidin RNA aptamer retained its binding of streptavidin and was stabile in HeLa cell extracts compared to the linear form of the streptavidin aptamer. The self-spliced circular RNA from the transcribed permuted intron–exon transcripts in E. coli cells was purified from a total RNA fraction using the solid-phase DNA probe method following anion exchange chromatography that excluded gel electrophoresis. This study provides an alternative method for designing and purifying useful RNA aptamers.

Vaginal swabs are appropriate specimens for the diagnosis of genital tract infection with Chlamydia trachomatis

Objectives. Self-administered vaginal swabs were used to determine the prevalence of Chlamydia trachomatis in a population under 25 years of age in order to evaluate if this is an acceptable and accurate method compared to the traditional cervical swabs. Methods. Patients were asked to perform a double self-administered vaginal swab prior to a traditional cervical swab. One self-collected vaginal swab was used with the AMPLIFIED CT DNA PROBING (GENPROBE bioMérieux) method that is based on a DNA probe against an amplified target for the identification of the Chlamydia trachomatis ribosomal RNA. The remaining two swabs were processed using BD PROBETEC ET (Becton Dickinson) for the determination of amplified DNA, using standard displacement amplification technology. Results. 8.2% of the 182 patients that were analysed between September 2005 and March 2006 tested positive. The two methods had an equivalent sensitivity and a specificity. It is important to concentrate prevention on young females who may have greater contacts with local specialists and could benefit from the self-administered vaginal swab technique.

Production of circular streptavidin RNA aptamer in vivo

RNA aptamers is one of highly hopeful candidates for RNA therapeutics. We previously reported an in vitro production of a circular streptavidin RNA aptamer. Here we show an application for producing the circular RNA aptamer in vivo. First, we constructed a circular RNA expression vector that contained self-splicing permuted intron-exon (PIE) sequences between T7 promoter and T7 terminator sequences so as to be transcribed by T7 RNA polymerase produced in JM109(DE3) cells. RNA expression driven by T7 RNA polymerase was triggered by addition of IPTG and the circularized RNA was generated from the resulting PIE transcripts. Circular streptavidin RNA aptamer generated in the JM109(DE3) cells was detected by a two dimensional denaturing PAGE analysis using the ethidium bromide staining. Northern blot analysis using a self-ligated sequence specific oligo DNA probe and sequencing analysis revealed that the self-splicing and circularization process precisely occurred in E. coli. The circular aptamer was easily purified by a solid phase DNA probe method from a partially purified total RNA fraction. This is the first demonstration of an in vivo expression of a circular RNA aptamer and its purification, paving the new way for inexpensive production of RNA aptamer.

Evaluation of an Alkaline Phosphatase–Labeled Oligonucleotide Probe for the Detection and Enumeration of the Thermostable-Related Hemolysin (trh) Gene of Vibrio parahaemolyticus

Reliable methods are needed to detect total and pathogenic Vibrio parahaemolyticus. One marker of V. parahaemolyticus virulence is the thermostable-related hemolysin. We developed an alkaline phosphatase–labeled DNA probe method for the specific detection and enumeration of trh-positive V. parahaemolyticus by colony hybridization. The probe was tested against a panel of 200 bacterial strains and determined to be specific for trh-positive V. parahaemolyticus. Additionally, the trh alkaline phosphatase probe colony hybridization was successfully used to detect and enumerate trh-positive V. parahaemolyticus in seafood and water samples collected from the United States and the United Kingdom.

Heterosigma akashiwo in central California waters

Heterosigma akashiwo (Hada) gives rise to red tides along the Atlantic and Pacific coasts and is known to produce brevetoxins. This investigation establishes baseline information showing the presence of H. akashiwo along the central California coast based on water samples collected from the Santa Cruz pier in Monterey Bay (on the open coast) and the Berkeley pier in San Francisco Bay. Light and electron microscopy as well as two species-specific DNA probe methods based on cell homogenates preparations were employed to detect H. akashiwo during the 2001–2002 field study. The DNA probe methods consisted of a sandwich hybridization assay (SHA), which targets ribosomal RNA (rRNA), and an end-point polymerase chain reaction (PCR) assay, which targets internal transcribed spacer (ITS) sequences of rRNA genes. The SHA was used to provide semi-quantitative data showing the intermittent presence of the species during a 13-month period in Monterey Bay. Samples that showed a variety of responses in the SHA (negative as well as the highest) were then subjected to the PCR assay in an attempt to confirm species identification using an independent DNA probe method that employs cell homogenates; samples included those from Monterey Bay and one from a red tide event in San Francisco Bay. SHA and PCR assays agreed on the presence or absence of H. akashiwo. Gene products from two field samples positive for H. akashiwo by PCR were cloned and sequenced and found to be identical to those of that species in GenBank. When the same samples were viewed by light microscopy, however, H. akashiwo cells were only seen in the sample with the highest abundance of that species, as evidenced by SHA. It was extremely difficult to recognize naturally occurring H. akashiwo using light microscopy in field samples that had been preserved with Lugol's iodine, including samples that gave positive results by cell homogenate methods. Results of this study indicate that H. akashiwo is present along the open California coast and could easily be missed in routine phytoplankton surveys. Despite its presence, H. akashiwo does not appear to routinely bloom with sufficient densities to cause harmful outbreaks of the frequency and severity documented in some other coastal environments. Molecular identification techniques may be the preferred approach over light microscopy when there is a need to rapidly screen many samples for fragile, harmful species and those that are otherwise problematic to identify based on their gross morphology alone.

Overlapping Gene Mutations of Hepatitis B Virus in a Chronic Hepatitis B Patient with Hepatitis B Surface Antigen Loss during Lamivudine Therapy

Disappearance of hepatitis B surface antigens (HBsAg) in chronic hepatitis B usually indicates clearance of hepatitis B virus (HBV) infection. However, false HBsAg negativity with mutations in pre-S2 and 'a' determinant has been reported. It is also known that YMDD mutations decrease the production of HBV and escape detection of serum HBsAg. Here, we report overlapping gene mutations in a patient with HBsAg loss during the lamivudine therapy. After 36 months of lamivudine therapy in a 44-yr-old Korean chronic hepatitis B patient, serum HBsAg turned negative while HBV DNA remained positive by a DNA probe method. Nucleotide sequence of serum HBV DNA was compared with the HBV genotype C subtype adr registered in NCBI AF 286594. Deletion of nucleotides 23 to 55 (amino acids 12 to 22) was identified in the pre-S2 region. Sequencing of the 'a' determinant revealed amino acid substitutions as I126S, T131N, M133T, and S136Y. Methionine of rtM204 in the P gene was substituted for isoleucine indicating YIDD mutation (rtM204I). We identified a HBV mutant composed of pre-S2 deletions and 'a' determinant substitutions with YMDD mutation. Our result suggests that false HBsAg negativity can be induced by combination of overlapping gene mutations during the lamivudine therapy.

Syndrome-related chromosome-specific radiation-induced break points of various inherited human metabolic disorders

The frequency, distribution pattern and localisation of gamma radiation-induced break points on the chromosomes of patients with various inherited metabolic disorders were studied to detect: (i) whether the break point distribution following irradiation is random and proportional to the length or the DNA content of the chromosome, or non-proportionally distributed on their length and at times clustering to form hot spots on certain region of the chromosomes; and (ii) to find whether there exists a syndrome-related chromosome-specific pattern of radiation-induced break points. Lymphocyte cultures from patients of haemophilia, ichthyosis, Duchenne muscular dystrophy, retinitis pigmentosa and α-thalassemia, whose defective gene loci were located by DNA probe method, were subjected to 3 Gy of gamma radiation at G 0. The chromosomal break point analysis was carried out on all the 23 types of chromosomes (excluding Y chromosome) using G banding and FISH painting. The exact location of the break points on G-banded chromosomes was identified using a semi-automated microscope densitometer system (Leitz MPV2). In normal individuals in all the chromosomes except the chromosome 1, a random distribution of break points proportional to their length based on their DNA content was observed. However, in all the syndromes studied a mixture of hypersensitive chromosomes with a non-random distribution pattern of chromosomal break points invariably clustering to form hot spots, and chromosomes with random distribution of break points proportional to their length were observed. The hypersensitive chromosomes and their hot spots were syndrome-specific.

자연 시료로부터 Alexandrium tamarense을 위한 종 특이적 DNA탐침의 응용

독성이 있는 특이한 편모충인 Alexandrium tamarense의 구별하기 위한 종특이적인 형광 DNA탐침 AT1이 서로 다른 3가지의 고정액에서, 그리고 배양기간의 차이와 whole cell hybridization을 사용하는 광학 현미경 또는 형광 현미경에 의한 세포밀도 측정에 있어서 hiding activity를 비교하는 방법으로 여러 다른 종의 실험에 사용되어졌고, 그의 결과를 보고하는 바이다. 형광 DNA탐침 AT1은 특이적으로, A. tamarense에 결합했지만, 형태학적으로 유사한 다른 편모충에는 결합하지 않았다. 특히 형태적으로 A. tamarense에 유사한 A. catenella는 형광 DNN탐침 AT1에 결합하지 않았다. A. tamarense은 다른 세가지 고정액을 처리하였을 때, 고정액과 상관없이 강한 형광신호를 발산하였다. 추가해서 말하면, 배양기간에 관계없이 형광 DNA탐침 AT1의 biding activity는 강했다. 광학 현미경에 의해서 측정 되어지기 보다는 형광현미경에 의해서 세포밀도가 측정되었다. 형광 DNA탐침 AT1을 이용한 A. tamarense의 동정과 계산은 이 분야에서 새로운 생물독성 감시와 예측 시스템에 기여 할 것이다. Fluorescent species-specific DNA probe (AT1) of toxic dinoflagellate Arexandrium tamarense was tested on several other species, on comparison of binding activity at different preservatives for fixation of the cells, at different culture age and estimation of cell density by light microscope or epifluorescent microscope using whole cell hybridization. Th AT1 probe specifically bound to Alexandrium tamarense, whereas it did not bind to other phytoplankton, in particular Alexandrium catenella, morphologically similar to Alexandrium tamarense, could not react to AT1 probe. When cells were fixed with all three preservatives, labeling cells of Alexandrium tamarense emitted strong fluorescent signal intensity. In addition, regardless culture days, binding activity with AT1 probe was strong. The tell densities estimated by epifluorescent microscope were than those estimated by light microscope. The enumeration and identifying of Arexandriurn tamarense using DNA probe method will be contributed to a new biotoxin monitoring and prediction system in field.

The toxR gene of Vibrio (Listonella) anguillarum controls expression of the major outer membrane proteins but not virulence in a natural host model.

To examine the hypothesis that the ancestral role of the toxR gene in the family Vibrionaceae is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathogen. The toxR gene of V. angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed. The toxR sequence was 63 to 51% identical to those reported for other species of the family Vibrionaceae. Distribution of the Va-toxR gene sequence in V. anguillarum strains of various serotypes was confirmed by using DNA probe and PCR methods. An isogenic toxR mutant of V. anguillarum PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method. The wild-type strain and the toxR mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods. A 35-kDa major OMP was not produced by the toxR mutant. However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the toxR mutant. For the toxR mutant, the MICs of two beta-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain. Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the toxR-regulated major OMPs of the family Vibrionaceae. The results indicate that the toxR gene is not involved in virulence expression in V. anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae. These results support the hypothesis that the ancestral role of the toxR gene is regulation of OMP gene expression and that only in some Vibrio species has ToxR been appropriated for the regulation of a virulence gene(s).

Identification of Streptococcus salivarius by PCR and DNA probe.

To establish species-specific PCR and DNA probe methods for Streptococcus salivarius and to clarify the distribution of dextranase in oral isolates of Strep. salivarius. A pair of PCR primers and a DNA probe were designed based on the nucleotide sequence of the dextranase gene of Strep. salivarius JCM5707. Both the PCR primer and the DNA probe specifically detected Strep. salivarius but none of the other oral streptococci (23 strains of 13 species). The primer and the probe were capable of detecting 1 pg and 1 ng of the genomic DNA, respectively, purified from Strep. salivarius JCM5707. All oral isolates (130 strains from 12 subjects) of Strep. salivarius from human saliva were positive by both methods. The present PCR and DNA probe methods are highly specific to Strep. salivarius and are useful for the its detection and identification of this bacterium. The dextranase widely distributes among oral isolates of Strep. salivarius. The DNA sequence of a dextranase gene present in the genome of Strep. salivarius is useful as the target DNA of the species-specific PCR and DNA probe.

Effects of continuous hepatitis with persistent hepatitis C viremia on outcome after resection of hepatocellular carcinoma.

The effect of persistent hepatitis C viremia on the outcome after resection of hepatocellular carcinoma (HCC) was investigated in 59 consecutive patients with a single small HCC (??3.0 cm in diameter). The presence of serum hepatitis C virus (HCV) RNA was evaluated using a reverse transcription polymerase chain reaction method as well as a branched DNA probe method. Clinicopathologic findings were compared between patients with and without viremia and the risk factors for poor outcome were evaluated. Hepatitis C virus (HCV) RNA was not detected in the sera from 7 patients (group 1), but was detected in the sera from the other 52 patients (group 2). Alanine a minotransferase (ALT) activity was significantly higher in group 2 than in group 1. The proportion of patients with active hepatitis was significantly higher in group 2. In group 2, new HCC often developed after the operation and four patients died of liver dysfunction. HCV viremia, high ALT activity, high concentration of total bilirubin, and liver cirrhosis were related to recurrence after the operation. Multivariate analysis indicated that HCV viremia and high ALT activity were independent risk factors for recurrence of HCC. Continuous hepatitis with persistent HCV viremia worsened the outcome after the resection of HCC by causing new development of HCC and deterioration of liver function. In patients with HCV‐related HCC, but without HCV viremia, satisfactory results can be expected after liver resection.

Potential for routine concurrent determination of chlamydia and cervical abnormalities by single fluid-based sampling

Objective: The FDA recently approved adjunctive HPV testing from the vial of PreservCyt® Solution used for the ThinPrep® Pap Test™. We have now evaluated the potential for routine chlamydia testing from cellular material collected for primary Pap testing using the fluid-based ThinPrep method. Methods: Cervical scrapings were collected for a conventional Pap smear and residual material adhering to the sampling implement after slide preparation was suspended in PreservCyt Solution. Chlamydia testing was performed on this residual material using two methods. 1) A slide was prepared using the ThinPrep 2000 System for a determination by direct fluorescence. 2) One mL of the cell suspension was removed and processed for determination by an amplified DNA probe technique. A separate conventional endocervical swab sampling from the same patient was used for testing by an independent direct DNA probe technique (reference method). One hundred specimens were selected and intentionally biased, 19 positive and 81 negative based on the independent swab results, for comparison to the slide and amplified DNA probe methods. Results: The slide-based method gave a sensitivity of 84% (16/19) and a specificity of 100% (81/81). Of 3 discrepant specimens, 2 tested positive by the amplified probe method, while all 3 remained negative by repeat DFA. The amplified probe method gave a sensitivity of 95% (18/19) and a specificity of 99% (80/81) after analysis of discrepant samples by repeat testing. Conclusions: Feasibility has been demonstrated for accurate and effective routine testing for chlamydia with either DFA or DNA probe methods, from a single, fluid-based sample already approved for primary Pap screening.