Polymerase Chain Reaction Approach Research Articles

Salinity significantly affects methane oxidation and methanotrophic community in Inner Mongolia lake sediments.

Methanotrophs oxidize methane (CH4) and greatly help in mitigating greenhouse effect. Increased temperatures due to global climate change can facilitate lake salinization, particularly in the regions with cold semiarid climate. However, the effects of salinity on the CH4 oxidation activity and diversity and composition of methanotrophic community in the sediment of natural lakes at a regional scale are still unclear. Therefore, we collected lake sediment samples from 13 sites in Mongolian Plateau; CH4 oxidation activities of methanotrophs were investigated, and the diversity and abundance of methanotrophs were analyzed using real-time quantitative polymerase chain reaction and high throughput sequencing approach. The results revealed that the diversity of methanotrophic community decreased with increasing salinity, and community structure of methanotrophs was clearly different between the hypersaline sediment samples (HRS; salinity > 0.69%) and hyposaline sediment samples (HOS; salinity < 0.69%). Types II and I methanotrophs were predominant in HRS and HOS, respectively. Salinity was significantly positively correlated with the relative abundance of Methylosinus and negatively correlated with that of Methylococcus. In addition, CH4 oxidation rate and pmoA gene abundance decreased with increasing salinity, and salinity directly and indirectly affected CH4 oxidation rate via regulating the community diversity. Moreover, high salinity decreased cooperative association among methanotrophs and number of key methanotrophic species (Methylosinus and Methylococcus, e.g). These results suggested that salinity is a major driver of CH4 oxidation in lake sediments and acts by regulating the diversity of methanotrophic community and accociation among the methanotrophic species.

RAPD-markers assisted genetic diversity analysis and Bt-Cry1Ac gene identification in eggplant (Solanum melongena L.)

Generic diversity (GD) screening helps to identify candidate genes involved in diseases, pests, or other stress resistance in plants. GD is crucial for maintaining vigor and healthy plant population. The random amplified polymorphic DNA (RAPD) is a rapid polymerase chain reaction (PCR)-based molecular marker, effective for screening genetic diversity in diverse plant species. In this study, we applied the RAPD-markers-assisted PCR approach to explore genetic diversity (GD) among 7 varieties of eggplant (Solanum melongena L.) at DNA level. Further, we assessed the presence of Bt-Cry1Ac gene in those varieties using the PCR approach. The eggplants showed genetic diversity at the DNA level. The amplification with two RAPD primers (OPA-02, OPA-04) produced a total of 41 bands. The highest similarity showed 78.57% among sample 4 (Eggplant China) vs sample 7 (Purple king F1) and sample 1 (Eggplant Srilanka) vs sample 4 (Eggplant China), while the lowest was 28.57% among sample 7 (Purple king F1) vs sample 3 (Eggplant Chittagong-1). In addition, the PCR results for the Bt Cry1Ac primer did not show a considerable band in the expected amplicon size. The dendrogram showed the genetic variability and relationship among all seven eggplant varieties at the genetic level. The study formulated a simple marker-based method for an effective molecular protocol to study genetics in eggplants. It also reported the presence of the Bt gene in popular eggplant varieties.

The Novel SLRP Family Member Lumican Suppresses Pancreatic Cancer Cell Growth.

The past studies clearly indicated that lumican was important in the context of pancreatic cancer (PC) onset and progression, but failed to clarify the underlying mechanistic basis for such activity. As such, we evaluated the functional importance of lumican in the context of pancreatic ductal adenocarcinoma (PDAC) to understand its mechanistic role in PC. Lumican levels were evaluated in PDAC patient tissues via quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry approaches. The role of lumican was additionally assessed via transfecting PDAC cell lines (BxPC-3, PANC-1) with lumican knockdown or overexpression constructs and treating PDAC cell lines with exogenous recombinant human lumican. Lumican expression levels were significantly higher in pancreatic tumor tissues relative to healthy paracancerous tissues. Lumican knockdown in BxPC-3 and PANC-1 enhanced their proliferation and migration, but reduced cellular apoptosis. Alternatively, lumican overexpression and exogenous lumican exposure failed to alter the proliferative activity of these cells. Further, lumican knockdown in BxPC-3 and PANC-1 cells results in marked P53 and P21 dysregulation. Lumican may suppress PDAC tumor growth by regulating P53 and P21, and the function of lumican sugar chains in the context of PC is worth studying in future studies.

Ultrafast Gene Fusion Assessment for Nonsquamous NSCLC

IntroductionGene fusion testing of ALK, ROS1, RET, NTRK, and MET exon 14 skipping mutations is guideline recommended in nonsquamous NSCLC (NS-NSCLC). Nevertheless, assessment is often hindered by the limited availability of tissue and prolonged next-generation sequencing (NGS) testing, which can protract the initiation of a targeted therapy. Therefore, the development of faster gene fusion assessment is critical for optimal clinical decision-making. Here, we compared two ultrafast gene fusion assays (UFGFAs) using NGS (Genexus, Oncomine Precision Assay, Thermo Fisher Scientific) and a multiplex reverse-transcriptase polymerase chain reaction (Idylla, GeneFusion Assay, Biocartis) approach at diagnosis in a retrospective series of 195 NS-NSCLC cases and five extrapulmonary tumors with a known NTRK fusion. MethodsA total of 195 NS-NSCLC cases (113 known gene fusions and 82 wild-type tumors) were included retrospectively. To validate the detection of a NTRK fusion, we added five NTRK-positive extrathoracic tumors. The diagnostic performance of the two UFGFAs and standard procedures was compared. ResultsThe accuracy was 92.3% and 93.1% for Idylla and Genexus, respectively. Both systems improved the sensitivity for detection by including a 5′-3′ imbalance analysis. Although detection of ROS1, MET exon 14 skipping, and RET was excellent with both systems, ALK fusion detection was reduced with sensitivities of 87% and 88%, respectively. Idylla had a limited sensitivity of 67% for NTRK fusions, in which only an imbalance assessment was used. ConclusionsUFGFA using NGS and reverse-transcriptase polymerase chain reaction approaches had an equal level of detection of gene fusion but with some technique-specific limitations. Nevertheless, UFGFA detection in routine clinical care is feasible with both systems allowing faster initiation of therapy and a broad degree of screening.

Association of xenobiotic-metabolizing genes polymorphisms with cervical cancer risk in the Tunisian population.

Host genetic characteristics and environmental factors interactions may play a crucial role in cervical carcinogenesis. We investigated the impact of functional genetic variants of four xenobiotic-metabolizing genes (AhR, CYP1A1, GSTM1, and GSTT1) on cervical cancer development in Tunisian women. The AhR gene polymorphism was analyzed using the tetra-primer ARMS-PCR, whereas the CYP1A1 polymorphism genotypes were identified by PCR-RFLP. A multiplex ligation-dependent polymerase chain reaction approach was applied for the analysis of GSTM1 and GSTT1 polymorphisms. The homozygous A/A genotype of the AhR gene (rs2066853) and the heterozygous T/C genotype of the CYP1A1 SNP (CYP1A1-MspI) appeared to be associated with an increased risk of cervical tumorigenesis (ORa = 2.81; ORa = 5.52, respectively). Furthermore, a significantly increased risk of cervical cancer was associated with the GSTT1 null genotype (ORa = 2.65). However, the null GSTM1 genotype showed any significant association with the risk of cervical cancer compared to the wild genotype (ORa = 1.18; p = 0.784). Considering the combined effect, we noted a significantly higher association with cancer risk for individuals with at least two high-risk genotypes of CYP1A1/GSTT1 (ORa = 4.2), individuals with at least two high-risk genotypes of CYP1A1/GSTT1/AhR (ORa = 11.3) and individuals with at least two high-risk genotypes of CYP1A1/GSTM1/GSTT1/AhR exploitation low-risk genotype as a reference. This study indicated that the single-gene contribution and the combined effect of xenobiotic-metabolizing gene polymorphisms (AhR, CYP1A1-MspI, GSTM1, and GSTT1) may have a considerable association with increased cervical cancer risk.

Heat-Treated Meat Origin Tracing and Authenticity through a Practical Multiplex Polymerase Chain Reaction Approach.

Meat adulteration have become a global issue, which has increasingly raised concerns due to not only economic losses and religious issues, but also public safety and its negative effects on human health. Using optimal primers for seven target species, a multiplex PCR method was developed for the molecular authentication of camel, cattle, dog, pig, chicken, sheep and duck in one tube reaction. Species-specific amplification from the premixed total DNA of seven species was corroborated by DNA sequencing. The limit of detection (LOD) is as low as 0.025 ng DNA for the simultaneous identification of seven species in both raw and heat-processed meat or target meat: as little as 0.1% (w/w) of the total meat weight. This method is strongly reproducible even while exposed to intensively heat-processed meat and meat mixtures, which renders it able to trace meat origins in real-world foodstuffs based on the authenticity assessment of commercial meat samples. Therefore, this method is a powerful tool for the inspection of meat adulterants and has broad application prospects.

Fast and noninvasive electronic nose for sniffing out COVID-19 based on exhaled breath-print recognition

The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approach has been widely used to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, instead of using it alone, clinicians often prefer to diagnose the coronavirus disease 2019 (COVID-19) by utilizing a combination of clinical signs and symptoms, laboratory test, imaging measurement (e.g., chest computed tomography scan), and multivariable clinical prediction models, including the electronic nose. Here, we report on the development and use of a low cost, noninvasive method to rapidly sniff out COVID-19 based on a portable electronic nose (GeNose C19) integrating an array of metal oxide semiconductor gas sensors, optimized feature extraction, and machine learning models. This approach was evaluated in profiling tests involving a total of 615 breath samples composed of 333 positive and 282 negative samples. The samples were obtained from 43 positive and 40 negative COVID-19 patients, respectively, and confirmed with RT-qPCR at two hospitals located in the Special Region of Yogyakarta, Indonesia. Four different machine learning algorithms (i.e., linear discriminant analysis, support vector machine, stacked multilayer perceptron, and deep neural network) were utilized to identify the top-performing pattern recognition methods and to obtain a high system detection accuracy (88–95%), sensitivity (86–94%), and specificity (88–95%) levels from the testing datasets. Our results suggest that GeNose C19 can be considered a highly potential breathalyzer for fast COVID-19 screening.

The effects of silver and copper nanoparticles and selenium on Salmo trutta hatchlings

AbstractWith the increase of nanotechnology applications, the risk of environmental adverse effects has emerged. It is known that nanoparticles (NPs) can induce oxidative stress, cause organ and DNA damage, or increase mortality rate in organisms, especially aquatic organisms. The present study aimed to investigate the potential genotoxicity of selenium, several nanoelements of copper, and a commercial product containing ionic silver colloids on Salmo trutta hatchlings based on the use of the polymerase chain reaction (PCR) approach and assess in a second step whether the addition of selenium and NPs of copper and silver improves PCR precision and efficiency. Sea trout eggs were incubated in NPs solutions. Before presenting the results, PCR analysis and agarose gel electrophoresis were made. DNA of hatchlings from the eggs incubated in copper NPs were less degraded than those incubated in silver colloids or selenium, and in the second phase of the study, a PCR product was only generated for samples incubated in selenium (Se). In agarose gel electrophoresis of isolates containing copper compounds, products were obtained in all samples, except for those containing 0.125 and 1.0 nM of Cu, and 0.25 and 1.0 nM of CuZnFe2O4 and CuFe2O4. It has been shown that genotoxic effects of tested NPs vary in a concentration‐dependent manner. Copper compounds did not interfere with PCR efficiency, except for CuFe2O4, which improved product quality in all tested concentrations. Presented results give an opportunity for further studies on the effects of NPs on PCR reaction and genotoxicity.

Multiplex PCR assay based on the citE2 gene and intergenic sequence for the rapid detection of Salmonella Pullorum in chickens

Salmonella is one of the most common Gram-negative pathogens and seriously threatens chicken farms and food safety. This study aimed to establish a multiplex polymerase chain reaction (PCR) approach for the identification of different Salmonella enterica subsp. enterica. The citE2 gene and interval sequence of SPS4_00301–SPS4_00311 existed in all S. enterica subsp. enterica serovars by genomic comparison. By contrast, a 76 bp deletion in citE2 was found only in Salmonella Pullorum. Two pairs of special primers designed from citE2 and interval sequence were used to establish the multiplex PCR system. The optimized multiplex PCR system could distinguish Salmonella Pullorum and non-Salmonella Pullorum. The sensitivity of the optimized multiplex PCR system could be as low as 6.25 pg/μL and 104 colony-forming units (CFU)/mL for genomic DNA and Salmonella Pullorum cells, respectively. The developed multiplex PCR assay distinguished Salmonella Pullorum from 33 different Salmonella enterica subsp. enterica serotypes and 13 non-target species. The detection of egg samples artificially contaminated with Salmonella Pullorum, Salmonella Enteritidis, and naturally contaminated 69 anal swab samples showed that results were consistent with the culture method. These features indicated that the developed multiplex PCR system had high sensitivity and specificity and could be used for the accurate detection of Salmonella Pullorum in clinical samples.

Detection of Streptococcus suis using the optimized real-time polymerase chain reaction protocol

The article presents the results of studies on the detection of Streptococcus suis by real-time polymerase chain reaction. Isolation and species identification of the studied isolates of streptococci was carried out according to morphological, cultural, biochemical and biological properties by conventional methods. The study of cultural characteristics of growth was carried out using conventional bacteriological methods on the brain heart infusion broth (BHI) and BHI agar with the addition of 5% sheep blood (blood BHI agar). To confirm biochemical properties as a confirmatory method, API 20 STREP test kit (bioMerieux, France) was used. In addition, to differentiate S. suis from the non-pathogenic species of streptococci, the hemolysis test was used. As a result of the studies, it was found that the use of the real-time PCR (polymerase chain reaction) method makes it possible to detect S. suis in an amount of 1 x 104 genome copies in the sample. All described validation parameters for the qualitative detection of S. suis DNA by real-time PCR meet international requirements, which guarantees accurate and reliable results. In Ukraine only a diagnostic test kit for convential PCR has been developed for the detection of swine streptococcosis. This approach is more time consuming and complex in comparison with the real-time PCR approach. We recommend that diagnostic laboratories implement this method in their practice. This will increase the number of effective diagnostic tools available to veterinarians on pig farms when they order laboratory tests. The high analytical sensitivity limit of a test is an essential parameter when screening is the focus, and obtaining false negative results causes a risk of the development of infection process among pig populations within infected herds. Our study showed that microbiological diagnostic methods to determine morphological and cultural properties can identify S. suis at the genus level. Determination of biochemical properties using the API 20 STREP test kit can be used to identify S. suis 1 and 2 serotypes. The conventional method and real-time PCR have 100% specificity and can be used to identify S. suis of different serotypes. Real-time PCR is a 2 to 4 times more sensitive limit than conventional PCR depending on the serotype being studied, and can be used to more accurately identify streptococcal DNA. It was found that the use of the real-time PCR method makes it possible to detect S. suis in an amount of 1 x 104 copies of the genome in the sample. Additionally, it was found that all the studied validation parameters of the qualitative method for determining S. suis DNA by real-time PCR meet international requirements, which guarantees accurate and reliable results.

Enhanced Detection of Viral RNA Species Using FokI-Assisted Digestion of DNA Duplexes and DNA/RNA Hybrids.

The accurate detection of nucleic acids from certain biological pathogens is critical for the diagnosis of human diseases. However, amplified detection of RNA molecules from a complex sample by direct detection of RNA/DNA hybrids remains a challenge. Here, we show that type IIS endonuclease FokI is able to digest DNA duplexes and DNA/RNA hybrids when assisted by a dumbbell-like fluorescent sensing oligonucleotide. As proof of concept, we designed a battery of sensing oligonucleotides against specific regions of the SARS-CoV-2 genome and interrogated the role of FokI relaxation as a potential nicking enzyme for fluorescence signal amplification. FokI-assisted digestion of SARS-CoV-2 probes increases the detection signal of ssDNA and RNA molecules and decreases the limit of detection more than 3.5-fold as compared to conventional molecular beacon approaches. This cleavage reaction is highly specific to its target molecules, and no detection of other highly related B-coronaviruses was observed in the presence of complex RNA mixtures. In addition, the FokI-assisted reaction has a high multiplexing potential, as the combined detection of different viral RNAs, including different SARS-CoV-2 variants, was achieved in the presence of multiple combinations of fluorophores and sensing oligonucleotides. When combined with isothermal rolling circle amplification technologies, FokI-assisted digestion reduced the detection time of SARS-CoV-2 in COVID-19-positive human samples with adequate sensitivity and specificity compared to conventional reverse transcription polymerase chain reaction approaches, highlighting the potential of FokI-assisted signal amplification as a valuable sensing mechanism for the detection of human pathogens.

Evolutionary history and introduction of SARS-CoV-2 Alpha VOC/B.1.1.7 in Pakistan through international travelers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge, and their identification is important for the public health response to coronavirus disease 2019 (COVID-19). Genomic sequencing provides robust information but may not always be accessible, and therefore, mutation-based polymerase chain reaction (PCR) approaches can be used for rapid identification of known variants. International travelers arriving in Karachi between December 2020 and February 2021 were tested for SARS-CoV-2 by PCR. A subset of positive samples was tested for S-gene target failure (SGTF) on TaqPathTM COVID-19 (Thermo Fisher Scientific) and for mutations using the GSD NovaType SARS-CoV-2 (Eurofins Technologies) assays. Sequencing was conducted on the MinION platform (Oxford Nanopore Technologies). Bayesian phylogeographic inference was performed integrating the patients’ travel history information. Of the thirty-five COVID-19 cases screened, thirteen had isolates with SGTF. The travelers transmitted infection to sixty-eight contact cases. The B.1.1.7 lineage was confirmed through sequencing and PCR. The phylogenetic analysis of sequence data available for six cases included four B.1.1.7 strains and one B.1.36 and B.1.1.212 lineage isolate. Phylogeographic modeling estimated at least three independent B.1.1.7 introductions into Karachi, Pakistan, originating from the UK. B.1.1.212 and B.1.36 were inferred to be introduced either from the UK or the travelers’ layover location. We report the introduction of SARS-CoV-2 B.1.1.7 and other lineages in Pakistan by international travelers arriving via different flight routes. This highlights SARS-CoV-2 transmission through travel, importance of testing, and quarantine post-travel to prevent transmission of new strains, as well as recording detailed patients’ metadata. Such results help inform policies on restricting travel from destinations where new highly transmissible variants have emerged.

Rapid membrane-based photothermal PCR for disease detection

The worldwide incidence of deadly infectious diseases has increased in recent years. Therefore, a rapid and accurate nucleic acid diagnostic system is in high demand. Polymerase chain reaction (PCR) is the standard method for amplifying and quantifying nucleic acid biomarkers for disease detection. Although several rapid PCR systems have been developed to overcome the limitations of conventional PCR, such as post-processing for analysis, complicated handling, and highly specialized instruments, a new PCR testing approach is still required. In this study, we developed a rapid membrane-based photothermal PCR that enables quantitative fluorescence detection. It requires no post-PCR procedures and provides user-friendly micro-scale solution control and sample loading. Au nanostructure was deposited on the reaction pad as a light-to-heat converter for PCR thermocycling. The automatically controlled 785 nm light source heated a solution in the reaction pad with photothermal effect. The temperature of the reaction solution, which was absorbed in a membrane, was cycled 25 times between 63 °C and 95 °C within 6 min with a photothermal cycler. The amplified samples on the membrane were then quantitatively analyzed by measuring the fluorescence signal of intercalating SYBR dye within 12 min. DNA samples extracted from Staphylococcus aureus and unpurified cells were amplified and analyzed. This membrane-based PCR approach can provide easy connectivity with various membrane-based sample pre-treatment processes, and therefore the proposed procedure could be used as the basis for an all-in-one molecular diagnosis platform. This simple, rapid, and sensitive PCR device could also serve as the basis for advanced molecular diagnostics.

Can periodontal pockets and caries lesions act as reservoirs for coronavirus?

The periodontal pocket and likely caries lesions may act as a reservoir and source of dissemination and development of systemic infections. While periodontal pockets have been found to harbor several viral species, there is no information on its ability to serve as a reservoir for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have used a real-time polymerase chain reaction (RT-PCR) approach to evaluate SARS-CoV-2 in periodontal pockets and cavitated caries lesions in a cross-sectional study of 72 participants who were divided into six groups: symptomatic positive COVID-19 cases with periodontal pockets, symptomatic positive with cavitated caries lesions, asymptomatic positive with periodontal pockets, asymptomatic positive with cavitated caries lesions, positive control, and negative control. A total of 180 samples were interrogated by RT-PCR to amplify the SARS-CoV-2 E and S genes. SARS-CoV-2 was present in 41.7% of symptomatic positive COVID-19 cases with periodontal pockets and 16.7% of symptomatic positive with cavitated caries lesions. The mean Ct value of E and S genes in periodontal pockets patients were 36.06±0.46 and 30.06±6.73, respectively, and the mean Ct value for both genes in caries lesions patients were 35.73±4.14, and 34.78±1.93, respectively. The sensitivity, specificity, and accuracy to detect SARS-CoV-2 among periodontal pockets were 20.8% (95% CI 7.13-42.15), 100% (95% CI 73.54-100.0), and 47.2% (95% CI 30.22-64.51), respectively. Among cavitated caries lesions patients, they were 8.3% (95% CI 1.03-27.0), 100% (95% CI 73.54-100.0), and 38.9% (95% CI 23.14-56.54), respectively. SARS-CoV-2 can be detected in periodontal pockets and caries lesions, and these sites may act as reservoirs for the virus. However, the sensitivity of SARS-CoV-2 detection is low compared with other methods. To our knowledge, this report is the first to investigate the relationship between SARS-CoV-2 and periodontal pockets and caries.

Meta-analysis of variable-temperature PCR technique performance for diagnosising Schistosoma japonicum infections in humans in endemic areas.

As China is moving onto schistosomiasis elimination/eradication, diagnostic methods with both high sensitivity and specificity for Schistosoma japonicum infections in humans are urgently needed. Microscopic identification of eggs in stool is proven to have poor sensitivity in low endemic regions, and antibody tests are unable to distinguish between current and previous infections. Polymerase chain reaction (PCR) technologies for the detection of parasite DNA have been theoretically assumed to show high diagnostic sensitivity and specificity. However, the reported performance of PCR for detecting S. japonicum infection varied greatly among studies. Therefore, we performed a meta-analysis to evaluate the overall diagnostic performance of variable-temperature PCR technologies, based on stool or blood, for detecting S. japonicum infections in humans from endemic areas. We searched literatures in eight electronic databases, published up to 20 January 2021. The heterogeneity and publication bias of included studies were assessed statistically. The risk of bias and applicability of each eligible study were assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 tool (QUADAS-2). The bivariate mixed-effects model was applied to obtain the summary estimates of diagnostic performance. The hierarchical summary receiver operating characteristic (HSROC) curve was applied to visually display the results. Subgroup analyses and multivariate regression were performed to explore the source of heterogeneity. This research was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines and was registered prospectively in PROSPERO (CRD42021233165). A total of 2791 papers were retrieved. After assessing for duplications and eligilibity a total of thirteen publications were retained for inclusion. These included eligible data from 4268 participants across sixteen studies. High heterogeneity existed among studies, but no publication bias was found. The pooled analyses of PCR data from all included studies resulted in a sensitivity of 0.91 (95% CI: 0.83 to 0.96), specificity of 0.85 (95% CI: 0.65 to 0.94), positive likelihood ratio of 5.90 (95% CI: 2.40 to 14.60), negative likelihood ratio of 0.10 (95% CI: 0.05 to 0.20) and a diagnostics odds ratio of 58 (95% CI: 19 to 179). Case-control studies showed significantly better performances for PCR diagnostics than cross-sectional studies. This was further evidenced by multivariate analyses. The four types of PCR approaches identified (conventional PCR, qPCR, Droplet digital PCR and nested PCR) differed significantly, with nested PCRs showing the best performance. Variable-temperature PCR has a satisfactory performance for diagnosing S. japonicum infections in humans in endemic areas. More high quality studies on S. japonicum diagnostic techniques, especially in low endemic areas and for the detection of dual-sex and single-sex infections are required. These will likely need to optimise a nested PCR alongside a highly sensitive gene target. They will contribute to successfully monitoring endemic areas as they move towards the WHO 2030 targets, as well as ultimately helping areas to achieve these goals.

Targeted Locus Amplification as Marker Screening Approach to Detect Immunoglobulin (IG) Translocations in B-Cell Non-Hodgkin Lymphomas.

Although MRD monitoring by the classic polymerase chain reaction (PCR) approach is a powerful outcome predictor, about 20% of mantle cell lymphoma (MCL) and 50% of follicular lymphoma (FL) patients still lack a molecular marker and are thus resulting not eligible for MRD monitoring. Targeted locus amplification (TLA), a new NGS technology, has been revealed as a feasible marker screening approach able to identify uncommon B-cell leukemia/lymphoma 1 (BCL1) and B-cell leukemia/lymphoma 2 (BCL2) rearrangements in MCL and FL cases defined as having "no marker" by the classic PCR approach.

Amplicon-based and metagenomic approaches provide insights into toxigenic potential in understudied Atlantic Canadian lakes

Cyanobacterial blooms and their toxigenic potential threaten freshwater resources worldwide. In Atlantic Canada, despite an increase of cyanobacterial blooms in the last decade, little is known about the toxigenic potential and the taxonomic affiliation of bloom-forming cyanobacteria. In this study, we employed polymerase chain reaction (PCR) and metagenomic approaches to assess the potential for cyanotoxin and other bioactive metabolite production in Harvey Lake (oligotrophic) and Washademoak Lake (mesotrophic) in New Brunswick, Canada, during summer and early fall months. The PCR survey detected the potential for microcystin (hepatotoxin) and anatoxin-a (neurotoxin) production in both lakes, despite a cyanobacterial bloom only being visible in Washademoak. Genus-specific PCR associated microcystin production potential with the presence of Microcystis in both lakes. The metagenomic strategy provided insight into temporal variations in the microbial communities of both lakes. It also permitted the recovery of a near-complete Microcystis aeruginosa genome with the genetic complement to produce microcystin and other bioactive metabolites such as piricyclamide, micropeptin/cyanopeptolin, and aeruginosin. Our approaches demonstrate the potential for production of a diverse complement of bioactive compounds and establish important baseline data for future studies of understudied lakes, which are frequently affected by cyanobacterial blooms.

Molecular diagnosis of three outbreaks during three successive years (2018, 2019, and 2020) of Lumpy skin disease virus in cattle in Sharkia Governorate, Egypt.

Background:Lumpy skin disease (LSD) is endemic in Egypt despite the Egyptian authorities’ annual mass vaccination of cattle with sheeppox vaccine (Veterinary Serum and Vaccine Research Institute, Egypt), and the LSD virus (LSDV) continues to thrive practically every summer. The disease has a huge economic impact on the trade of the animal and its by-product.Aim:This paper study the molecular characterization of LSDV strains that have been circulating in Sharkia Governorate, Egypt, for three successive years (2018, 2019, and 2020).Methods:A total of 61 specimens (26 skin nodules and 35 oculonasal swabs) were collected from a clinic in the hospital of veterinary medicine, Zagazig University, during the summer months (July, August, and September) of three outbreaks in 2018, 2019, and 2020. These were examined by polymerase chain reaction (PCR) based on the open reading frame 103 (ORF103) gene to confirm the suspected cases and determine the degree of homology between the three different outbreaks during three successive years between each other and between the derived sequences of GenBank.Results:Cattle is thought to be infected with LSDV due to the presence of scattered local or diffuse circumscribed skin nodules along with fever and lymph node enlargement and sometimes leg edema. The PCR approach proved rapid, sensitive, and specific for the detection of LSDV and confirmative diagnosis of the disease. Forty-six were detected to be positive by PCR (75.4%). The seven sequenced samples were translated to amino acid and registered in GenBank under accession number MW357655-MW357661. A single nucleotide mutation and amino acid variation were observed at positions 161 C (2020)/T (2018&2019) and consequently, a change in the amino acid at position 54 P (2020)/L (2018&2019) between the outbreak in 2020 and those in 2018 and 2019, respectively. The field LSDV isolates from Egypt cattle were more closely related to other LSDV sequences from Africa (Kenya), Asia, Europe, and the United States.These findings highlight the necessity of ongoing surveillance and characterization of circulating strains and the need to improve procedures for distinguishing vaccine strains from field viruses.

Relationship between the monoamine oxidase gene overactivity and the other pathophysiological and behavioral parameters implicated in memory deficiency in albino Winstar rats as Alzheimer’s disease model

The current study aimed to assess the pathophysiology mechanisms that mediate the effect on albin winstar rats' memory induced by the co -administration of fluoride and aluminum sulfate, as a model of Alzheimer's disease. This was done by assessing monoamine oxidase-A (MAO-A) activity, antioxidant activity, H2O2 and amyloid-β concentration in the hippocampus, embedded deep into the brain's temporal lobe, and level of cytokines in serum. The polymerase chain reaction approach was used to genotyping MAO-A, followed by single -stranded conformational polymorphism (SSCP) coupled with sequencing technique. The experimental animals were divided into two groups: control and treated groups. The uptake of heavy metals led to significantly increased MAO-A activity, amyloid -β deposition, H2O2 and cytokines levels in the treated group. However, the finding showed a significant decrease in antioxidant activity in the treated group. The results indicated that metals caused memory and learning impairments. PCR -SSCP genotyping showed many SNPs and haplotypes of the MAO-A exon 2 region, which showed the MAO-A gene polymorphism changes associated with Alzheimer's disease. The overall results indicated a role of metals to induce oxidative stress stimulating pathophysiological hallmarks in the hippocampus due to an increase in the influx of monoamine oxidase expression, which has been implicated in impaired memory, this study focused on the genetic variation of the exon 2 in monoamine oxidase-A gene and its relationship to Alzheimer's disease with the presence of several single nucleotide polymorphisms that may be related to Alzheimer's disease model in rats.

Expression of Phytase gene in transgenic maize with seed-specific promoter 27-kDa γ Zein and constitutive promoter CaMV 35S

Phytase enzymes are applied to animal feed to help animals absorb more nutrients. The use of feed raw materials containing phytase enzymes is expected to reduce the cost of animal feed production. Efforts to increase the phytase content in maize were carried out by improving genetics, in the way of assembling transgenic plants containing high phytase content. The 27-kDa γ Zein promoter is a specific promoter that expresses genes in caryopsis, and promoter CaMV 35S is a constitutive promoter that controls gene expression in all tissues and generally does not depend on the growth phase. Transgenic maize was transformed using Agrobacterium tumefacien infection method on maize B104. The reverse transcriptase polymerase chain reaction (RT-PCR) approach was used to examine the expression of phytase genes in leaves, roots, and caryopsis was done 10, 20, and 30 days after pollination (DAP). The phytase enzyme activity test was also carried out by using the colorimetric phosphomolybdate analysis method to see the phytase enzyme activity in unit µg-1. The results showed that the phytase gene in transgenic plants with the 27-kDa γ Zein promoter was highly expressed in maize caryopsis, but in line Z6.10 was also expressed in leaves, while in the CaMV 35S promoter the phytase gene was only expressed on the leaves. Phytase enzyme activity showed that transgenic maize was higher than non-transgenic maize.