Polymerase Chain Reaction Amplification Assay Research Articles

Comparison of PCR and Loop Mediated Isothermal Amplification Method for Detection of Salmonella spp. in Chicken Meat

The aim of the study was to compare Polymerase Chain Reaction (PCR) and Loop Mediated Isothermal Amplification (LAMP) assay for detection of Salmonella in Chicken Meat. Food safety remains a major concern worldwide, with outbreaks of foodborne illness resulting in significant costs to individuals and to the food industry. The Salmonella genus is the most prevalent group of foodborne pathogens that are regularly isolated from food-producing animals and thus are one of the leading causes of food borne diseases. Out of total 40 Salmonella isolates, LAMP and PCR assay detected 36 (90%) and 32 (80%) isolates, respectively. The sensitivity of the LAMP assay was noted to be 10-fold higher than that of PCR whereas the specificity of both LAMP and PCR was same.

Current State of Molecular and Serological Methods for Detection of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of an acute and devastating enteric disease that causes moderate-to-high mortality in suckling piglets. The accurate and early detection of PEDV infection is essential for the prevention and control of the spread of the disease. Many molecular assays have been developed for the detection of PEDV, including reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR (qRT-PCR) and loop-mediated isothermal amplification assays. Additionally, several serological methods have been developed and are widely used for the detection of antibodies against PEDV. Some of them, such as the immunochromatography assay, can generate results very quickly and in field conditions. Molecular assays detect viral RNA in clinical samples rapidly, and with high sensitivity and specificity. Serological assays can determine prior immune exposure to PEDV, can be used to monitor the efficacy of vaccination strategies and may help to predict the duration of immunity in piglets. However, they are less sensitive than nucleic acid-based detection methods. Sanger and next-generation sequencing (NGS) allow the analysis of PEDV cDNA or RNA sequences, and thus, provide highly specific results. Furthermore, NGS based on nonspecific DNA cleavage in clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems promise major advances in the diagnosis of PEDV infection. The objective of this paper was to summarize the current serological and molecular PEDV assays, highlight their diagnostic performance and emphasize the advantages and drawbacks of the application of individual tests.

A Pan-Global Study of Bacterial Leaf Spot of Chilli Caused by Xanthomonas spp.

Bacterial Leaf Spot (BLS) is a serious bacterial disease of chilli (Capsicum spp.) caused by at least four different Xanthomonas biotypes: X. euvesicatoria pv. euvesicatoria, X. euvesicatoria pv. perforans, X. hortorum pv. gardneri, and X. vesicatoria. Symptoms include black lesions and yellow halos on the leaves and fruits, resulting in reports of up to 66% losses due to unsalable and damaged fruits. BLS pathogens are widely distributed in tropical and subtropical regions. Xanthomonas is able to survive in seeds and crop residues for short periods, leading to the infections in subsequent crops. The pathogen can be detected using several techniques, but largely via a combination of traditional and molecular approaches. Conventional detection is based on microscopic and culture observations, while a suite of Polymerase Chain Reaction (PCR) and Loop-Mediated Isothermal Amplification (LAMP) assays are available. Management of BLS is challenging due to the broad genetic diversity of the pathogens, a lack of resilient host resistance, and poor efficacy of chemical control. Some biological control agents have been reported, including bacteriophage deployment. Incorporating stable host resistance is a critical component in ongoing integrated management for BLS. This paper reviews the current status of BLS of chilli, including its distribution, pathogen profiles, diagnostic options, disease management, and the pursuit of plant resistance.

Diagnostic Techniques for Soil-Transmitted Helminths - Recent Advances.

Soil-transmitted helminth (STH) infections (hookworms, Trichuris, Ascaris) and Strongyloides spp. are associated with a substantial global burden and high morbidity. Sensitive and specific methods for diagnosis of these infections are essential for mapping the burden in communities, accurate assessment of infection levels, to guide interventions and monitoring the success of STH control programs. Despite considerable progress to control STH over several decades, we are still far from identifying a fully adequate diagnostic test. Conventional microscopy-based methods such as direct Kato–Katz smear or mounts after stool centrifugation/flotation-based concentration techniques have been the mainstay of diagnosis, especially in resource-poor countries where these infections abound. However, recently, these are being adapted to closed, easy to perform, digital formats, thereby improving the sensitivity as well as applicability in a remote, resource-limited setting. The use of image analysis systems to identify and quantify helminth eggs, with potential adaptation to smartphones, is also promising. Antibody detection tests have a limited role mostly in the case of Strongyloides hyperinfection. Coproantigen detection tests have been developed and used in veterinary practice for detection of STH, but these have not been evaluated for use in humans. More sensitive molecular diagnostics, including assays developed with new bioinformatic tools and techniques such as polymerase chain reaction (PCR), quantitative PCR (qPCR) and loop-mediated amplification assay, can help in the clear and precise assessment of STH burden during elimination phase and are of immense value for diagnosis in areas with low endemicity and in travelers to endemic regions. Moreover, the molecular techniques will help detect new species that may emerge. Sample preservation and efficient DNA extraction are critical and significantly affect the efficiency of molecular diagnostic tests. In addition to the diagnosis of clinical or asymptomatic infection in humans, detection of STH eggs in environmental samples is imperative to boost STH control efforts. Overall the diagnostic performance, cost-effectiveness, ease of performance, rapidity and in-field applicability of any test should be considered when choosing from the various diagnostic assays in areas with different endemicity, in addition to striving towards the development of novel technologies and optimization of existing methods.

Quantitative Proteomic Analysis Revealed Broad Roles of N6-Methyladenosine in Heat Shock Response.

As optimum temperature is essential for all living organisms, heat shock represents a challenging problem for their survival. Therefore, cellular response to heat shock is among the most extensively investigated stress response pathways; however, how the human proteome responds to heat shock has not been comprehensively investigated. In this study, we employed stable isotope labeling by amino acids in cell culture (SILAC), together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, to fulfill an in-depth analysis of the alterations in the human proteome in M14 human melanoma cells in response to heat shock stress. We found that, after heat shock, 284 and 278 out of the 4319 quantified proteins were with substantially diminished and elevated expressions, respectively. We also examined the alterations in human kinome after heat shock by using our recently developed targeted proteomic method relying on parallel-reaction monitoring. Our results showed that the expression levels of 11 and 22 kinase proteins were increased and decreased, respectively, by at least 1.5-fold upon heat shock. By interrogating publicly available RNA-seq and m6A sequencing data, we observed that the elevated expression of more than 30 proteins, including CHEK1 and CCND3 kinases, could occur via an m6A-mediated mechanism. Furthermore, our results from single-base elongation and ligation-based quantitative polymerase chain reaction (qPCR) amplification (SELECT) and luciferase reporter assays revealed that heat shock gave rise to elevated m6A levels at A280 and A286 sites in the 5'-untranslated region of HSPH1 mRNA, thereby leading to increased translation of HSPH1 protein. Together, our discovery and targeted proteomic methods revealed the reprogramming of human proteome and kinome upon heat shock stress and provided insights into cellular responses toward heat shock stress.

Development of a novel loop-mediated isothermal amplification assay for rapid detection of Mycobacterium leprae in clinical samples.

Sensitive and definitive diagnostic tests are required for timely treatment of leprosy and to control its transmission. In the present study, we report the development of loop-mediated isothermal amplification assay using six primers targeting the RLEP gene sequence uniquely present in Mycobacterium leprae. Tissue punch samples (n = 50) and slit aspirates (n = 50) from confirmed cases of leprosy (M. leprae positive by quantitative polymerase chain reaction), reporting at the Department of Dermatology, Safdarjung Hospital, New Delhi, were analyzed using newly developed closed tube loop-mediated isothermal amplification assay. The sensitivity and specificity; positive predictive value, negative predictive value and accuracy were calculated using MedCalc statistical software. The loop-mediated isothermal amplification assay specifically amplified M. leprae genomic DNA with an analytical sensitivity of 100 fg. About 47 Out of the 50 quantitative polymerase chain reactions confirmed M. leprae positive tissue samples, 47 were positive by loop-mediated isothermal amplification assay (sensitivity 94%; 95% confidence interval 83.5%-98.8%) while only 31/50 were positive by histopathology (sensitivity 62%; 95% confidence interval 47.2%-75.4%) . Using slit aspirate samples of these 50 patients, 42 were positive by both quantitative polymerase chain reaction and loop-mediated isothermal amplification assay (sensitivity 84%; 95% confidence interval 70.9%-92.8%) while only 23/50 (sensitivity 46%; 95% confidence interval 31.8%-60.7%) were positive by microscopy. In the present study, the leprosy patient cohort was not uniform, as it comprised a lower number of paucibacillary cases (22%) compared to multibacillary (78%) cases. Loop-mediated isothermal amplification assay established here provides a rapid and accurate diagnostic test for leprosy in terms of sensitivity and specificity. The assay is simple to perform in comparison with other molecular techniques (polymerase chain reaction/quantitative polymerase chain reaction) and has potential for field applicability.

LAMP-based foldable microdevice platform for the rapid detection of Magnaporthe oryzae and Sarocladium oryzae in rice seed

Rice blast (caused by Magnaporthe oryzae) and sheath rot diseases (caused by Sarocladium oryzae) are the most predominant seed-borne pathogens of rice. The detection of both pathogens in rice seed is essential to avoid production losses. In the present study, a microdevice platform was designed, which works on the principles of loop-mediated isothermal amplification (LAMP) to detect M. oryzae and S. oryzae in rice seeds. Initially, a LAMP, polymerase chain reaction (PCR), quantitative PCR (qPCR), and helicase dependent amplification (HDA) assays were developed with primers, specifically targeting M. oryzae and S. oryzae genome. The LAMP assay was highly efficient and could detect the presence of M. oryzae and S. oryzae genome at a concentration down to 100 fg within 20 min at 60 °C. Further, the sensitivity of the LAMP, HDA, PCR, and qPCR assays were compared wherein; the LAMP assay was highly sensitive up to 100 fg of template DNA. Using the optimized LAMP assay conditions, a portable foldable microdevice platform was developed to detect M. oryzae and S. oryzae in rice seeds. The foldable microdevice assay was similar to that of conventional LAMP assay with respect to its sensitivity (up to 100 fg), rapidity (30 min), and specificity. This platform could serve as a prototype for developing on-field diagnostic kits to be used at the point of care centers for the rapid diagnosis of M. oryzae and S. oryzae in rice seeds. This is the first study to report a LAMP-based foldable microdevice platform to detect any plant pathogens.

Detection of PMS2 Mutations by Screening Hereditary Nonpolyposis Colon Cancer Families from Denmark and Sweden.

Background and Aims: Hereditary nonpolyposis colon cancer (HNPCC) and Lynch syndrome (LS) are characterized by defects in the mismatch repair (MMR) system, which protects the integrity of the genome. Pathogenic variants in four MMR genes (MLH1, MSH2, MSH6, and PMS2) are responsible for LS, an autosomal, dominant hereditary disease that occurs with a frequency of 2-5% among all colorectal cancer cases. It has been estimated that ∼2-5% of all pathogenic variants found in the four MMR genes in LS cases are detected in the PMS2 gene. An overview of detected variants is presented here. Materials and Methods: Long-range (LR) PMS2 polymerase chain reaction (PCR) and PMS2 multiplex ligation probe amplification (MLPA) assays were used to detect PMS2 variants in ∼1500 probands. In a subset of the probands, pathogenic PMS2 variants were detected by next-generation sequencing, and all detected variants were confirmed by LR-PCR combined with an MLPA assay. Results: A summary of PMS2 mutation analyses performed on colon cancer patients from molecular diagnostic laboratories in Denmark and Sweden is presented. By screening ∼1500 HNPCC probands, a total of 40 different PMS2 variants were detected in 71 probands (5%); 20 variants were classified as pathogenic (C5), 2 variants as likely pathogenic (C4), 15 variants as variants of unknown significance (VUSs) (C3), 1 variant as likely benign (C2), and 2 variants as benign (C1). In total, 22/71 (31%) of the probands carried a pathogenic sequence variant. Among the probands with isolated loss of pPMS2 expression, the fraction of pathogenic variants was 20/35 (55%). Conclusions: Approximately 5% of the probands found in the Danish and Swedish populations presented here carried a PMS2 variant. In this study, six novel pathogenic variants and seven VUSs are reported.

Structurally Defined Ru(II) Metallointercalators for Real-Time Monitoring of DNA Amplification Reactions.

The low cost and convenience of fluorescent DNA binding dyes make them widely used for real-time DNA analysis. Even though several dyes for real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assays are commercially available, most of them rely on organic parent molecules with an indefinite chemical structure, making it difficult to predict their behavior in nucleic acid amplification and to get the best performance. Herein, we demonstrate for the first time the use of the structurally defined dipyridophenazine complexes of ruthenium(II) for real-time monitoring of PCR and LAMP. These inorganic metallointercalators exhibit less inhibition on DNA amplification. They can quantify a range of initial template concentrations and provide the product melting curve to differentiate the unspecific amplification and even perform multiplex assays. These ruthenium(II) complexes have the advantages of the defined structure, nontoxicity, good stability, and facile synthesis, and they may act as excellent alternative DNA binding dyes with wide applications in the field of nucleic acid research.

Electrochemical Methodologies for the Detection of Pathogens

Bacterial infections remain one of the principal causes of morbidity and mortality worldwide. The number of deaths due to infections is declining every year by only 1% with a forecast of 13 million deaths in 2050. Among the 1400 recognized human pathogens, the majority of infectious diseases is caused by just a few, about 20 pathogens only. While the development of vaccinations and novel antibacterial drugs and treatments are at the forefront of research, and strongly financially supported by policy makers, another manner to limit and control infectious outbreaks is targeting the development and implementation of early warning systems, which indicate qualitatively and quantitatively the presence of a pathogen. As toxin contaminated food and drink are a potential threat to human health and consequently have a significant socioeconomic impact worldwide, the detection of pathogenic bacteria remains not only a big scientific challenge but also a practical problem of enormous significance. Numerous analytical methods, including conventional culturing and staining techniques as well as molecular methods based on polymerase chain reaction amplification and immunological assays, have emerged over the years and are used to identify and quantify pathogenic agents. While being highly sensitive in most cases, these approaches are highly time, labor, and cost consuming, requiring trained personnel to perform the frequently complex assays. A great challenge in this field is therefore to develop rapid, sensitive, specific, and if possible miniaturized devices to validate the presence of pathogens in cost and time efficient manners. Electrochemical sensors are well accepted powerful tools for the detection of disease-related biomarkers and environmental and organic hazards. They have also found widespread interest in the last years for the detection of waterborne and foodborne pathogens due to their label free character and high sensitivity. This Review is focused on the current electrochemical-based microorganism recognition approaches and putting them into context of other sensing devices for pathogens such as culturing the microorganism on agar plates and the polymer chain reaction (PCR) method, able to identify the DNA of the microorganism. Recent breakthroughs will be highlighted, including the utilization of microfluidic devices and immunomagnetic separation for multiple pathogen analysis in a single device. We will conclude with some perspectives and outlooks to better understand shortcomings. Indeed, there is currently no adequate solution that allows the selective and sensitive binding to a specific microorganism, that is fast in detection and screening, cheap to implement, and able to be conceptualized for a wide range of biologically relevant targets.

Pesticide behavior in paddy fields and development of azole-resistant Aspergillus fumigatus: Should we be concerned?

Tricyclazole as a common fungicide wildly used to control rice blast disease in the Asian country may induce azole resistance in Aspergillus fumigatus isolates. The main reason of the acquired azole resistance is probably environmental exposure through wide fungicide use in agriculture. The present study was conducted with the aim of investigating the current status of the azole-resistant A. fumigatus obtained from the paddy fields with exposure to tricyclazole. A total of 108 soil samples were collected from four different locations of paddy fields in Mazandaran Province, Iran. Pure fungal colonies were initially identified based on the conventional tools, and then reconfirmed by using DNA sequencing of the partial ß-tubulin gene. In addition, the in vitro antifungal susceptibility was determined using the Clinical and Laboratory Standards Institute document (CLSI) M38-A2. The identification of the mutations in the CYP51A gene was accomplished by the implementation of the polymerase chain reaction amplification assay on the selected isolates. Overall, 31 of 108 (28.7%) isolates were identified as A.fumigatus, four (3.7%) of which were recognized as azole-resistant with MICs of itraconazole ≥8μg/ml and voriconazole ≥4μg/ml. Only two out of the four azole-resistant A. fumigatus isolates harboured TR34/L98H variant and the other two isolates were identified as azole-resistant without any CYP51A gene mutations. However, other point mutations (TR46/Y121F/T289A) were not detected in the CYP51A gene. The high molecular structure similarity between environmental and medical triazoles may result in the selection of resistance mechanisms. Nonetheless, one might conclude that tricyclazole with different molecular structures against medical azoles induces azole-resistance in A.fumigatus isolates. The behavior of such pesticides as tricyclazole in the rice paddy fields would have an effective role in the development of azole-resistance that requires detailed information.

Long-term low dissolved oxygen accelerates the removal of antibiotics and antibiotic resistance genes in swine wastewater treatment

In this study, a novel process for swine wastewater treatment, anoxic and four-stage micro oxygen aerated sequence coupled with a coagulation process (AO4) was investigated in the pilot scale to determine its capacity in the removal of antibiotics and antibiotic resistance genes (ARGs). Results showed that long-term low dissolved oxygen (DO) operational condition played a significant role in decreasing the antibiotics and relative abundance of ARGs. Around 98% of the total antibiotics were removed along with 4–6 orders magnitude reductions of the ARGs copy numbers in AO4 process. Reduction of bacterial endogenous decay rate in AO4 process with low DO (0.15mg/L and 0.10mg/L) might result in the increase in the total bacterial population size and the concentration of sludge. As a result, high biomass accelerated the accumulation of antibiotics in sludge. In addition, real-time polymerase chain reaction amplification (qPCR) assays indicated that the long-term low DO could reduce the relative abundance of ARGs via enriching the 16S rDNA in activated sludge and inhibiting the emergence or distribution of ARGs. Our findings imply that the AO4 process could not only achieve a significant reduction of conventional contaminants such as chemical oxygen demand (COD), chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) but also antibiotics and ARGs.

Characteristics of Plasmids Coharboring 16S rRNA Methylases, CTX-M, and Virulence Factors in Escherichia coli and Klebsiella pneumoniae Isolates from Chickens in China.

The objective of this study was to characterize plasmids coharboring 16S rRNA methylases, blaCTX-M and virulence-associated genes in Escherichia coli and Klebsiella pneumoniae isolates from chickens in China. A total of 32 positive transconjugants exhibited coresistance to amikacin and cefotaxime in E. coli (24/281) and K. pneumoniae (8/93), and were identified by conjugation experiments and S1-pulsed-field gel electrophoresis. Polymerase chain reaction amplification assay detecting resistance genes showed that rmtB or armA gene accompanied with different blaCTX-M genes coexisted on 32 transferred plasmids. The blaCTX-M-98b gene was identified in chicken-derived E. coli and K. pneumoniae for the first time. The association between resistance genes and virulence genes was observed in the transferred plasmids; 68.8% (22/32) transferred resistance plasmids coharboring various virulence genes including traT, iutA, fyuA, msbB, and vagC genes with diverse proportions. Genetic stability tests revealed that 93.8% (30/32) transferred plasmids continued to exist in the host strain after continuous passage of 30 times in 15 days. Furthermore, 87.5% (28/32) conjugants showed no significant differences in growth rates compared with E. coli J53. Results of the growth competition assay showed that conjugants have low fitness cost, which indicated that there were no obvious negative effects on the host's growth. The combination of blaCTX-M-98b-rmtB-traT on 85-kb transferred IncF plasmids in E. coli, and blaCTX-M-14-rmtB-traT on 95-kb transferred IncF plasmids in K. pneumoniae were first identified in this study. These features of plasmids may contribute to the successful spread of resistance and virulence among pathogens of different sources and geographical origins.

An integrated closed-tube 2-plex PCR amplification and hybridization assay with switchable lanthanide luminescence based spatial detection.

Switchable lanthanide luminescence is a binary probe technology that inherently enables a high signal modulation in separation-free detection of DNA targets. A luminescent lanthanide complex is formed only when the two probes hybridize adjacently to their target DNA. We have now further adapted this technology for the first time in the integration of a 2-plex polymerase chain reaction (PCR) amplification and hybridization-based solid-phase detection of the amplification products of the Staphylococcus aureus gyrB gene and an internal amplification control (IAC). The assay was performed in a sealed polypropylene PCR chip containing a flat-bottom reaction chamber with two immobilized capture probe spots. The surface of the reaction chamber was functionalized with NHS-PEG-azide and alkyne-modified capture probes for each amplicon, labeled with a light harvesting antenna ligand, and covalently attached as spots to the azide-modified reaction chamber using a copper(i)-catalyzed azide-alkyne cycloaddition. Asymmetric duplex-PCR was then performed with no template, one template or both templates present and with a europium ion carrier chelate labeled probe for each amplicon in the reaction. After amplification europium fluorescence was measured by scanning the reaction chamber as a 10 × 10 raster with 0.6 mm resolution in time-resolved mode. With this assay we were able to co-amplify and detect the amplification products of the gyrB target from 100, 1000 and 10,000 copies of isolated S. aureus DNA together with the amplification products from the initial 5000 copies of the synthetic IAC template in the same sealed reaction chamber. The addition of 10,000 copies of isolated non-target Escherichia coli DNA in the same reaction with 5000 copies of the synthetic IAC template did not interfere with the amplification or detection of the IAC. The dynamic range of the assay for the synthetic S. aureus gyrB target was three orders of magnitude and the limit of detection of 8 pM was obtained. This proof-of-concept study shows that the switchable lanthanide luminescent probes enable separation-free array-based multiplexed detection of the amplification products in a closed-tube PCR which can enable a higher degree of multiplexing than is currently feasible by using different spectrally separated fluorescent probes.

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Comparative Study of Sensitivity, Linearity, and Resistance to Inhibition of Digital and Nondigital Polymerase Chain Reaction and Loop Mediated Isothermal Amplification Assays for Quantification of Human Cytomegalovirus

Performing nucleic acid amplification techniques (NAATs) in digital format using limiting dilution provides potential advantages that have recently been demonstrated with digital polymerase chain reaction (dPCR). Key benefits that have been claimed are the ability to quantify nucleic acids without the need of an external calibrator and a greater resistance to inhibitors than real-time quantitative PCR (qPCR). In this study, we evaluated the performance of four NAATs, qPCR, dPCR, real-time quantitative loop mediated isothermal amplification (qLAMP), and digital LAMP (dLAMP), for the detection and quantification of human cytomegalovirus (hCMV). We used various DNA templates and inhibitors to compare the performance of these methods using a conventional real-time thermocycler platform (Bio-Rad CFX96) and a chip based digital platform (Fluidigm Biomark 12.765 Digital Array). dPCR performed well and demonstrated greater resistance to inhibitors than the other methods although this resistance did not apply equally to all inhibitors tested. dLAMP was found to be less sensitive than dPCR, but its quantitative performance was better than qLAMP, the latter being unable to quantify below 1000 copies. dLAMP was also more resistant to inhibitors than qLAMP. Unlike qPCR, both digital methods were able to quantify viral genomes without requiring a calibrator; however, neither can currently compete with the large reaction volumes, and thus the greater absolute sensitivity, of qPCR. With the introduction of digital instrumentation that will enable larger reaction volumes, digital amplification methods such as those evaluated in this study could potentially offer a robust alternative to qPCR for nucleic acid quantification.

Production of homozygous transgenic rainbow trout with enhanced disease resistance.

Previous studies conducted in our laboratory showed that transgenic medaka expressing cecropin B transgenes exhibited resistant characteristic to fish bacterial pathogens, Pseudomonas fluorescens and Vibrio anguillarum. To confirm whether antimicrobial peptide gene will also exhibit anti-bacterial and anti-viral characteristics in aquaculture important fish species, we produced transgenic rainbow trout expressing cecropin P1 or a synthetic cecropin B analog, CF-17, transgene by sperm-mediated gene transfer method. About 30 % of fish recovered from electroporation were shown to carry the transgene as determined by polymerase chain reaction (PCR) amplification assay. Positive P1 transgenic fish were crossed to non-transgenic fish to establish F1 transgenic founder families, and subsequently generating F2, and F3 progeny. Expression of cecropin P1 and CF-17 transgenes was detected in transgenic fish by reverse transcription (RT)-PCR analysis. The distribution of body sizes among F1 transgenic fish were not significantly different from those of non-transgenic fish. Results of challenge studies revealed that many families of F2 and F3 transgenic fish exhibited resistance to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). All-male homozygous cecropin P1 transgenic families were produced by androgenesis from sperm of F3 heterozygous transgenic fish in one generation. The resistant characteristic to A. salmonicida was confirmed in progeny derived from the outcross of all-male fish to non-transgenic females. Results of our current studies confirmed the possibility of producing disease-resistant homozygous rainbow trout strains by transgenesis of cecropin P1 or CF-17 gene and followed by androgenesis.

Molecular characterization and phylogenetic study of African swine fever virus isolates from recent outbreaks in Uganda (2010-2013).

BackgroundAfrican swine fever (ASF) is a highly lethal and economically significant disease of domestic pigs in Eastern Africa particularly in Uganda where outbreaks regularly occur. Sequence analysis of variable genome regions have been extensively used for molecular epidemiological studies of African swine fever virus (ASFV) isolates. By combining p72, P54 and pB602L (CVR), a high level resolution approach is achieved for viral discrimination. The major aim of this study therefore, was to investigate the genetic relatedness of ASF outbreaks that occurred between 2010 and 2013 in Uganda to contribute to the clarification of the epidemiological situation over a four year period.MethodsTissue samples from infected domestic pigs associated with an ASF outbreak from 15 districts in Uganda were confirmed as being infected with ASFV using a p72 gene-based polymerase chain reaction amplification (PCR) assay recommended by OIE. The analysis was conducted by genotyping based on sequence data from three single copy ASFV genes. The E183L gene encoding the structural protein P54 and part of the gene encoding the p72 protein was used to delineate genotypes. Intra-genotypic resolution of viral relationships was achieved by analysis of tetramer amino acid repeats within the hypervariable CVR of the B602L gene.ResultsTwenty one (21) ASF outbreaks were confirmed by the p72 ASF diagnostic PCR, however; only 17 isolates were successfully aligned after sequencing. Our entire isolates cluster with previous ASF viruses in genotype IX isolated in Uganda and Kenya using p72 and P54 genes. Analysis of the CVR gene generated three sub-groups one with 23 tetrameric amino acid repeats (TRS) with an additional CAST sequence, the second with 22 TRS while one isolate Ug13. Kampala1 had 13 TRS.ConclusionWe identified two new CVR subgroups different from previous studies. This study constitutes the first detailed assessment of the molecular epidemiology of ASFV in domestic pigs in the different regions of Uganda.

Molecular Diagnosis and Ocular Imaging of West Nile Virus Retinitis and Neuroretinitis

To describe the ocular features of West Nile virus (WNV) infection proven by serology and molecular diagnostic techniques. Prospective case series. Fifty-two patients who presented to the uveitis clinic with ocular inflammatory signs and history of fever preceding ocular symptoms between January 2010 and January 2012 were enrolled for laboratory diagnosis. Serum samples were collected from 30 healthy controls from the same geographic area. Patients were tested for all endemic infectious diseases that can cause ocular inflammation by serology or molecular diagnostics. When patients had positive antibodies for WNV, serum/plasma samples were tested by real-time reverse transcription (RT) polymerase chain reaction (PCR) and RT loop-mediated isothermal gene amplification assays. The PCR product was subjected to nucleotide sequencing. Fundus fluorescence angiography (FFA), optical coherence tomography (OCT), and indocyanine green angiography were performed. Visual prognosis was analyzed. Clinical signs (retinitis, neuroretinitis, and choroiditis) and ocular complications (decrease in vision). A total of 37 of 52 patients (71%) showed positive results for at least 2 laboratory tests for WNV. Fundus examination revealed discrete, superficial, white retinitis; arteritis; phlebitis; and retinal hemorrhages with or without macular star. The FFA revealed areas of retinal inflammation with indistinct borders, vascular and optic disc leakage, vessel wall staining, or capillary nonperfusion. Indocyanine green angiography confirmed choroidal inflammation in 1 of the patients who was diabetic. The OCT scan of the macula revealed inner retinal layer edema in active inflammation and retinal atrophy in late stage. At the final visit, 43% of patients had visual acuity better than 6/12. In addition to previously reported clinical signs, retinitis, neuroretinitis, and retinal vasculitis were seen in this population. Atrophy of the inner retinal layer was seen on OCT after resolution of inflammation. Visual prognosis was good in patients with focal retinitis and poor in patients with occlusive vasculitis. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Corticobasal and ataxia syndromes widen the spectrum of C9ORF72 hexanucleotide expansion disease

Recently, a hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 was reported as the cause of chromosome 9p21-linked frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS). We here report the prevalence of the expansion in a hospital-based cohort and associated clinical features indicating a wider clinical spectrum of C9ORF72 disease than previously described. We studied 280 patients previously screened for mutations in genes involved in early onset autosomal dominant inherited dementia disorders. A repeat-primed polymerase chain reaction amplification assay was used to identify pathogenic GGGGCC expansions. As a potential modifier, confirmed cases were further investigated for abnormal CAG expansions in ATXN2. A pathogenic GGGGCC expansion was identified in a total of 14 probands. Three of these presented with atypical clinical features and were previously diagnosed with clinical olivopontocerebellar degeneration (OPCD), atypical Parkinsonian syndrome (APS) and a corticobasal syndrome (CBS). Further, the pathogenic expansion was identified in six FTD patients, four patients with FTD-ALS and one ALS patient. All confirmed cases had normal ATXN2 repeat sizes. Our study widens the clinical spectrum of C9ORF72 related disease and confirms the hexanucleotide expansion as a prevalent cause of FTD-ALS disorders. There was no indication of a modifying effect of the ATXN2 gene.