Conventional Real-time Polymerase Chain Reaction Research Articles

Treponema pallidum ssp. pallidum identification by real-time PCR targetting the polA gene in paraffin-embedded samples positive by immunohistochemistry.

Syphilis is a systemic and sexually transmitted infection caused by Treponema pallidum ssp. pallidum. This spirochete causes different clinical and subclinical stages depending on the duration of infection and immune status of the host. Several tests have been developed for diagnosis, and are classified into direct and indirect methods. The first one includes dark field microscopy, direct fluorescent antibody test in fluids or tissue, and molecular biology techniques. In the indirect method (serologic), the routine tests are used, and are divided in two categories: non-treponemal and treponemal ones. The objective of this work was to identify T. pallidum ssp. pallidum in paraffin-embedded skin biopsies positive by immunohistochemistry, using conventional polymerase chain reaction (PCR) and quantitative real time PCR (qPCR). We included a sample of 17 paraffin-embedded biopsies. DNA was extracted and processed by conventional PCR and real-time PCR with a TaqMan® probe to identify the polA gene. Using PCR, 11 tested positive (64.7%) and 6 (35.3%) were negative. With qPCR and TaqMan® probe, 100% of samples tested positive. The minimum number of spirochetes detected in each sample was 2. With this work, we can conclude that qPCR is a fast and very accurate method for diagnosis of syphilis in tissue specimens.

English

The identification of bee viruses is of great importance where there is no information on their natural occurrence in honeybee populations. Honeybee samples (Apis mellifera L.) obtained from Giza Governorate, Egypt were evaluated for total RNA from the head, thorax and abdomen, and varroa mites. Two fragments of 250 and 540 bp of deformed wing (DWV) and kakugo viruses (KV), respectively, were amplified using conventional real time-polymerase chain reaction (RT-PCR), and were sequenced and analyzed. All bee body parts resulted in a strongly positive DWV sequence except asymptomatic honeybee samples that were negative to the DWV infection. The analysis of varroa mite groups (n = 50) also revealed the presence of DWV which is the first report on this virus in Egypt. Homology search of DWV-PCR amplified fragments revealed 99 and 98% similarities with DWV isolate Warwick-2009 poly-protein gene (accession number: GU109335.1) and the Chilensis DWV isolate, respectively (accession number: JQ413340.1). Also, homology search of KV-PCR amplified fragments using the Genius program 7.1.2 revealed 97 and 90.0% similarities with the KV isolate_Ox genomic sequence (accession number: KC786224.1) and the DWV_Ox genomic sequence (accession number: KC786223.1), respectively. For this study data, a real-time SG RT-Qpcr was found as a fast, simple, sensitive and useful technique to detect and quantify low viral loads in honeybees and varroa mites.   Key words: Deformed wing virus (DWV), kakugo virus (KV), mite, real time-polymerase chain reaction (RT-PCR), Apis mellifera.

Pathways to false-positive diagnoses using molecular genetic detection methods; Phytophthora cinnamomi a case study.

Phytophthora cinnamomi is one of the world's most invasive plant pathogens affecting ornamental plants, horticultural crops and natural ecosystems. Accurate diagnosis is very important to determine the presence or absence of this pathogen in diseased and asymptomatic plants. In previous studies, P. cinnamomi species-specific primers were designed and tested using various polymerase chain reaction (PCR) techniques including conventional PCR, nested PCR and quantitative real-time PCR. In all cases, the primers were stated to be highly specific and sensitive to P. cinnamomi. However, few of these studies tested their primers against closely related Phytophthora species (Phytophthora clade 7). In this study, we tested these purported P. cinnamomi-specific primer sets against 11 other species from clade 7 and determined their specificity; of the eight tested primer sets only three were specific to P. cinnamomi. This study demonstrated the importance of testing primers against closely related species within the same clade, and not just other species within the same genus. The findings of this study are relevant to all species-specific microbial diagnosis.

Development of a PCR Assay for Diagnosing Trematode (Opisthorchis and Haplorchis) Infections in Human Stools.

We developed a combined conventional polymerase chain reaction (PCR) and real-time PCR (qPCR)-based assay for detecting and discriminating between Opisthorchis viverrini and Haplorchis taichui parasite infections. The first PCR amplifies the mitochondrial cytochrome c oxidase subunit I (COI) genes of parasites, and differential diagnosis is achieved by performing qPCR with specific primers and SYBR Green I. The detection limit of the assay was found to be 2.0 × 102 plasmid copies in a test in which a stool sample was spiked with a single egg, which is equivalent to 5 eggs per gram (EPG). The testing of 34 clinical stool samples that had been demonstrated to contain "Opisthorchis-like" eggs by microscopy showed that the novel assay exhibited a sensitivity of 100% for "Opisthorchis-like" parasitic infections, and 71% and 91% of these samples were found to be infected with O. viverrini and H. taichui, respectively. A further four parasitic infections were diagnosed in the 16 negative samples, and the microscopic findings of these samples were confirmed to be false negatives by sequencing analysis. The assay also displayed high specificity during the testing of 10 samples containing other common parasites. The fact that our qPCR SYBR Green I-based assay detected submicroscopic traces of parasitic DNA and was able to differentiate between parasites that produce eggs with similar morphologies indicates that it has a good potential for development of diagnostic application to use in areas where multiple parasites coexist.

Expression of complement C5a receptor and the viability of 4T1 tumor cells following agonist-antagonist treatment.

Complement system is theoretically believed to halt the progression of tumor by the activity of C5a/CD88. Protein C5a is a potent pro.inflammatory mediator that activates the complement system by binding to its receptor. The purpose of this study is to determine the expression of the anaphylatoxin C5a receptor on 4T1 cell line and to study the viability of the cells after being treated with the C5a peptides. The cells 4T1 had undergone immunofluorescence staining, conventional polymerase chain reaction (PCR) and real-time PCR for the expression of determination part. Whereas Alamar Blue and MTT assays were conducted for the viability study of the cells. The cells showed positive result in expressing the receptor of the C5a through immunostaining and PCR. The CT value recorded at initial dilution was 22.24. In cell viability assay, the cell was treated with C5a peptides, PMX205 and EP54. The purpose of this treatment was to see whether C5a had a direct effect on the cell itself using both assays. The result showed that PMX205, which is an antagonist, gave more effects towards the cell as compared with the treatment of EP54. This experiment shows the presence of C5a receptor on 4T1 cell line. We believe that the antagonist peptide is eligible to be used widely in cancer immunotherapy field; but in vivo studies need to be carried out first in the future, as it will determine how these drugs affect the tumor cell growth.

A simple method for extracting DNA from rhododendron plants infected with Phytophthora spp. for use in PCR

AbstractAmong the numerous protocols that describe the extraction of DNA, those relating to the isolation of DNA from infected plants, are rare. This study describes a rapid and reliable method of extracting a high quality and quantity of DNA from rhododendron leaves artificially infected with Phytophthora cactorum, P. cambivora, P. cinnamomi, P. citrophthora, and P. plurivora. The use of the modified Doyle and Doyle protocol (1987) allowed us to obtain high quantity and quality DNA (18.26 μg from 100 mg of the fresh weight of infected leaves at the ratios of A260/280and A260/230- 1.83 and 1.72, respectively), suitable for conventional polymerase chain reaction (PCR) and real-time PCR amplifications.

Development of highly sensitive detection for BCR-ABL transcripts in chronic myeloid leukemia by droplet digital PCR

BCR-ABL tyrosine kinase inhibitors (TKIs) have been the standard treatment for chronic myeloid leukemia (CML) patients. It is known that complete molecular response (CMR) is necessarily equated with eradication of the CML clone, until 50% of patients with stable CMR relapse when imatinib (the first-line TKI treatment) is withdrawn. More sensitive assays for minimal residual disease (MRD) are required to identify patients who are more likely to remain in remission without therapy. In order to improve the performance of conventional real-time polymerase chain reaction (RQ-PCR), it is necessary to consider the factors that limit of detection and specificity of the method. However, droplet digital PCR (ddPCR) approaches this problem in a different way. Using nanofluidic technology, the nucleic acid sample is divided into hundreds of individual replicate PCR reactions. We demonstrated a high sensitive method of ddPCR with lower limit of detection using BCR-ABL p210 plasmid and BCR-ABL transcripts from patient sample dilutions, and compared the results with conventional RQ-PCR method. The results from ddPCR showed successful detection of BCR-ABL transcripts in undetectable samples by RQ-PCR, and sensitivity limit by detecting down to 10 copies per microliter. The correlation of measurements between RQ-PCR and ddPCR from plasmid and clinical samples indicated remarkable correlation between both techniques. Moreover, we used ddPCR to investigate MRD in 3 specimens from one CML patient who sustained CMR by conventional RQ-PCR before relapsed. This approach results in more efficient amplification of BCR–ABL transcripts that may provide an accurate measurement of MRD in CML patients with CMR. Application of this approach in prospective samples enrolled in clinical trials will shed light on its role in predicting relapse. We anticipate that the method will be employed in multiple applications in the clinic, including diagnosis, cancer recurrence monitoring, and treatment management.

Utility of the MALDI-TOF MS method to identify nontuberculous mycobacteria.

In comparison to the conventional real-time polymerase chain reaction method (PCR method) or the DNA-DNA hybridization method (DDH method), the utility of NTM identification by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method has seldom been reported. In this study, 75 clinical NTM isolates from our hospital between April 2013 and July 2014 were identified and analyzed using PCR, DDH, and MALDI-TOF MS methods, and the results for the MALDI-TOF MS method were compared with the others. Identification at the species level was in agreement for 71 (94.5%) of the 75 isolates. For further details, identification was possible for 23 (95.8%) of 24 Mycobacterium avium, 11 (100%) of 11 Mycobacterium intracellulare, and 1 (50%) of 2 isolates mixed with M.avium and M. intracellulare. Mycobacterium ksansasii, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium gordonae, and Mycobacterium chelonae identified by DDH method were same result by MALDI-TOF MS. Additionally, Mycobacterium mucogenicum, which could not be identified by the DDH method, was identified by the MALDI-TOF MS method. However, two isolates identified as Mycobacterium terrae by DDH method could not be identified by the MALDI-TOF MS method and were determined to be Mycobacterium arupense by 16S ribosomal RNA (rRNA) sequence analysis. The present findings show that, for rare bacterial species, identification is sometimes not possible, but, in most cases, the results of identification by the MALDI-TOF MS method have a high concordance rate with the results of the PCR and DDH methods.

Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis

This study is a comparative evaluation (Chi-square test) of a closed tube loop mediated isothermal amplification assay using hydroxy naphthol blue dye (HNB-LAMP), real-time polymerase chain reaction (PCR) and conventional PCR in the diagnosis of intraocular tuberculosis. Considering clinical presentation as the gold standard in 33 patients, the sensitivity of HNB-LAMP assay (75.8%) was higher (not significant, P value 0.2) than conventional PCR (57.6%) and lower than real-time PCR (90.9%). Specificity was 100% by all three methods. No amplification was observed in negative controls (n = 20) by all three methods. The cost of the HNB-LAMP assay was Rs. 500.00 and it does not require thermocycler, therefore, it can be used as an alternative to conventional PCR in resource-poor settings.

An in vitro method for detecting genetic toxicity based on inhibition of RNA synthesis by DNA lesions.

IntroductionA wide variety of DNA lesions such as ultraviolet light-induced photoproducts and chemically induced bulky adducts and crosslinks (intrastrand and interstrand) interfere with replication and lead to mutations and cell death. In the human body, these damages may cause cancer, inborn diseases, and aging. So far, mutation-related actions of DNA polymerases during replication have been intensively studied. However, DNA lesions also block RNA synthesis, making the detection of their effects on transcription equally important for chemical safety assessment. Previously, we established an in vivo method for detecting DNA damage induced by ultraviolet light and/or chemicals via inhibition of RNA polymerase by visualizing transcription.ResultsHere, we present an in vitro method for detecting the effects of chemically induced DNA lesions using in vitro transcription with T7 RNA polymerase and real-time reverse transcription polymerase chain reaction (PCR) based on inhibition of in vitro RNA synthesis. Conventional PCR and real-time reverse transcription PCR without in vitro transcription can detect DNA lesions such as complicated cisplatin DNA adducts but not UV-induced lesions. We found that only this combination of in vitro transcription and real-time reverse transcription PCR can detect both cisplatin- and UV-induced DNA lesions that interfere with transcription.ConclusionsWe anticipate that this method will be useful for estimating the potential transcriptional toxicity of chemicals in terminally differentiated cells engaged in active transcription and translation but not in replication.

Dopamine-assisted synthesis of carbon-coated silica for PCR enhancement.

Polymerase chain reaction (PCR) has become one of the most popular methods to identify genomic information on cells and tissues as well as to solve crimes and check genetic diseases. Recently, the nanomaterials including nanocomposite and nanoparticles have been considered as a next generation of solution to improve both quality and productivity of PCR. Herein, taking into these demands, carbon-coated silica was synthesized using silica particles via polymerization of biocompatible dopamine (PD) to form polydopamine (PDA) film and carbonization of PDA into graphitic structures. For further investigation of the effects of as-prepared silica, PDA-coated silica (PDA silica), and carbonized PDA silica (C-PDA silica), two different types of genes were adopted to investigate the influences of them in the PCR. Furthermore, the strong interaction between the nanocomposites and PCR reagents including polymerase and primers enables regulation of the PCR performance. The effectiveness of the nanocomposites was also confirmed through adopting the conventional PCR and real-time PCR with two different types of DNA as realistic models and different kinds of analytical methods. These findings could provide helpful insight for the potential application in biosensors and biomedical diagnosis.

Enrichment of bacteria samples by centrifugation improves the diagnosis of orthopaedics-related infections via real-time PCR amplification of the bacterial methicillin-resistance gene.

BackgroundTo effectively treat orthopaedic infections by methicillin-resistant strains, an early diagnosis is necessary. Bacterial cultures and real-time polymerase chain reaction (PCR) have been used to define methicillin-resistant staphylococci. However, even when patients display clinical signs of infections, bacterial culture and real-time PCR often cannot confirm infection. The aim of this study was to prospectively compare the utility of real-time PCR for the mecA gene detection following centrifugation of human samples with suspected orthopaedic infections.ResultsIn addition to the conventional real-time PCR method, we performed real-time PCR following centrifugation of the sample at 4,830×g for 10 min in a modified real-time PCR (M-PCR) method. We suspended cultured methicillin-resistant Staphylococcus aureus and generated standard dilution series for in vitro experiments. The in vitro detection sensitivity of the M-PCR method was approximately 5.06 times higher than that of the conventional real-time PCR method. We performed bacterial culture, pathological examination, real-time PCR, and M-PCR to examine the infectious fluids and tissues obtained from 36 surgical patients at our hospital. Of these, 20 patients who had undergone primary total hip arthroplasty were enrolled as negative controls. In addition, 15 patients were examined who were clinically confirmed to have an infection, including periprosthetic joint infection (eight patients), pyogenic spondylitis (two patients), infectious pseudoarthrosis (two patients), and after spine surgery (three patients). In one sample from a patient who developed infectious pseudoarthrosis and two samples from surgical site infections after spine surgery, the mecA gene was detected only by the M-PCR method. In one patient with infectious pseudoarthrosis, one patient with infection after arthroplasty, and two patients with purulent spondylitis, the detection sensitivity of the M-PCR method was increased compared with PCR (clinical sample average: 411.6 times).ConclusionsThese findings suggest that the M-PCR method is useful to detect methicillin-resistant strains infections. In addition, the centrifugation process only takes 10 min longer than conventional real-time PCR methods. We believe that the M-PCR method could be clinically useful to detect orthopaedic infections caused by methicillin-resistant strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1180-2) contains supplementary material, which is available to authorized users.

Fluorescence melting curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences

Previous fluorescence melting curve analysis (FMCA) used intercalating dyes, and this method has restricted application. Therefore, FMCA methods such as probe-based FMCA and molecular beacons were studied. However, the usual dual-labeled probes do not possess adequate fluorescence quenching ability and sufficient specificity, and molecular beacons with the necessary stem structures are hard to design. Therefore, we have developed a peptide nucleic acid (PNA)-based FMCA method. PNA oligonucleotide can have a much higher melting temperature (Tm) value than DNA. Therefore, short PNA probes can have adequate Tm values for FMCA, and short probes can have higher specificity and accuracy in FMCA. Moreover, dual-labeled PNA probes have self-quenching ability via single-strand base stacking, which makes PNA more favorable. In addition, this method can facilitate simultaneous identification of multiple DNA templates. In conventional real-time polymerase chain reaction (PCR), one fluorescence channel can identify only one DNA template. However, this method uses two fluorescence channels to detect three types of DNA. Experiments were performed with one to three different DNA sequences mixed in a single tube. This method can be used to identify multiple DNA sequences in a single tube with high specificity and high clarity.

Development of a TaqMan MGB RT-PCR for the rapid detection of H3 subtype avian influenza virus circulating in China

Previous studies demonstrated that the H3 avian influenza virus (AIV) in China is isolated most frequently from wild birds and live poultry markets. However, there is no subtype-specific real-time polymerase chain reaction (RT-PCR) available for the rapid and highly sensitive identification of H3 AIV. In this study, a TaqMan minor groove binder (MGB) probe and a pair of primers were designed based on a conserved region in the hemagglutinin gene of H3 AIV. These were used to generate an H3-MGB RT-PCR assay that recognizes only H3 AIV. The detection limit of the H3-MGB RT-PCR was 10 copies of DNA per reaction when 10-fold serial dilutions of T-H3HA plasmid were used as the template. This was 1000-times more sensitive than conventional RT-PCR. In experimental samples obtained from oropharyngeal swabs or cloacal swabs, the virus was detected in all ducks using H3-MGB RT-PCR, whereas only one duck tested positive for the virus in oropharyngeal swabs tested using conventional RT-PCR. The H3-MGB RT-PCR assay developed in this study is a sensitive and rapid tool for screening H3 AIV in China.

Understanding trophic interactions of Orius spp. (Hemiptera: Anthocoridae) in lettuce crops by molecular methods.

The aphid Nasonovia ribisnigri (Mosley) (Hemiptera: Aphididae) and the thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) are common pests in Mediterranean lettuce crops, where Orius spp. are common generalist predators. Predation by Orius spp. was studied in a lettuce plot by conventional polymerase chain reaction (PCR) and real-time PCR analyses using specific primers of both main pests. Also, high-throughput sequencing was used to have a wider approach of the diet of these predators in natural field conditions. Molecular analyses indicated a higher predation on N. ribisnigri in spring and on F. occidentalis in summer. Predation on alternative prey, like Collembola, was also found in both seasons. Real-time PCR was more sensitive than conventional PCR in showing the target trophic links, whereas high-throughput sequencing revealed predation on other natural enemies - intraguild predation (IGP), showing other trophic interactions of Orius majusculus within the studied ecosystem. This study gives important information about the trophic relationships present in Mediterranean lettuce crops in different periods of the year. The detected predation by Orius spp. on alternative prey, as well as on other natural enemies, should be further investigated to clarify whether it adds or detracts to the biological control of N. ribisnigri and F. occidentalis.

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.

Monitoring and identification of Cynanchum wilfordii and Cynanchum auriculatum by using molecular markers and real-time polymerase chain reaction

Cynanchum wilfordii (Asclepiadaceae) is widely distributed throughout Korea, Japan, and China. Dried roots of this plant have been used as a tonic to promote renal function. Due to the morphological similarities of the dried roots of this plant to those of Cynanchum auriculatum, which is used as a substitute herbal medicine for C. wilfordii, distinguishing these two species is extremely difficult. The present study was conducted to develop molecular markers to distinguish C. wilfordii and C. auriculatum by using conventional polymerase chain reaction (PCR) and realtime PCR analyses. Comparative analysis based on the sequence of the trnL-trnF intergenic spacer revealed 4 base-pair variations, and the inter-individual sequences of the 2 species separately showed 100% homology. According to these results, the variations were divided into 2 groups. The 2 species were further distinguished using a sequence-characterized amplified region marker developed based on a randomly amplified polymorphic DNA-PCR product, and then a single nucleotide polymorphism marker was designed based on the trnL-trnF intergenic spacer for more efficient detection in real-time PCR. The results showed that speciesspecific molecular markers might allow accurate discrimination of C. wilfordii and C. auriculatum.

The detection of T-Nos, a genetic element present in GMOs, by cross-priming isothermal amplification with real-time fluorescence.

An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06 × 10(3) copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO.

MP47-09 HKLK1 IMPROVES THE ERECTILE FUNCTION IN AGED RATS BY ENHANCING ENOS AND INOS EXPRESSON AND ANTI-TISSUE FIBROSIS EFFECT

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2014MP47-09 HKLK1 IMPROVES THE ERECTILE FUNCTION IN AGED RATS BY ENHANCING ENOS AND INOS EXPRESSON AND ANTI-TISSUE FIBROSIS EFFECT Yang Luan, Tao Wang, Li Zhuan, Yan Zhang, Ya-Jun Ruan, Shao-Gang Wang, Ji-Hong Liu, and Zhang-Qun Ye Yang LuanYang Luan More articles by this author , Tao WangTao Wang More articles by this author , Li ZhuanLi Zhuan More articles by this author , Yan ZhangYan Zhang More articles by this author , Ya-Jun RuanYa-Jun Ruan More articles by this author , Shao-Gang WangShao-Gang Wang More articles by this author , Ji-Hong LiuJi-Hong Liu More articles by this author , and Zhang-Qun YeZhang-Qun Ye More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1462AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Age is the most common risk factor for erectile dysfunction (ED). Human tissue kallikrein1 (hKLK1), as a pleiotropic agent, plays an important role in vasodilation, endothelial cell protection, anti-tissue fibrosis, etc. We aim to explore the effect and preliminarily test the mechanisms of hKLK1 on age-related erectile dysfunction with transgenic rats harboring hKLK1gene (TGR(hKLK1). Methods Four groups of adult male Sprague-Dawley rats were involved in this study: 1) young wild type rats (yWTR, 4 months, n = 10) as control group; 2) young TGR(hKLK1) (yTGR, 4 months, n = 10) group; 3) aged wild type rats (aWTR, 27 months, n = 10) group; 4) aged TGR (hKLK1) (aTGR, 27 months, n = 26) group. Extraction of genome DNA followed by conventional polymerase chain reaction (PCR) and real-time PCR after reverse transcription-PCR of tissue RNA were performed to identify the existence of hKLK1gene. The ratio of peak intracavernous pressure (ICP) to systemic mean arterial pressure (MAP) were measured at various voltages of electrical stimulation to the cavernous nerve (CN) to evaluate the erectile function of rats in each group. The eNOS and iNOS expression in penis were detected by real-time PCR. The ratios of smooth muscle to collagen and apoptosis within the corporal tissue were analyzed by masson's trichrome staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively. Results All TGR (hKLK1) carried hKLK1gene while wild type rats negtively contained it. No conspicuous diversity was found between yWTR and yTGR groups at electrostimulation to CN, real-time PCR, masson's trichrome staining and TUNEL assays. The ICP/MAP ratio of aWTR group was decreased compared with the control group (P<0.05), but the erectile function was greatly improved in aTGR group relative to aWTR group (P<0.05, respectively) and reached the level of control group at all kinds of voltages of 2.5v, 5.0v and 7.5v. The expression of mRNA for eNOS in aTGR was markedly higher than that in aWTR (P<0.05), although it was still lower than yWTR (P<0.01). Moreover, the iNOS mRNA expression in aTGR was obviously increased than that in aWTR and yWTR (P<0.05 of both). The reduction of the smooth muscle/collagen in aWTR, relative to yWTR, was significantly attenuated in aTGR (P<0.01). Meanwhile, the apoptosis data showed the same tendency towards the results of masson's trichrome staining (P<0.001). Conclusions Our results suggest that hKLK1 may improve ED in aged rats via promoting eNOS and iNOS expression and anti-tissue fibosis as well as anti-apoptosis effect. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e521 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yang Luan More articles by this author Tao Wang More articles by this author Li Zhuan More articles by this author Yan Zhang More articles by this author Ya-Jun Ruan More articles by this author Shao-Gang Wang More articles by this author Ji-Hong Liu More articles by this author Zhang-Qun Ye More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture. To prevent and control outbreaks, a rapid, reproducible, sensitive, and effective diagnostic method is needed. We evaluated a new conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) protocol using a primer set (VohB_Fw-VohB_Rv) designed to amplify a 112 bp fragment flanking the vohB gene (coding for hemolysin production), against 24 V. ordalii strains isolated from different fish species, the V. ordalii type strain, and 42 representative related and unrelated bacterial species. The primer set was species-specific, recognizing all V. ordalii strains evaluated, with no cross-reaction with the other bacterial species. A sensitivity of 103 copies of the vohB gene was obtained with a standard curve. When the VohB_Fw-VohB_Rv qPCR protocol was applied to Atlantic salmon seeded tissues (kidney, liver, spleen, and muscle), the detection limit ranged from 5.27 × 102 to 4.13 × 103 V. ordalii CFU ml-1, i.e. 62 to 145 copies of the vohB gene, using the previously calculated standard curve. The conventional PCR also detected V. ordalii, but the total reaction time was 1 h longer. When the qPCR protocol was applied to naturally infected cage-cultured Atlantic salmon samples, 5 of 8 fish tested positive for V. ordalii, but only one of them was diagnosed as positive by direct cultivation on agar. We conclude that the PCR protocol evaluated is fast, specific, and sensitive enough to detect V. ordalii in infected tissues and is an important tool for secure diagnosis of atypical vibriosis, and is therefore helpful for the control of the disease through the prompt detection within fish populations.