Results Of Polymerase Chain Reaction Assay Research Articles

An Automatic Implementation of Oropharyngeal Swab Sampling for Diagnosing Respiratory Infectious Diseases via Soft Robotic End-Effectors

The most widely adopted method for diagnosing respiratory infectious diseases is to conduct polymerase chain reaction (PCR) assays on patients’ respiratory specimens, which are collected through either nasal or oropharyngeal swabs. The manual swab sampling process poses a high risk to the examiner and may cause false-negative results owing to improper sampling. In this paper, we propose a pneumatically actuated soft end-effector specifically designed to achieve all of the tasks involved in swab sampling. The soft end-effector utilizes circumferential instability to ensure grasping stability, and exhibits several key properties, including high load-to-weight ratio, error tolerance, and variable swab-tip stiffness, leading to successful automatic robotic oropharyngeal swab sampling, from loosening and tightening the transport medium tube cap, holding the swab, and conducting sampling, to snapping off the swab tail and sterilizing itself. Using an industrial collaborative robotic arm, we integrated the soft end-effector, force sensor, camera, lights, and remote-control stick, and developed a robotic oropharyngeal swab sampling system. Using this swab sampling system, we conducted oropharyngeal swab-sampling tests on 20 volunteers. Our Digital PCR assay results (RNase P RNA gene absolute copy numbers for the samples) revealed that our system successfully collected sufficient numbers of cells from the pharyngeal wall for respiratory disease diagnosis. In summary, we have developed a pharyngeal swab-sampling system based on an “enveloping” soft actuator, studied the sampling process, and implemented whole-process robotic oropharyngeal swab-sampling.

First detection of bovine tuberculosis by Ziehl-Neelsen staining and polymerase chain reaction at dairy farms in the Lekok Sub-District, Pasuruan Regency, and Surabaya region, Indonesia.

Bovine tuberculosis (TB) is a zoonotic disease of great public health importance, particularly in Indonesia, where control measures are limited or are not implemented. This study aimed to detect the presence of Mycobacterium pathogens in milk samples from dairy cattle in Pasuruan regency and Surabaya City, East Java, using Ziehl-Neelsen acid-fast staining and polymerase chain reaction (PCR). Milk samples were aseptically collected from 50 cattle in the Lekok Subdistrict, Pasuruan Regency, and 44 from dairy farms in the Lakarsantri Subdistrict, Wonocolo Subdistrict, Mulyorejo Subdistrict, and Kenjeran Subdistrict, Surabaya, East Java. To detect Mycobacteria at the species level, each sample was assessed by Ziehl-Neelsen staining and PCR using the RD1 and RD4 genes. The results of PCR assay from 50 samples in Lekok Subdistrict, Pasuruan Regency showed that 30 samples (60%) were positive for Mycobacterium tuberculosis and two samples (4%) were positive for Mycobacterium bovis, although Ziehl-Neelsen staining did not show the presence of Mycobacterium spp. In the Surabaya region, 31 samples (70.45%) were positive for M. tuberculosis and three samples (6.8%) were positive for M. bovis. Six samples (13.63%) from all PCR-positive samples could be detected microscopically with Ziehl-Neelsen. The presence of bovine TB in this study supports the importance of using a molecular tool alongside routine surveillance for a better understanding of the epidemiology of bovine TB in East Java.

Multiplex Polymerase Chain Reaction Assay to Detect Nasopharyngeal Viruses in Immunocompromised Patients With Acute Respiratory Failure

In immunocompromised patients with acute respiratory failure (ARF), the clinical significance of respiratory virus detection in the nasopharynx remains uncertain. Is viral detection in nasopharyngeal swabs associated with causes and outcomes of ARF in immunocompromised patients? This preplanned post hoc analysis of a randomized controlled trial enrolled immunocompromised patients admitted to 32 ICUs for ARF between May 2016 and December 2017. Nasopharyngeal swabs sampled at inclusion were assessed for 23 respiratory pathogens using multiplex polymerase chain reaction (PCR) assay. Causes of ARF were established by managing physicians and were reviewed by three expert investigators masked to the multiplex PCR assay results. Associations between virus detection in nasopharyngeal swabs, causes of ARF, and composite outcome of day 28 mortality, invasive mechanical ventilation (IMV), or both were assessed. Among the 510 sampled patients, the multiplex PCR assay results were positive in 103 patients (20.2%), and a virus was detected in 102 samples: rhinoviruses or enteroviruses in 35.5%, coronaviruses in 10.9%, and flu-like viruses (influenza virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus) in 52.7%. The cause of ARF varied significantly according to the results of the multiplex PCR assay, especially the proportion of viral pneumonia: 50.0%with flu-like viruses, 14.0%with other viruses, and 3.6%when no virus was detected (P< .001). No difference was found in the composite outcome of day 28 mortality, IMV, or both according to positive assay findings (54.9%vs54.7%; P= .965). In a pre-established subgroup analysis, flu-like virus detection was associated with a higher rate of day 28 mortality, IMV, or both among recipients of allogeneic hematopoietic stem cell transplantation compared with those without detected virus. In immunocompromised patients with ARF, the results of nasopharyngeal multiplex PCR assays are not associated with IMV or mortality. A final diagnosis of viral pneumonia is retained in one-third of patients with positive assay results and in one-half of the patients with a flu-like virus.

Microarray Profile of Circular RNAs Identifies hsa_circ_0001583 as A New Circular RNA Biomarker for Breast Cancer: A Retrospective Study.

Objective Breast cancer (BC) is the most common cancer, which is currently the leading cause of cancer death. Circular RNAs (circRNAs) play important roles in cancer, however, circRNAs serving as vital index in BC for guiding treatment have not yet been identified. The aim of our study is to explore a novel kind of potential biomarker for BC. Materials and Methods In this retrospective study, the samples used for assays were two groups of breast tumor tissue obtained from four BC patients, including four pairs of tumor tissues and adjacent nontumor samples. The circRNA expression profiles were detected via microarray and validated by real-time quantitative polymerase chain reaction (PCR). Results The differentially expressed circRNAs in tested samples were screened and analyzed by using human circRNA microarray. After analysis, considering a fold gene expression change of ≥2.0 and P<0.05, results suggested that 256 circRNAs were significantly up-regulated and 277 circRNAs were significantly down-regulated. Besides, the results of the real-time quantitative PCR assay showed that the expression of hsa_circ_0001583 was significantly up-regulated in BC groups (P<0.05) by real-time quantitative PCR. Therefore, we thought hsa_circ_0001583 might serve as a novel kind of biomarker for BC. ConclusionHsa_circ_0001583 showed significant up-regulation in BC patients with paired adjacent tissues. Many cancer immune pathways were related to has_circ_0001583, including autoimmune thyroid disease, chemokine and T-cell receptor signaling pathways.

Antimicrobial resistance profiling and burden of resistance genes in zoonotic Salmonella isolated from broiler chicken.

Background Salmonella is frequently found in poultry of which only motile serovars have zoonotic significance due to their potential to induce human gastrointestinal infections. Antimicrobial resistance, being a public health concern, the emergence of multidrug‐resistant (MDR) Salmonella serotypes affecting food chain has greater impact worldwide.AimInformation on circulation of zoonotic Salmonella strains in commercial poultry farm level is limited in many parts of the world. This cross‐sectional study was aimed to investigate the zoonotic Salmonella strains circulating in the broiler farm environment with their detailed antimicrobial resistance profiling.MethodsPooled faecal samples were collected randomly from commercial broiler farms of Chattogram district, Bangladesh. Standard bacteriological procedure was followed to isolate Salmonella, and identification was confirmed by genus specific polymerase chain reaction (PCR). After phenotypic characterisation of resistance profile against eight antimicrobials by disc diffusion technique, all strains were screened by PCR for some selected resistance genes. ResultsOut of the 350 samples, Salmonella was isolated and identified from 86 samples. In antimicrobial sensitivity testing, more than 98.8% isolates showed resistance to ampicillin and 94.2% to tetracycline followed by enrofloxacin (56%) and ciprofloxacin (50%). Notably, 94% isolates were found to be MDR. The results of PCR assays revealed that 81.4% of the isolates were carrying the tetA gene, 19.8% the tetB and 10.47% the tetC gene. The prevalence of the isolates bearing the bla TEM, bla CTX‐M and Sul‐I gene were 95.4%, 7.0 % and 37.2 %, respectively.ConclusionThere is a great risk to secure healthy poultry products due to the circulation of these MDR zoonotic Salmonella

Simultaneous detection of feline parvovirus and feline bocavirus using SYBR Green I-based duplex real-time polymerase chain reaction.

Since both feline parvovirus (FPV) and feline bocavirus (FBoV) can cause diarrhea in cats, it is difficult to distinguish them clinically. This study aimed to develop a SYBR Green I-based duplex real-time polymerase chain reaction (PCR) assay for distinguishing FPV and FBoV-1 on the basis of the melting temperature of the PCR product. A total of 132 fecal samples from different domestic and feral cats were collected, and the results of SYBR Green I-based duplex real-time PCR assay were compared with those of the traditional PCR assay for a comprehensive evaluation. The melting temperatures were found to be 86 °C and 77.5 °C for FBoV-1 and FPV, respectively, and no specific melting peaks for other non-targeted feline viruses were observed. The data obtained from this assay had a good linear relationship; the detection limits of FPV and FBoV-1 were 2.907 × 101 copies/μL and 3.836 × 101 copies/μL, respectively. In addition, the experiment exhibited high reproducibility. The positive detection rates of the SYBR Green I-based duplex real-time PCR assay for FPV and FBoV-1 were 16.67% (22/132) and 6.82% (9/132), respectively, and the positive detection rate for co-infection with FPV and FBoV-1 was 3.03% (4/132). This result was much more sensitive than that of the traditional PCR method. Thus, the developed SYBR Green I-based assay is a sensitive, rapid, specific, and reliable method for the clinical diagnosis of FPV and FBoV-1 and can provide technical support for the simultaneous detection of co-infection with these viruses in the future.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13205-021-02947-w.

Experience of an Orthopaedic Surgery Department Early During the COVID-19 Outbreak in Japan Including Real-Time Polymerase Chain Reaction Assay Results for SARS-CoV-2.

IntroductionThe coronavirus disease 2019 (COVID-19) epidemic beginning December 2019 in China has now become a worldwide pandemic. With the need to develop an approach to manage orthopaedic surgeries, we aimed to evaluate the most current data on all the surgical cases in our department including the results of the reverse-transcriptase polymerase chain reaction (RT-PCR) assay for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).MethodsThe monthly number of surgical cases from 2016 were reviewed, and compared the numbers of surgical cases both in elective and emergency surgery during the pandemic with the pre-pandemic period. The results of RT-PCR for SARS-CoV-2 in 94 orthopaedic surgery cases from May 13 to June 30, 2020, and clinical signs/symptoms, and laboratory data of 48 consecutive cases within a month from May 13 were also evaluated.ResultsThe mean monthly number of surgeries from January to May 2020 was significantly lower than the mean number in 2019 (73.8 vs 121.9, respectively, p=0.01). The proportion of emergency surgeries in all surgeries performed in May 2020 was 35.5%, which is significantly more than the mean rate of 20.4% in 2019 (p=0.04). Hip arthroplasties and spine surgeries showed the greatest reduction, at greater than 80% and 65%, respectively. Although none of the 94 patients were positive for SARS-CoV-2, 66.7% showed signs/symptoms typical of COVID-19. The most frequent signs/symptoms were production of nasal mucus (25.5%), followed by dry cough (19.1%); and fatigue, headache, and dizziness (17.0% each). The incidence of abnormal values, which are commonly noted in COVID-19 patients, were eosinopaenia 37.5%; lymphopaenia 18.8%; thrombocytopaenia 8.3%; and elevated prothrombin time 10.4%.ConclusionsOur results show that our RT-PCR negative patients showed signs/symptoms and abnormal laboratory values typical of COVID-19, indicating surgeons should be aware of these abnormalities in patients and the need to rule out COVID-19 before proceeding with surgery.

Molecular Characterization of Methicillin-Resistant Enterotoxin-Producing Staphylococcus aureus Isolated From Samosa and Falafel in Iran

Objective: This study aimed to determine the contamination rate of Staphylococcus aureus in Samosa and falafel as most popular snacks, detect the classic enterotoxins, mecA, and tst genes and investigate antimicrobial resistance in the isolates. Materials and Methods: The samples were examined using bacterial culture and the suspected isolates were characterized by biochemical tests. The identity of S. aureus isolates and the presence of enterotoxin-encoding genes were assessed using a multiplex polymerase chain reaction (PCR) assay and antibiotic resistance of the isolates was determined. Results: The results revealed that 56 (46.67%) samples were contaminated with S. aureus, among which 45 isolates (80.35%) were characterized as enterotoxigenic S. aureus. The highest prevalence rate belonged to sea encoding gene as 20 isolates (35.71%) were positive for this gene followed by sed gene which was detected in 14 S. aureus isolates (25%). Most isolates (75%) were resistant to cefoxitin. Moreover, the results of PCR assays indicated that 10 (17.58%) and 7 (12.5%) isolates were positive for mecA and tst genes, respectively. Conclusion: The results of the present study demonstrated that staphylococcal contamination of Samosa and falafel should be considered as a potential health risk for consumers.

Phenotypic and genotypic characterization of antibiotic-resistant in Escherichia coli isolates from patients with diarrhea

Escherichia coli is a common enteric pathogen of human and livevestock. Antibiotic resistance is the main concern of public health. The aim of this study was to detect this bacterium in stool samples of diarrheal patients and identify the phenotypic and genotypic characterizations of antibiotic-resistant isolates such as dfrA1, sul1, citm, tetA, qnr, aac(3)-IV in Shahrekord. Two hundred fifty diarrheal stool samples from patients were collected. Microbiological and biochemical examinations were done to detect E. coli. Phenotypic and genotypic antibiotic resistance of the isolates were determined using dick diffusion method and polymerase chain reaction (PCR), respectively. Among 114 E. coli isolates, the least resistance was for gentamicin (0%) and the most resistance was for trimethoprim (79.8%). The resistance to sulfamethoxazole, ciprofloxacin, ampicillin, and tetracycline were 71.05%, 10.5%, 52.63%, and 3.5% respectively. The results of PCR assay revealed that 10 isolates contain sul1, 49 isolates harbor citm, 8 isolates tetA, 36 isolates dfrA1, 11 isolates qnr genes but there was no isolate with aac(3)-IV gene. In comparison between phenotypic and genotypic of the isolates revealed that citm, tetA, dfrA1, qnr, sul1, aac(3)-IV genes covered 42.98%, 7.01%, 31.57%, 9.64%, 8.7%, 0% of the antibiotic resistance, respectively. Our results revealed that all isolates harbor one or more antibiotic resistance genes and that the PCR is a fast practical and appropriate method to determine the presence of antibiotic resistance genes.

EFFECT OF EXTRACTION METHOD AND DNA QUALITY ON THE RELIABILITY OF MOLECULAR DETECTION OF BRADYRHIZOBIUM JAPONICUM IN SOYBEAN RHIZOSPHERE

Several protocols of DNA extraction from the soil with commercial kits and CTAB based method were tested to develop relatively easy procedure of reliable molecular detection of B. japonicum introduced with coated soybean seeds. Method of DNA extraction clearly influences the quality and quantity of DNA and thus directly affects the results of PCR (Polymerase Chain Reaction) and qPCR (quantitative Polymerase Chain Reaction) assays and the possibility of bacteria identification. Obtaining reliable results in PCR does not guarantee the same results in qPCR with SYBR Green. The use of DNA extraction commercial kits and successful DNA extraction may in some cases lead to inability to detect bacteria, although it is present in the sample. To avoid such problems, the extraction methods should be selected and, if necessary, modified individually for different soil samples. In this study, DNA extraction with Genomic MINI AX Soil Spin showed to be the most appropriate method for obtaining reliable results in both PCR and real-time PCR. However, for real-time PCR, strong dilution of DNA and the addition of reagents that relieve inhibition of amplification were necessary.

Heterothallic sexual reproduction in three canker-inducing tree pathogens within the Fusarium torreyae species complex

ABSTRACTFusarium zanthoxyli and F. continuum are sister taxa that are the etiological agents of canker disease of prickly ash (Zanthoxylum bungeanum) in northern China. These two pathogens, together with F. torreyae, the causal agent of canker disease of the critically endangered conifer Florida torreya (Torreya taxifolia) from northern Florida and southwestern Georgia, constitute a novel clade, the F. torreyae species complex. To assess their reproductive mode, a polymerase chain reaction (PCR) assay targeting the MAT1-1 and MAT1-2 idiomorphs was designed and validated, using MAT sequences mined from the whole-genome sequence of the three F. torreyae clade pathogens and several closely related fusaria. Results of the MAT idiomorph PCR assay indicated that isolates of the three pathogens were MAT1-1 or MAT1-2. When MAT1-1 and MAT1-2 isolates of each species were crossed on carrot agar, all of the F. zanthoxyli (N = 30) and F. continuum (N = 3) isolates tested were female fertile, yielding mature perithecia with viable ascospores. By comparison, only one pairing of the five different isolates of F. torreyae produced perithecia; however, the majority of the asci in this cross aborted or produced fewer than eight ascospores. Of the three temperatures tested (i.e., 22, 25, and 27 C), the optimal temperature for perithecium production was 22–25 C in F. zanthoxyli and 25 C in F. continuum and F. torreyae. Ascospore progeny from three separate crosses of F. zanthoxyli and F. continuum and one cross of F. torreyae were genotyped to assess whether they were the products of genetic recombination and sexual reproduction. Genotyping of 34–40 progeny from the F. zanthoxyli and F. continuum crosses confirmed that they were the products of sexual reproduction. However, only 36% of the progeny in the F. torreyae cross were recombinant, which was roughly half of the nonparental progeny expected with three markers segregating.

Feline Herpesvirus 1 and Mycoplasma spp. Conventional PCR Assay Results From Conjunctival Samples From Cats in Shelters With Suspected Acute Ocular Infections

Signs of ocular infections like discharge and conjunctivitis occur commonly in cats in shelters and feline herpesvirus 1 (FHV-1), Chlamydia felis, Mycoplasma spp, and feline calicivirus (FCV) are thought to be the most common causes. While molecular assays are available to amplify nucleic acids of each of these agents as single tests or in panels, additional information is needed concerning whether the assay results can be used to predict response to treatment. The objectives of this study were to report results for conventional polymerase chain reaction (PCR) assays that amplify nucleic acids of FHV-1, Mycoplasma spp., C. felis, and FCV from cats with signs of acute ocular and upper respiratory infections in an animal shelter and to determine whether the results are associated with treatment responses to topical administration of cidofovir (anti-FHV-1) or oxytetracycline (anti-Mycoplasma spp. and C. felis). Conjunctival samples were collected from both eyes of 60 cats with ocular signs of disease. Total deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) were extracted from each sample and assayed for DNA of FHV-1, Mycoplasma spp., and C. felis and RNA of FCV by conventional PCR assays. Cats were randomized to be administered either oxytetracycline ointment or cidofovir drops in both eyes and a standardized ocular disease score system was used to determine a total ocular score for each cat prior to treatment on Day 0 and on Day 7. Nucleic acids of one or more agents were amplified from one or both eyes from 39 of 60 cats (65%). FHV-1 DNA (21 cats), Mycoplasma spp. DNA (25 cats) or FCV RNA (2 cats) were amplified most commonly. After treatment for 7 days, 32 of 60 cats (53.3%) were considered improved with 27 of 32 cats (84.4%) having ocular scores of 0 (21 cats) or 1 (6 cats). When the results of the FHV-1 PCR assay were compared to cidofovir treatment responses, the positive and negative predictive values of the assay were shown to be 29.4% and 60%, respectively. When the results of the Mycoplasma spp. PCR assay were compared to oxytetracycline treatment responses, the positive and negative predictive values of the assay were shown to be 40% and 38.5%, respectively. The predictive value of conventional PCR assay results for FHV-1 or Mycoplasma spp. DNA was low, suggesting that performing these tests to formulate a treatment protocol has minimal clinical utility in cats with suspected acute ocular infections.

Study of Human Cytomegalovirus nucleic acid in specimens of colorectal adenocarcinoma and villous adenoma using Polymerase chain reaction (PCR) technique

Although the rapid advance in the understanding of molecular pathways underlying human tumorigenesis causes that initiate dysregulation of the pathways remain largely unknown. Human cytomegalovirus has been shown to transform cultured cells, however, viral DNA is not detected in most transforming cells, and the mechanism by which HCMV might contribute to oncogenesis has remained obscure. For further investigation of HCMV role in human colorectal tumorigenesis, we have tested 13 specimens of colorectal adenocarcinoma &amp; 5 specimens of villous adenoma using polymerase chain reaction (PCR) techniques. The results of PCR assay showed specific single band in 9 of 13 specimens (69.23%) of colorectal adenocarcinoma, and 3 of 5 (60%) of villous adenoma, by using specific primer targeted directly to the early gene (pp65) that expressed within host DNA, the results read by agarose gel electrophoresis &amp; UV – transilluminator. The presence of HCMV genetic information in human tumors is a hot point because HCMV can infect the organs latently in a high percentage of normal individuals, it also remains possible that HCMV modulates the biologic properties of malignant cells without being directly involved in carcinogenesis.

Loop-Mediated Isothermal Amplification-Based Detection of Canine Parvoviral DNA in Faecal Samples of Diarrhoeic Dogs

The present study was undertaken to validate loop-mediated isothermal amplification (LAMP) assay based on PCR (Polymerase Chain Reaction)for the detection of canine parvovirus (CPV) and to compare the diagnostic potential of LAMP. A total of 54 faecal samples collected from diarrheic dogs suspected for CPV infection were subjected to PCR, using the primer amplifying the gene coding for the VP2 protein. These samples were also tested by LAMP assay using primer sets, and the results were statistically analysed using McNemar test. The sensitivity and specificity of LAMP assay based on PCR assay was analysed. The specificity of the LAMP primers was determined by cross-examination of templates extracted from canine adenovirus 1, canine coronavirus and canine distemper virus and Escherichia coli. Sensitivity of both assays was identified by subjecting a serial ten-fold dilution of the DNA (Deoxyribo Nucleic Acid) of CPV-2 vaccine strain (Megavac-P) to LAMP and PCR assays for the determination of their detection limits. A total of 32 samples (59.25 per cent) were detected as positive by PCR, which yielded a specific amplicon of 699 bp. In LAMP assay, 39 (72.2 per cent) samples were found to be positive. The sensitivity of the test as compared with PCR was found to be 100 per cent and specificity was 68.2. Statistical analysis of the results of PCR and LAMP assay using McNemar test revealed that there exists significant difference between these two tests (P < 0.05). The specificity determination of the LAMP primers showed that no amplification products were detected in the template DNA extracted from canine adenovirus 1, canine coronavirus and canine distemper virus vaccines and E. coli, whereas only CPV-2 vaccine strain showed positive reactions. The detection limit of LAMP was up to 10-7 in the place of 10-3 for PCR. This study showed that LAMP assay could be employed as a rapid field level diagnostic tool for the diagnosis of canine parvoviral diarrhoea.

Utility of a rapid immunochromatographic strip test in detecting canine parvovirus infection compared with polymerase chain reaction.

Aim:The present study was undertaken to detect the presence of canine parvovirus (CPV) in fecal samples of diarrheic dogs by conventional polymerase chain reaction (PCR) and immunochromatographic (IC) strip test and to compare the diagnostic potential of these tests.Materials and Methods:A total of 50 fecal samples collected from diarrheic dogs suspected for CPV infection were subjected to PCR using CPV-555 primer amplifying the gene coding for the VP1 protein. These samples were also tested by IC strip test using a commercial rapid Ag test kit. The results were statistically analyzed using McNemar test.Results:A total of 22 samples (44%) were detected as positive by PCR, which yielded a specific amplicon of 583 bp. In IC strip test, 18 (36%) samples were found to be positive. The sensitivity of the test as compared to PCR was found to be 72.22% and specificity was 92.86%. Positive predictive value and negative predictive value of IC strip test was found to be 88.89% and 81.25%, respectively. Statistical analysis of the results of PCR and IC assay using McNemar test revealed no significant difference (p>0.05).Conclusion:The IC strip test could be employed as a rapid field level diagnostic tool for the diagnosis of canine parvoviral diarrhea.

Development of real-time PCR for detection and quantitation of Streptococcus parauberis.

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples.

Serum galactomannan versus a combination of galactomannan and polymerase chain reaction-based Aspergillus DNA detection for early therapy of invasive aspergillosis in high-risk hematological patients: a randomized controlled trial.

The benefit of the combination of serum galactomannan (GM) assay and polymerase chain reaction (PCR)-based detection of serum Aspergillus DNA for the early diagnosis and therapy of invasive aspergillosis (IA) in high-risk hematological patients remains unclear. We performed an open-label, controlled, parallel-group randomized trial in 13 Spanish centers. Adult patients with acute myeloid leukemia and myelodysplastic syndrome on induction therapy or allogeneic hematopoietic stem cell transplant recipients were randomized (1:1 ratio) to 1 of 2 arms: "GM-PCR group" (the results of serial serum GM and PCR assays were provided to treating physicians) and "GM group" (only the results of serum GM were informed). Positivity in either assay prompted thoracic computed tomography scan and initiation of antifungal therapy. No antimold prophylaxis was permitted. Overall, 219 patients underwent randomization (105 in the GM-PCR group and 114 in the GM group). The cumulative incidence of "proven" or "probable" IA (primary study outcome) was lower in the GM-PCR group (4.2% vs 13.1%; odds ratio, 0.29 [95% confidence interval, .09-.91]). The median interval from the start of monitoring to the diagnosis of IA was lower in the GM-PCR group (13 vs 20 days; P = .022), as well as the use of empirical antifungal therapy (16.7% vs 29.0%; P = .038). Patients in the GM-PCR group had higher proven or probable IA-free survival (P = .027). A combined monitoring strategy based on serum GM and Aspergillus DNA was associated with an earlier diagnosis and a lower incidence of IA in high-risk hematological patients. Clinical Trials Registration. NCT01742026.

Transient Expression of Foot and Mouth Disease Virus (FMDV)Coat Protein in Tobacco (Nicotiana tabacom) via Agroinfiltration

Background: Transient and stable transformation of host plants are the common techniques to produce transgenic plants. However, the main drawback of stable transformation is the fact that it takes quite a long time to produce a transgenic line. While, transient gene expression is a quick method to produce recombinant proteins in plants. Objective: The main goal of the present study was to evaluate efficient agroinfiltration as an efficient and rapid method for production of recombinant antigen of FMDV. Materials and Methods: Tobacco leaves were transformed via agroinfiltration using a needle-free syringe. Presence of the gene cassette was verified by polymerase chain reaction (PCR). Expression of the foreign gene was evaluated using Real Time PCR, protein dot blot and enzyme-linked immunosorbent assay (ELISA). Results: PCR analysis confirmed successful transformation of plant leaves. Expression of foreign protein was confirmed at both transcription and translation levels. Results of Real Time PCR assay indicated that the foreign gene was transcribed in transformed leaves. ELISA results showed that the foreign gene was expressed in the transformed leaves in high level. Conclusion: Here, the efficacy of agroinfiltration for transient expression of FMDV coat protein in tobacco was illustrated. Accordingly, transient agroinfilteration expedites the process of recombinant antigens expression in plant tissues.

English

Molecular identification and antimicrobial susceptibility of enteroaggregative Escherichia coli (EAEC) associated with childhood diarrhea was done out in Egypt. The usefulness of quantitative biofilm assay in detection of EAEC was compared with multiplex polymerase chain reaction (PCR). One hundred and fifty cases of childhood diarrhea were divided into three groups; 50 cases of acute diarrhea (group I), 50 cases of persistent diarrhea (group II) and 50 cases of healthy subjects of matched age and sex as a control group (group III). E. coli was isolated and identified by conventional microbiological methods. EAEC was detected by multiplex PCR and quantitative biofilm assay. Antimicrobial susceptibility profile of the isolated EAEC strains was done using disc diffusion method. E. coli was isolated from 78% (39/50) cases of acute diarrhea and 76% (38/50) cases of chronic diarrhea. The results show no significant difference between the results of multiplex PCR and quantitative biofilm assay; in 77 E. coli isolates, 15 (19.5 %) generated positive results for EAEC with multiplex PCR for two the specific genes AggR and EAST and 12 (15.6 %) strains showed positive results for EAEC by quantitative biofilm assay. As regard the antimicrobial susceptibility profile of the isolated EAEC strains, the results show that 85.7 and 87.5% of the EAEC strains isolated from cases of acute and persistent diarrhea, respectively were sensitive to amikacin, 47.1 and 62.5% were sensitive to cefoperazone, 28.5 and 50.00% were sensitive to ceftriaxone and 42.8% and 62.5% were sensitive to imepenem, 28.5 and 12.5 % of the EAEC strains isolated from cases of acute and persistent diarrhea respectively were sensitive to Amoxicillin-Clavulanic. All the isolated EAEC strains (100.00%) were resistant to sulphamethoxole/trimethoprim. High incidence of EAEC associated diarrhea among pediatric cases in Egypt must be considered before decision of antimicrobial therapy. Quantitative biofilm assay can be simple, rapid and convenient method for detection of EAEC in comparison with molecular methods and can therefore be recommended as a rapid screening test for EAEC in clinical laboratories. &nbsp; Key words: Enteroaggregative Escherichia coli (EAEC), infantile diarrhea, multiplex polymerase chain reaction (PCR), quantitative biofilm.

High glucose concentration up-regulates the expression of matrix metalloproteinase-9 and -13 in tendon cells

BackgroundDiabetes mellitus is associated with tendinopathy or tendon injuries. However, the mechanism underlying diabetic tendinopathy is unclear. The purpose of this study was to examine the effects of high glucose concentrations on the activity and expression of matrix metalloproteinases, type I collagen, and type III collagen in tendon cells.MethodsTendon cells from rat Achilles tendons were treated with 6 mM, 12 mM, and 25 mM glucose, and then cell proliferation was evaluated by the 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Messenger RNA (mRNA) expression of MMP-2, MMP-8, MMP-9, and MMP-13 and type I and type III collagen was assessed by quantitative real-time polymerase chain reaction (PCR). The enzymatic activity of MMP-2 and MMP-9 was measured by gelatin zymography.ResultsThe MTT assay results showed that the glucose concentration did not affect tendon cell proliferation. The results of the real-time PCR assay revealed that the mRNA expression of MMP-9 and MMP-13 was up-regulated by treatment with 25 mM glucose, whereas the mRNA expression of type I and III collagen was not affected. Gelatin zymography showed that 25 mM glucose increased the enzymatic activity of MMP-9.ConclusionsHigh glucose concentration up-regulates the expression of MMP-9 and MMP-13 in tendon cells, which may account for the molecular mechanisms underlying diabetic tendinopathy.