Dual Priming Oligonucleotide Research Articles

A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis.

Establishment and application of DPO-based quadruple real-time PCR for detection of mycobacteria

Objective To establish a rapid, accurate and specific method to detect the common mycobacteria based on multiplex real-time PCR. Methods The dual priming oligonucleotide （DPO）primers and TaqMan probes labeled with FAM，ROX，HEX or JOE fluoresceins at 5′ end and eclipse at 3′ end respectively were designed to detect the 16S rRNA of mycobacteria.Both specificity and sensitivity were estimated on multiplex real-time PCR detecting genome DNA from 4 mycobacterial model species.Sixty eight early morning sputum specimens collected from hospitalized patients in the Red Cross Hospital of Hangzhou were detected by multiplex real-time PCR, bacterial culture and smear microscopy simultaneously.The positive rates were analyzed by chi-square. Results Mycobacteria including Mycobacterium tuberculosis and three common non-tuberculosis mycobacteria spp.were identified by multiplex real-time PCR accurately and specifically, with the limited load at 101 cfu/ml.In 68 sputum specimens，31 were positive (positive rate 45.6%) by this method, which was significant higher than that by smear microscopy (positive rate 14.7%, χ2=15.4,P<0.05).The positive cases were identified as 28 Mycobacterium tuberculosis, 1 Mycobacterium avium and 2 Mycobacterium intracellulare in agreement with the culture results.One case，which is detected by culture，but not by PCR，was identified as Mycobacterium chelonae by sequencing. Conclusion The multiplex real-time PCR characterizing as sensitive, specific and time-saving for Mycobacterium tuberculosis and common non-tuberculosis mycobacteria could be chosen as the rapid laboratory test of mycobacterial infection.（Chin J Lab Med,2012,35:722-725） Key words: Mycobacterium; Multiplex polymerase chain reaction; DNA primers

“Spoligoriftyping,” a Dual-Priming-Oligonucleotide-Based Direct-Hybridization Assay for Tuberculosis Control with a Multianalyte Microbead-Based Hybridization System

We developed "spoligoriftyping," a 53-plex assay based on two preexisting methods, the spoligotyping and "rifoligotyping" assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical Specimens

Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens.

Evaluation of Seeplex® STD6 ACE Detection kit for the diagnosis of six bacterial sexually transmitted infections

Traditionally, the diagnosis of bacterial sexually transmitted infection (STI) has been dependent on the isolation of the causative pathogens by culturing endocervical or urethral swab specimens on selective media. While such procedures typically provide excellent diagnostic accuracy, they are often time-consuming and expensive. A multiplex polymerase chain reaction (PCR) assay, based on a semi-automated detection system, was evaluated for the detection of six STI causative organisms. The Seeplex(®) STD6 ACE (auto-capillary electrophoresis) Detection assay employed six pairs of dual priming oligonucleotide (DPO™) primers specifically targeted to unique genes of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Mycoplasma hominis, and Trichomonas vaginalis. A total of 739 specimens (304 cervical swabs and 435 urine samples) collected for 4 months were tested, and results were compared to those obtained with a combined monoplex PCR. The concordance between the multiplex PCR and monoplex PCR assay was 100% for both sensitivity and specificity. We also tested for the presence of two pathogenic bacteria (C. trachomatis and N. gonorrhoeae) and compared the results obtained with the multiplex PCR and BD ProbeTec duplex strand displacement amplification (SDA). The results of the multiplex PCR and duplex SDA were 99.7% concordant for C. trachomatis and 100% concordant for N. gonorrhoeae. The multiplex PCR assay using the Seeplex(®) STD6 ACE Detection kit proved to be a novel cost-effective and fast diagnostic tool with high sensitivity and specificity for the simultaneous detection of six STI pathogens.

Evaluation of the Seeplex® Meningitis ACE Detection Kit for the Detection of 12 Common Bacterial and Viral Pathogens of Acute Meningitis

BackgroundBacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis.MethodsAnalytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis.ResultsThe lower detection limits ranged from 101 copies/µL to 5×101 copies/µL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent.ConclusionsThe Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens.

Pre‐existing YMDD mutants in treatment‐naïve patients with chronic hepatitis B are not selected during lamivudine therapy

Although the rate at which mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of hepatitis B virus polymerase form is high during prolonged lamivudine (LAM) therapy, these mutations sometimes occur naturally in treatment-naïve patients with chronic hepatitis B. The prevalence of natural YMDD mutants differs geographically, and its clinical significance during LAM therapy is unknown. This study aimed to investigate whether pre-existing YMDD mutants were selected during LAM therapy. It included 14 treatment-naïve patients who were treated with LAM for at least 9 months. LAM resistance was evaluated before and at 3-month intervals during treatment. Mutations were analyzed by direct sequencing, restriction fragment mass polymorphism (RFMP) assays, and a single-step multiplex polymerase chain reaction (PCR) test using dual-priming oligonucleotide (DPO) primers. DPO-based multiplex PCR showed two YMDD mutations in two patients before LAM therapy; rtM204V and rtL180M + rtM204V/I. Further, two patients had an rtL180M mutation without an accompanying rtM204V/I mutation. No mutant was detected in any patient by direct sequencing or the RFMP assay before LAM therapy. A virological response was observed at 3 months in all patients with pre-existing YMDD mutants. All mutations disappeared after 3 months of LAM therapy, and during the follow-up period, no re-emergence was detected by any of the three methods. Further, the viral load was suppressed optimally. In conclusion, pre-existing YMDD mutants were cleared early during the course of LAM therapy, which produced a consistent virological response, and the mutants were not selected by LAM therapy.

DPO multiplex PCR as an alternative to culture and susceptibility testing to detect Helicobacter pylori and its resistance to clarithromycin

BackgroundMacrolide resistance in Helicobacter pylori is the major risk factor for treatment failure when using a proton pump inhibitor-clarithromycin containing therapy. Macrolide resistance is due to a few mutations on the 23S ribomosal subunit encoded by the 23S rRNA gene. The present study aimed at investigating the performance of the dual priming oligonucleotide (DPO)-PCR kit named Seeplex® ClaR-H. pylori ACE detection designed to detect H. pylori and two types of point mutations causing clarithromycin resistance in H. pylori.MethodsThe performance of Seeplex® ClaR-H. pylori ACE detection was evaluated on 127 gastric biopsies in comparison to conventional bacterial culture followed by the determination of susceptibility to clarithromycin by E-test, as well as by an in-house real-time PCR using a fluorescence resonance energy transfer (FRET) technology.ResultsConsidering culture as the reference test, the sensitivity of DPO-PCR and real-time FRET-PCR was 97.7% and 100% while specificity was 83.1% and 80.7%, respectively. However, both PCR were concordant in detecting 14 H. pylori positive cases which were negative by culture. Globally, E-test and DPO-PCR were concordant with regard to clarithromycin susceptibility in 95.3% of the cases (41/43), while real-time FRET-PCR and DPO-PCR were concordant in 95% (57/60).ConclusionThe DPO-PCR is an interesting tool to detect H. pylori on gastric biopsies and to study its susceptibility to clarithromycin in laboratories that cannot perform real-time PCR assays.

Pyrosequencing cut‐off value identifying BRAFV600E mutation in fine needle aspiration samples of thyroid nodules

Recently, tremendous efforts have been made towards the development of sensitive techniques to detect the BRAF(V600E) mutation in fine needle aspiration biopsy (FNAB) samples. However, newly developed quantitative and semi-quantitative methods, such as dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR), have the potential to generate false-positive (FP) results. To eliminate the possibility of FP results, we generated a receiver operating characteristic (ROC) curve to investigate the diagnostic accuracy of pyrosequencing using quantitative data. Cytological diagnoses of 983 thyroid nodules were made according to the Bethesda System 2007. The BRAF(V600E) mutation was analysed by pyrosequencing, and statistical analyses were performed. Of the 983 nodules, 902 were adopted to evaluate the diagnostic value of pyrosequencing. The number of pathologically confirmed malignancies was 192, of which 182 were papillary thyroid cancer (PTC). By generating an ROC curve, we defined the optimal cut-off value of the mutant allele peak as 5·95% (area under the curve, 0·849; sensitivity, 0·55; 1-specificity, 0). When we applied this selective cut-off value, the number of PTCs positive for BRAF(V600E) was 99 (54·4% of the total number of PTCs). With cytology alone, the diagnostic sensitivity and specificity of detecting malignancy were 71·2% and 100%, respectively. Pyrosequencing improved the diagnostic sensitivity from 71·2% to 78·5% (McNemar's test, P < 0·001), without any change in the diagnostic specificity. When 'suspicious for malignancy' was considered a positive cytological outcome, pyrosequencing increased the diagnostic sensitivity of cytology from 95·8% to 96·9%; however, this improvement did not show statistical significance (McNemar's test, P > 0·05). Pyrosequencing is an effective method for detecting the BRAF(V600E) mutation in FNAB samples. By allowing the optimal cut-off value to be determined, pyrosequencing improves the diagnostic sensitivity while eliminating the possibility of FP results.

Simultaneous Detection of Rift Valley Fever, Bluetongue, Rinderpest, and Peste des Petits Ruminants Viruses by a Single-Tube Multiplex Reverse Transcriptase-PCR Assay Using a Dual-Priming Oligonucleotide System

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions.

Evaluation of multiplex PCR using dual-priming oligonucleotide for the detection of vanA and vanB in vancomycin-resistant enterococci

Evaluation of multiplex PCR using dual-priming oligonucleotide for the detection of <i>vanA</i> and <i>vanB</i> in vancomycin-resistant enterococci

Novel Multiplex PCR Using Dual-Priming Oligonucleotides for Detection and Discrimination of the Mycobacterium tuberculosis Complex and M. bovis BCG

We developed a novel multiplex PCR assay using dual-priming oligonucleotide primers targeting the RD1 gene for simultaneous identification of the Mycobacterium tuberculosis complex and M. bovis bacillus Calmette-Guérin (BCG). This assay would be useful both for detection of the M. tuberculosis complex and for differentiation of M. bovis BCG from pathogenic M. tuberculosis complex species.

Comparison of two commercial molecular assays for simultaneous detection of respiratory viruses in clinical samples using two automatic electrophoresis detection systems

Two molecular assays were compared with real-time RT-PCR and viral culture for simultaneous detection of common viruses from respiratory samples: a multiplex ligation-dependant probe amplification (MLPA) and a dual priming oligonucleotide system (DPO). In addition, the positive detections of MLPA and DPO were identified using two different automatic electrophoresis systems. A panel of 168 culture-positive and negative samples was tested by the molecular assays for the presence of influenza A and B virus, respiratory syncytial virus, human metapneumovirus, rhinovirus, coronaviruses, parainfluenza viruses and adenovirus.One hundred and twenty-nine (77%) samples were positive as detected by at least one method. Sixty-nine (41%) samples were positive by cell culture (excluding human metapneumovirus and coronaviruses), 116 (69%) by RT-PCR, 127 (76%) by MLPA and 100 (60%) by DPO. The MLPA yielded results in one attempt for all samples included while 12 (7.2%) samples had to be repeated by the DPO assay due to inconclusive results. The MLPA assay performed well in combination with either electrophoresis system, while the performance of the DPO assay was influenced by the electrophoresis systems.Both molecular assays are comparable with real-time RT-PCR, more sensitive than viral culture and can detect dual infections easily. Results can be obtained within 1 day.

A Comparison Analysis on the Diagnosis of Helicobacter pylori Infection and the Detection of Clarithromycin Resistance according to Biopsy Sites

This study was performed to determine the biopsy sites that are suitable for the diagnosis of Helicobacter pylori infection and to assess the sensitivity of culture, histology, and dual-priming oligonucleotide (DPO)-based multiplex PCR. Moreover, we evaluated the usefulness of PCR for the detection of 23S rRNA mutations, which are responsible for the clarithromycin resistance of H. pylori. From 90 patients, we obtained biopsy specimens for culture, histology, and Seeplex ClaR-H. pylori PCR (Seegene Inc., Korea). Phenotypic susceptibility to clarithromycin was evaluated using the E-test (AB Biodisk, Sweden). H. pylori was detected in 48 of 90 patients. The positive rates of infection in the antrum and body were higher than those in the biopsies obtained from the duodenal bulb. The positive rates in histology, PCR, and culture were 46.7%, 42.2%, and 34.4%, respectively. Using histology or PCR, we identified H. pylori in 46 of the 48 patients. 23S rRNA mutations were detected in 8 patients. The clarithromycin E-test showed that all the 10 wild-type patients were susceptible. However, the results of the PCR and E-test of 3 of the 8 mutation-positive patients were discrepant. We observed that a combination of histology and PCR affords a high detection rate of H.pylori infection and that DPO-based PCR can be practically used for the diagnosis of H. pylori infection and the determination of clarithromycin resistance. These techniques were useful for biopsy sampling simultaneously from the antrum and body for the detection of clarithromycin resistance of multiple strain infection or heteroresistance.

BRAFV600E Mutation Analysis in Fine-Needle Aspiration Cytology Specimens for Evaluation of Thyroid Nodule: A Large Series in aBRAFV600E-Prevalent Population

The BRAFV600E mutation is highly specific to papillary thyroid carcinoma. A test for this mutation may increase the diagnostic accuracy of fine-needle aspiration cytology (FNAC), especially in a BRAFV600E mutation-prevalent population. This prospective study enrolled 1074 patients with thyroid nodules who underwent both FNAC and BRAFV600E mutation analysis by dual-priming oligonucleotide (DPO)-based multiplex PCR in FNA specimens. The ancillary test for BRAFV600E significantly improved the sensitivity of FNA procedure, from 67.5% with FNAC alone to 89.6% with FNAC and the DPO-based multiplex PCR analysis combined. Diagnostic accuracy increased from 90.9 to 96.6%. Nine cases of papillary thyroid carcinoma were detected only by BRAFV600E mutation analysis. Unexpectedly, the preoperative DPO-based multiplex PCR produced five false-positive results, which surgery showed to represent benign nodules. Molecular testing for the BRAFV600E mutation in FNA thyroid nodule specimens increases diagnostic value when applied in a BRAFV600E mutation-prevalent population. However, when using this potentially powerful technique, we must consider both its strengths and its weaknesses.

Detection ofBRAFMutations in Thyroid Nodules by Allele-Specific PCR Using a Dual Priming Oligonucleotide System

With various methods, BRAF mutations have been detected in 73.4% to 87.1% of papillary thyroid carcinomas (PTCs) in Korea. We assessed the ability of allele-specific polymerase chain reaction using a dual priming oligonucleotide system, compared with direct sequencing and pyrosequencing, to detect BRAF mutations in fine-needle aspiration specimens from 85 patients undergoing thyroidectomy. Final pathologic diagnoses were 55 malignant lesions and 30 benign lesions. We detected BRAF mutations in 47 (90%) of 52 PTCs by at least 1 method. The sensitivity of the dual priming oligonucleotide system (88.5%) was slightly higher than that of direct sequencing (82.7%) and pyrosequencing (86.5%). The specificity and positive predictive value of all 3 methods were 100%. The dual priming oligonucleotide system is a simple, rapid, and reliable method for detecting BRAF mutations. This method may be a useful adjunct tool to improve the preoperative diagnostic accuracy of PTC in fine-needle aspiration biopsy specimens.

Simultaneous detection and subtyping of porcine endogenous retroviruses proviral DNA using the dual priming oligonucleotide system

The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes.

Dual priming oligonucleotide–based multiplex PCR analysis for detection of BRAFV600E mutation in FNAB samples of thyroid nodules in BRAFV600E mutation–prevalent area

To evaluate the diagnostic value of dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) for the detection of BRAF(V600E) mutations in ultrasound-guided fine-needle aspiration biopsy (US-FNAB) of thyroid nodules. Our institutional review board approved this retrospective study, and informed consent was not required from patients. The 130 patients underwent US-FNAB to evaluate BRAF status in thyroid nodules. In FNAB washouts, DPO-based multiplex PCR, direct DNA sequencing, and PCR-restriction fragment length polymorphism (RFLP) were used to detect BRAF(V600E). The diagnostic performance of these methods was calculated. We compared cytologic results by BRAF status. Diagnostic accuracy and sensitivity were highest when screening with DPO-based multiplex PCR. BRAF(V600E) positivity was a useful marker at thyroid nodules with "suspicious for papillary thyroid carcinoma" or "inadequate" cytological result. DPO-based multiplex PCR may be an alternative to direct DNA sequencing because of its high sensitivity, high accuracy, and simplicity. BRAF(V600E) may be a useful additional diagnostic marker in BRAF(V600E)-prevalent areas.

Derangements in Endothelial Cell-to-Cell Junctions Involved in the Pathogenesis of Hypercholesterolemia-Induced Erectile Dysfunction

Endothelial cell-to-cell junctions are crucial for vascular formation, networking, and remodeling of blood vessels as well as for inducing and integrating intracellular signals. We investigated the differential expression and distribution of endothelial cell-to-cell junction proteins in the penis of mice with hypercholesterolemia-induced erectile dysfunction. Two-month-old C57BL/6J mice were fed a diet containing 4% cholesterol and 1% cholic acid, and age-matched control animals were fed a normal diet, for 3 months. We performed dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) (Seegene, Seoul, Korea) to screen the differential gene expression of 21 endothelial cell-to-cell junctions. At 5 months, erectile function was measured by electrical stimulation of the cavernous nerve, and the penis was harvested and stained with antibody to claudin-5, vascular endothelial (VE)-cadherin, and platelet/endothelial cell adhesion molecule (PECAM)-1 (N = 8 per group). Cavernous specimens from a separate group of animals were used for claudin-5, VE-cadherin, and PECAM-1 reverse transcriptase-PCR and Western blot analysis. Erectile function was significantly lower in hypercholesterolemic mice than in controls. DPO-based multiplex PCR revealed a profound decrease in the gene expression of endothelium-specific cell-to-cell junction proteins, including claudin-5, VE-cadherin, and PECAM-1, in hypercholesterolemic mice compared with that in controls. The expression of claudin-5, VE-cadherin, and PECAM-1 protein evaluated by Western blot or immunohistochemistry was significantly lower in hypercholesterolemic mice than in controls. These endothelial cell-to-cell junction proteins were more sparsely distributed in the endothelium of cavernous sinusoids than in the endothelium of cavernous artery and dorsal blood vessels. Down-regulation of the endothelial cell-to-cell junctions and decreased endothelial content in the corpus cavernosum might play a major role in the deterioration of erectile function in hypercholesterolemic mice.

Use of a dual priming oligonucleotide system to detect multiple sexually transmitted pathogens in clinical specimens

To evaluate a new dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) assay for detection of six sexually transmitted pathogens, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum and Trichomonas vaginalis. Using 130 clinical specimens, the results obtained by the multiplex PCR, previously established in-house PCR and COBAS Amplicor PCR assays were compared. The specimens frequently contained multiple pathogens (34/130 specimens). The multiplex PCR assay had an overall sensitivity of 96% and specificity of 100% compared to the in-house PCR assay at >20 microg ml(-1) of DNA concentrations in samples and there was no cross-reaction with nonpathogenic Neisseria species that cause the majority of false-positive results with the COBAS Amplicor PCR assay. The DPO-based multiplex PCR assay detected the six sexually transmitted pathogens in clinical specimens with a high sensitivity and specificity, although its sensitivity was dependent on the DNA content of the samples. It is the first report about the new DPO-based technique to detect multiple sexually transmitted pathogens in a single assay, which has considerable potential to diagnose the infections accurately and rapidly.