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The analysis of nucleotide sequences of the Argemone genus samples by bioinformatics methods was aimed at the study of phylogenetic relationships of species within the genus, at the development of genus-specific primer pairs, and TaqMan-probe and their verification in silico. Molecular genetic studies by the polymerase chain reaction method in the "real-time" mode (RT-PCR) were devoted to checking the "performance" and specificity of the developed system of primers and TaqMan-probe using samples of eight species of Argemone and 11 species of other plants. Phylogenetic analysis using internal transcribed spacer 1 (ITS1) sequences confirmed previous phylogenetic studies and improved understanding of relationships within the genus. Before the in vitro test of the developed system of primers and TaqMan-probe, the ability of the extracted DNA to be amplified by the RT-PCR method using the system of primers and the TaqMan-probe to the 18S rRNA gene of eukaryotes was checked to exclude pseudo-negative results during the further verification of specificity. The conditions for RT-PCR were selected, namely, temperatures and times of denaturation, hybridization, elongation, and the number of cycles. The developed system of primers and TaqMan-probe for the RT-PCR method demonstrated high species specificity and sensitivity. Amplification was noted only in DNA samples of eight Argemone species, while PCR analysis of other plant species and negative controls showed no amplification. The detection limit of the developed system was determined to be 0.01%. It is proposed that this marker system be used to detect falsification in food and other products, particularly Argemone contamination in mustard or coconut oil. [ABSTRACT FROM AUTHOR]

Olive oil is appreciated worldwide for its unique nutritional and organoleptic properties. It is rich in unsaturated fatty acids and antioxidants, which are well-known for their health benefits. The qualitative characteristics of olive oil can be adversely affected by various biotic and abiotic factors. Particularly, microbial pathogens, such as mold fungi, can cause the deterioration of the oil and, thus, be a serious risk to consumer health. In this study, the effectiveness of DNA-based methods, i.e., endpoint PCR, Real-Time PCR (RT-PCR), and loop-mediated isothermal amplification (LAMP), all based on the ITS2-28S region, were used to evaluate the fungal contamination of samples of extra virgin olive oil. All the DNA techniques were able to detect, albeit at different levels, fungal infections affecting some of the basic quality parameters of the olive oils analyzed. However, compared to endpoint PCR and/or RT-PCR, the LAMP assay greatly simplified and accelerated the identification of pathogenic mold in the oil samples. This may encourage the olive oil industry to adopt this method in order to offer the consumer an oil with specific health parameters and therefore guarantee the safety and quality of this precious food product. [ABSTRACT FROM AUTHOR]

We performed genotyping analysis of human biallelic polymorphisms (single nucleotide polymorphisms) for the detection of homologous blood transfusion in sports doping. DNA was extracted from dried blood spots and quantified real‐time fast PCR. The method was proven to allow the detection of transfusions up to a donor percentage of 1%, with a significant improvement in terms of sensitivity with respect to both the reference cytofluorimetric method and a previously proposed strategy based on the DNA STR‐based strategy. [ABSTRACT FROM AUTHOR]

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Objectives: The study investigates how antibiotics affect biofilm formation and toxin gene expression in Clostridium difficile, which is essential for its survival and persistence. Methods: The study confirmed 25 strains of C. difficile and assessed biofilm formation. The MIC of metronidazole and vancomycin was determined through agar dilution, and the impact of sub-MIC levels on biofilm formation and eradication was investigated. Additionally, Real-time PCR was used to analyze the expression levels of target genes related to antibiotic treatment. Results: We found that certain genes, such as the ImmA/IrrE system, were associated with increased biofilm formation in isolates. Sub-MIC antibiotic levels influenced gene expression related to biofilm activities, particularly emphasizing the importance of toxin-antitoxin systems. The results suggest that antibiotics at sub-MIC levels may play a signaling role in promoting biofilm formation and gene expression in C. difficile. Conclusion: Our study suggests that toxin and antitoxin genes may impact C. difficile biofilm formation, while antibiotics could signal biofilm strengthening and gene expression increase. [ABSTRACT FROM AUTHOR]

Background: In kidney transplant recipients prone to infections like cytomegalovirus (CMV), a vital need for an accurate diagnostic method is evident. This study at Khorshid Laboratory in Tehran rigorously compares real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) for CMV detection in transplant patients. Methods: In January to March 1400, 70 kidney transplant recipients were assessed for CMV DNA using RT-PCR and concurrent ELISA tests, with statistical analysis aided by SPSS. Results: In kidney transplant patients (average age: 49.40 ± 13.64 years), 4.3% tested positive for CMV via PCR. Strong correlation between serological and molecular methods. IgG and IgM antibody detection both showed high sensitivity and specificity, advocating for efficient CMV diagnosis at Khorshid Laboratory, Tehran. Conclusion: This study emphasizes the need for a quick and efficient CMV diagnostic approach in kidney transplant patients. The strong correlation between molecular and serological methods favors using the faster RT-PCR method, crucial for timely management, especially with the increasing agerelated CMV incidence. The findings strongly recommend RT-PCR integration for enhanced sensitivity in kidney transplant patients at Khorshid Laboratory, Tehran. [ABSTRACT FROM AUTHOR]

Prosthetic joint infection is a serious complication of total joint arthroplasty that causes great morbidity in affected individuals. The most common cause of prosthesis associated infections are members of Staphylococcus spp., including Staphylococcus epidermidis. Culture has served as the gold standard for diagnosis, despite obvious shortcomings in terms of sensitivity and time. Bacterial genomic DNA extraction methodologies were evaluated for optimal recovery of genomic DNA from sterilised wash-out samples, spiked with S. epidermidis. Real time Polymerase chain reaction (PCR) assays targeting the S. epidermidis specific gseA gene were designed to reliably detect and quantify S. epidermidis. Sixty post-operative wash-out samples from primary hip and knee arthroplasties were taken aseptically. All were shown to be culture negative using the culture-dependent approach. These were samples were subjected to S. epidermidis-specific real time PCR. Standard curve showed good linearity. Sensitivity limit of the assay was <10 CFU S. epidermidis per sample. Reproducibility of the assay was confirmed. S. epidermidis was not identified in any of these samples using the novel species specific SYBR Green real time PCR technique. Results indicated that wash-out samples were true negatives and did not harbour S. epidermidis. To support this, patients displayed no symptoms of infection. To illustrate the full effectiveness of the novel real time PCR assay, a larger number of samples need to be tested (>1,000 patients). [ABSTRACT FROM AUTHOR]

The monitoring of mosquitoes is of great importance due to their vector competence for a variety of pathogens, which have the potential to imperil human and animal health. Until now mosquito occurrence data is mainly obtained with conventional monitoring methods including active and passive approaches, which can be time- and cost-consuming. New monitoring methods based on environmental DNA (eDNA) could serve as a fast and robust complementary detection system for mosquitoes. In this pilot study already existing marker systems targeting the three invasive mosquito species Aedes (Ae.) albopictus, Ae. japonicus and Ae. koreicus were used to detect these species from water samples via microfluidic array technology. We compared the performance of the high-throughput real-time PCR (HT-qPCR) system Biomark HD with real-time PCR (qPCR) and also tested the effect of different filter media (Sterivex® 0.45 µm, Nylon 0.22 µm, PES 1.2 µm) on eDNA detectability. By using a universal qPCR protocol and only 6-FAM-MGB probes we successfully transferred these marker systems on the HT-qPCR platform. All tested marker systems detected the target species at most sites, where their presence was previously confirmed. Filter media properties, the final filtration volume and observed qPCR inhibition did not affect measured Ct values via qPCR or HT-qPCR. The Ct values obtained from HT-qPCR were significantly lower as Ct values measured by qPCR due to the previous preamplification step, still these values were highly correlated. Observed incongruities in eDNA detection probability, as manifested by non-reproducible results and false positive detections, could be the result of methodological aspects, such as sensitivity and specificity issues of the used assays, or ecological factors such as varying eDNA release patterns. In this study, we show the suitability of eDNA-based detection of mosquito species from water samples using a microfluidic HT-qPCR platform. HT-qPCR platforms such as Biomark HD allow for massive upscaling of tested species-specific assays and sampling sites with low time- and cost-effort, thus this methodology could serve as basis for large-scale mosquito monitoring attempts. The main goal in the future is to develop a robust (semi)-quantitative microfluidic-based eDNA mosquito chip targeting all haematophagous culicid species occurring in Western Europe. This chip would enable large-scale eDNA-based screenings to assess mosquito diversity, to monitor species with confirmed or suspected vector competence, to assess the invasion progress of invasive mosquito species and could be used in pathogen surveillance, when disease agents are incorporated. [ABSTRACT FROM AUTHOR]

Monitoring, management and conservation of rare and elusive species often requires early detection of individuals, especially for re-introduced and endangered taxa. Environmental DNA (eDNA) approaches can enhance the detection power of traditional biomonitoring methods for low-density, newly-established populations. In this study, we used species-specific Real Time PCR TaqMan assays to assess the presence of two endangered freshwater species, the white-clawed crayfish Austropotamobius pallipes and the Eurasian otter Lutra lutra at eight sites in four river catchments in Liguria (northwestern Italy). The Eurasian otter was considered extinct in the study area since the 1980s. However, recent, although scattered sightings indicated a recolonisation by a few individuals. The white-clawed crayfish populations declined drastically and became increasingly dispersed in the western part of Liguria. Our eDNA analysis confirmed the presence of both species in some of the selected rivers and detected Eurasian otter DNA where the species was not recorded through traditional monitoring methods. This study confirms eDNA-based monitoring approaches as valuable tools to assess the presence of rare and elusive species and help implement protection plans at a local scale. [ABSTRACT FROM AUTHOR]

Background:Hand foot mouth disease (HFMD), a disease of childhood is also reported in adults caused by Coxsackie virus A type 16. Clinical condition is characterized by vesicular eruptive lesion in mouth, hand, foot, buttock, or genitalia. Coxsackie virus is a member of picoranaviridae family that includes non-enveloped SSRNA viruses. Spread in humans in the case of HFMD is through oral route, by the shed virus from intestine of infected person or through upper respiratory tract by the secretions or vesicle fluid of the diseased individual. Incubation period is 3 to 6 days. Diagnosis of HFMD most of the time is based clinically depending on the patient’s age, symptoms and clinical presentation of rash. Samples include stool, oral samples, biopsy & vesicular scrapings. Viral culture is the gold standard. Treatment is mainly supportive, to maintain hydration, acetaminophen and NSAIDS to reduce temperature. Novel agents like pleconaril play a vital role in the management of viral infection cases. Hand hygiene is the main stay of prevention. [ABSTRACT FROM AUTHOR]

Background: Herpes simplex type 2 is a common infection worldwide. This disease is common in both developed and developing countries. Early detection of infection is very important to reduce the risk of infection. Real-time reliable PCR is a very sensitive and specific method that can be used as the best marker in determining the therapeutic effect by identifying a viral genome in an individual. The prevalence of herpes simplex virus type 2 in women with genital herpes was evaluated by Real time PCR method. Methods: From January 1999 to March 2010, 45 samples of vaginal swabs and cervix of women with genital herpes were examined for HSV virus DNA detection using Real Time PCR. Results: The mean age of the patients was 35.9 + 5.9. The percentage of positive cases of herpes simplex virus type 2 in the studied women was 22.2% and the history of infection with hpv was 33.3% vs. 12.5%. = 0.094 which was significant. Conclusion: Clinical specimens of vaginal swabs from genital herpes caused by herpes simplex virus 2 can be quantitatively analyzed instead of nucleic acid extraction and amplification by PCR. [ABSTRACT FROM AUTHOR]

Psoriasis is a chronic, immune-mediated, hyperproliferative skin disease. Etiopathogenesis of psoriasis is not well understood. Plexin B2 was found to have effects on CD100-mediated T-cell morphology and expressed in the immune system. It may play a role in the pathogenesis of psoriasis. To assess the tissue level of plexin-B2 and plexin B2 related gene polymorphism which is signal regulatory protein gamma (SIRPγ-rs71212732) in psoriatic patients before and after NB-UVB, acitretin therapy alone or in combination and to detect correlation between level of tissue plexin B2 and disease severity and improvement. This single blinded randomized controlled trial was carried on 50 psoriatic patients and 50 healthy controls. Psoriasis Area and Severity Index score (PASI) was used to evaluate the disease severity. Tissue plexin-b2 level was measured using ELISA and SIRPγ-rs71212732 (T\C) was assessed using TaqMan™ assays and real-time PCR. A significant lower tissue plexin-B2 level was observed in control group (2.9 ± 0.6 pg/g) than cases (25.8 ± 2.8, pg/g) (p < 0.001). Also, a significantly higher tissue plexin-B2 level was observed in sever psoriasis (32.7 ± 3.8 pg/ml) in than moderate psoriasis (13.6 ± 2.1 pg/ml, p = 0.001). Tissue plexin B2 was positively correlated with diseases severity. Significantly higher (TC& TT) genotypes and mutant (C) allele among patients compared to the controls, p < 0.001 for all. Tissue plexin-b2 level was high in psoriasis vulgaris with positive correlation with disease severity and decreased after treatment. This may indicate a role of plexin-b2 in psoriasis vulgaris pathogenesis. [ABSTRACT FROM AUTHOR]

This study aimed to determine the presence of Brucella spp. in 73 abortion materials collected from various provinces of Turkey. Culture isolation and Real-time PCR methods were used for this purpose, and the laboratory diagnosis was compared. The clinical samples were taken from aborted cattle. A total of 79 samples were used for this study, consisting of 28 fetal stomach contents and 51 vaginal swabs. The presence of Brucella spp. was investigated using both culture isolation and real-time PCR methods. Of the collected samples, eight (10.12%) were found to be positive for Brucella spp. through culture isolation. These same samples were then subjected to Real Time PCR testing for comparison. Thirteen point nine two percent (11/79) of the samples tested positive for Brucella spp. using real-time PCR. This suggests that inhibitors, bacterial load in clinical samples, and possible contamination may reduce the chance of isolating the bacteria in culture or lead to false negative results. Therefore, it can be concluded that real-time PCR is a fast and reliable alternative to culture for diagnosing brucellosis. [ABSTRACT FROM AUTHOR]

Sonchus arvensis is recognized for its high content of phenolic compounds and antioxidants, exhibiting various medicinal benefits, including anti-diabetic, anti-depressant, and anti-cancer properties. This has positioned the plant as a significant candidate for commercial food, medicinal, and antioxidant supplements. Salinity may enhance the level of chlorogenic and caffeic acid, which are key secondary metabolites in S. arvensis. To investigate this, a completely randomized design experiment with three replications was implemented in a greenhouse to examine the impact of salinity on the expression of six genes responsible for the biosynthesis of chlorogenic acid. Additionally, the study examined how salinity affects the accumulation of chlorogenic acid, caffeic acid, chicoric acid, and apigenin in the lower and middle leaves of plants. Salinity stress treatments were applied at four different levels: 0 (control), 50, 100, and 150 mM of NaCl. The results indicated that the expression levels of phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), and p-coumaroyl ester 3′-hydroxylase (C3′H) were highest in the middle leaves at a concentration of 150 mM NaCl. Notably, there was an eight-fold increase in C4H expression in these leaves under the same salinity conditions. Conversely, the expression of shikimate/quinate O-hydroxycinnamoyl transferase (HCT) and quinate O-hydroxycinnamoyl transferase (HQT) genes decreased across all salinity treatments. Additionally, the levels of chlorogenic acid, caffeic acid, and chicoric acid were significantly elevated at 50 mM NaCl in both the lower and middle leaves, suggesting that cultivating S. arvensis in mildly saline environments could be beneficial. Furthermore, the findings from this study may serve as a preliminary step towards the cloning and full characterization of the genes examined in S. arvensis.

This study aimed to investigate the effect of different pH (7 and 4) and temperatures (4 and 40°C) on bacteriocin activity and plantaricin C (plnC) gene expression of Lactobacillus plantarium. Six strains of L. plantarum were used in these trials. The bacteriocin activity was measured after the different treatments by well diffusion test using indicator bacteria (Staphylococcus aureus and Escherichia coli O157:H7). Moreover, the plnC gene expression was determined by real-time PCR using 16S rRNA primers for universal bacteria and plantacirin C primers. The results declared a significant difference between the different pH and temperatures. In addition, the downregulation of plnC gene expression in pH 4. The upregulation of the same gene was applied to 40 °C during bacteria incubation. Also, there is no correlation between the bacteriocin activity and plnC gene expression after applying different pH and temperatures in L. plantarum bacteria. [ABSTRACT FROM AUTHOR]

Abstract Background Enterocytozoon bieneusi is the most common species found in humans. Although E. bieneusi has been investigated in humans, genotype profile of E. bieneusi is not known in Türkiye. Methods In this study, we screened E. bieneusi in patients (n = 94) with different types of malignant solid tumors by Real Time PCR and then sequenced E. bieneusi positive samples. All cancer patients were undergoing chemotherapy and had diarrhea. Moreover, as control groups, we also screened E. bieneusi in patients with diarrhea (n = 50) and without diarrhea (n = 50). Results Among all patients analyzed, 33 (17%) were found to be E. bieneusi-positive. As the patients were categorized, the molecular prevalence of E. bieneusi increased to 25.5% among cancer patients with diarrhea. However, the molecular prevalence of E. bieneusi was found to be lower in patients with presenting only diarrhea (8%) and patients without diarrhea (10%). The high molecular prevalence value detected among cancer patients with diarrhea was also statistically significant compared to other patient groups (P = 0.00112 and P = 0.0269). Among the 33 Real Time PCR positive samples, 10 of them were amplified by nested PCR and among these 10 samples, 6 of them were successfully genotyped. The phylogenetic tree showed the presence of D and Type IV which were also identified in stray cats living in İzmir in our previous study. Conclusions High molecular prevalence value indicates the importance of screening stool samples of cancer patients with diarrhea for E. bieneusi and genotyping results indicate that D and Type IV are circulating between humans and cats.

Late blight, caused by the pathogen Phytophthora infestans is an economical disease of potato worldwide. To better understand mechanisms of potato to resist this fungus, pathway signaling of TUB and PR5 were monitored using quantitative real-time PCR (qPCR) across four time points post infection. Results demonstrated that significant variance in the expression profiles of both genes in infected potato plants as compared to the non-infected controls. It is also notable that TUB and PR5 genes have a higher and faster expression in the resistant cultivar ‘Spunta’ as compared to the susceptible one ‘Draga’ with a maximum expression for TUB (1.8 and 1.4-fold) and PR5 (3.5 and 1.2-fold) respectively, at 48 hours post infection. Our data suggest that TUB and PR5 genes, positively regulate P. infestans—resistance in potato plants during disease progress, which can provide testable hypotheses that will need direct future experiments to define how these pathway signaling of both genes may be specified in potato defense system.

Abstract Background Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. Material & methods The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. Results All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. Conclusion Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

Background: Enterocytozoon bieneusi is the most common species found in humans. Although E. bieneusi has been investigated in humans, genotype profile of E. bieneusi is not known in Türkiye. Methods: In this study, we screened E. bieneusi in patients (n = 94) with different types of malignant solid tumors by Real Time PCR and then sequenced E. bieneusi positive samples. All cancer patients were undergoing chemotherapy and had diarrhea. Moreover, as control groups, we also screened E. bieneusi in patients with diarrhea (n = 50) and without diarrhea (n = 50). Results: Among all patients analyzed, 33 (17%) were found to be E. bieneusi-positive. As the patients were categorized, the molecular prevalence of E. bieneusi increased to 25.5% among cancer patients with diarrhea. However, the molecular prevalence of E. bieneusi was found to be lower in patients with presenting only diarrhea (8%) and patients without diarrhea (10%). The high molecular prevalence value detected among cancer patients with diarrhea was also statistically significant compared to other patient groups (P = 0.00112 and P = 0.0269). Among the 33 Real Time PCR positive samples, 10 of them were amplified by nested PCR and among these 10 samples, 6 of them were successfully genotyped. The phylogenetic tree showed the presence of D and Type IV which were also identified in stray cats living in İzmir in our previous study. Conclusions: High molecular prevalence value indicates the importance of screening stool samples of cancer patients with diarrhea for E. bieneusi and genotyping results indicate that D and Type IV are circulating between humans and cats. [ABSTRACT FROM AUTHOR]

Acute Oak Decline (AOD) is an oak disease that was first described in the United Kingdom. From the time the first symptoms appear, the disease can kill trees even within 6 years. One of the factors causing this disease is the action of the pathogenic bacteria Brenneria goodwinii, Rahnella victoriana and Gibbsiella quercinecans. In recent years, a deterioration in the condition of oak stands has been observed throughout Slovakia. These trees show exudates characteristic of AOD, which may indicate the presence of pathogenic bacteria. Samples of such symptomatic exudates from tree trunks were collected and analysed on 20 different plots in central and western Slovakia. Using real-time PCR, the presence of Brenneria goodwinii and Gibbsiella quercinecans was confirmed in ten and three stands, respectively. This report is the first information about the observation of these bacteria in weakened stands of Quercus robur in Slovakia. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. Material & methods: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. Results: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. Conclusion: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups. [ABSTRACT FROM AUTHOR]

Herein we evaluate the effect of secondary metabolites from the cyanobacteria Anabaena sp. on the expression of vancomycin resistance genes in Staphylococcus Saprophyticus.. This study is motivated by the growing concerns of multi-drug resistant S. saprophyticus strains and the need for alternative antimicrobial agents. of During a two-month period, 120 clinical samples were collected from elderly patients referred to Tehran hospitals. S. saprophyticus isolates were identified using both phenotypic and chemical tests. Subsequently, molecular methods were employed to detect the presence of van genes. The microbroth dilution method used to determine the sensitivity of the bacterial isolates to the methanolic extract of Anabaena sp. . Finally, real-time PCR was employed to measure the expression level of van genes compared to the 16S rRNA gene in S. saprophytic isolates treated with the cyanobacterial secondary metabolite. Among the 83 bacterial isolates obtained from the clinical samples, only eight were identified as S. saprophyticus. One isolate harbored the vanA gene, while two isolates possessed the vanB gene. The minimum inhibitory concentration (MIC) of the cyanobacterial secondary metabolite against S. saprophyticus was 750 μg/mL, with a sub-MIC of 325 μg/mL. Importantly, the expression level of vanA and vanB genes in the strains treated with the cyanobacterial secondary metabolite down-regulated compared to the reference gene (16S rRNA). The findings suggest that the secondary metabolites from Anabaena sp. possess a potent antimicrobial effect and can potentially reduce the expression of antibiotic resistance genes in S. saprophyticus. This paves the way for the development of a new generation of anti-staphylococcal drugs. [ABSTRACT FROM AUTHOR]

Background: In 2019, the first cases of an acute respiratory infectious disease were announced in the city of Wuhan, China. In places where the speed of transmission and the resulting high prevalence and death from this virus is high, it is important to find things that help prevent and reduce the symptoms and complications of the disease, one of these things. Things are serum levels of vitamin D. As a result of this study, serum levels of vitamin D were measured in patients with Covid-19. Methods: From December to March of 1400, 100 samples of people hospitalized in Khurshid laboratory were examined to identify the RNA of the Covid-19 virus by Real Time PCR method and at the same time to check the serum levels of vitamin D. The data was statistically analyzed using SPSS software. Results: The average age of the patients was between 12.45 and 40.8 years. Comparison the two Covid-19 positive and negative groups in terms of symptoms, it was found that the patients with RH positive had more positive PCR percentage and a significant difference was reported between RH positive and Covid-19 (p=0.006). In comparing the relationship between disease symptoms and the rate of PCR positive reports of gastrointestinal symptoms, history of significant disease, cough, fever, a significant difference was reported (p<0.001). Also, 50% of the samples were PCR positive. Based on the t-test, a significant difference was reported between the serum levels of vitamin D and Covid-19 (p<0.001). Conclusion: The results showed that the vitamin D is an acceptable protective factor against Covid19 infection and its deficiency increases the probability of infection. [ABSTRACT FROM AUTHOR]

The monitoring of mosquitoes is of great importance due to their vector competence for a variety of pathogens, which have the potential to imperil human and animal health. Until now mosquito occurrence data is mainly obtained with conventional monitoring methods including active and passive approaches, which can be time- and cost-consuming. New monitoring methods based on environmental DNA (eDNA) could serve as a fast and robust complementary detection system for mosquitoes. In this pilot study already existing marker systems targeting the three invasive mosquito species Aedes (Ae.) albopictus, Ae. japonicus and Ae. koreicus were used to detect these species from water samples via microfluidic array technology. We compared the performance of the high-throughput real-time PCR (HT-qPCR) system Biomark HD with real-time PCR (qPCR) and also tested the effect of different filter media (Sterivex® 0.45 µm, Nylon 0.22 µm, PES 1.2 µm) on eDNA detectability. By using a universal qPCR protocol and only 6-FAM-MGB probes we successfully transferred these marker systems on the HT-qPCR platform. All tested marker systems detected the target species at most sites, where their presence was previously confirmed. Filter media properties, the final filtration volume and observed qPCR inhibition did not affect measured Ct values via qPCR or HT-qPCR. The Ct values obtained from HT-qPCR were significantly lower as Ct values measured by qPCR due to the previous preamplification step, still these values were highly correlated. Observed incongruities in eDNA detection probability, as manifested by non-reproducible results and false positive detections, could be the result of methodological aspects, such as sensitivity and specificity issues of the used assays, or ecological factors such as varying eDNA release patterns. In this study, we show the suitability of eDNA-based detection of mosquito species from water samples using a microfluidic HT-qPCR platform. HT-qPCR platforms such as Biomark HD allow for massive upscaling of tested species-specific assays and sampling sites with low time- and cost-effort, thus this methodology could serve as basis for large-scale mosquito monitoring attempts. The main goal in the future is to develop a robust (semi)-quantitative microfluidic-based eDNA mosquito chip targeting all haematophagous culicid species occurring in Western Europe. This chip would enable large-scale eDNA-based screenings to assess mosquito diversity, to monitor species with confirmed or suspected vector competence, to assess the invasion progress of invasive mosquito species and could be used in pathogen surveillance, when disease agents are incorporated.

Background: Herpes simplex type 2 is a common infection worldwide. This disease is common in both developed and developing countries. Early detection of infection is very important to reduce the risk of infection. Real-time reliable PCR is a very sensitive and specific method that can be used as the best marker in determining the therapeutic effect by identifying a viral genome in an individual. The prevalence of herpes simplex virus type 2 in women with genital herpes was evaluated by Real time PCR method. Methods: From January 1999 to March 2010, 45 samples of vaginal swabs and cervix of women with genital herpes were examined for HSV virus DNA detection using Real Time PCR. Results: The mean age of the patients was 35.9 + 5.9. The percentage of positive cases of herpes simplex virus type 2 in the studied women was 22.2% and the history of infection with hpv was 33.3% vs. 12.5%. = 0.094 which was significant. Conclusion: Clinical specimens of vaginal swabs from genital herpes caused by herpes simplex virus 2 can be quantitatively analyzed instead of nucleic acid extraction and amplification by PCR.

This experiment established a relative quantitative detection method for bovine derived components in milk powder based on Real time PCR. The specificity, sensitivity, and stability of cattle-specific primers and probes were tested. By simulating mixed samples of cow’s milk powder and horse’s milk powder with different concentrations, linear fitting was performed based on the functional relationship of their △Ct values, and a standard curve was drawn to establish a relative quantitative detection of bovine derived components in milk powder. The results showed that the minimum detection limit of this method was 0.000 01 mg/mL. The recovery rate was 91.11%~119.2%, with an inter group coefficient of variation of ≤0.58% and an intra group coefficient of variation of ≤1.44%. This method is suitable for detecting the adulteration of bovine derived components and content in milk powder in terms of specificity and stability.

Olive oil is appreciated worldwide for its unique nutritional and organoleptic properties. It is rich in unsaturated fatty acids and antioxidants, which are well-known for their health benefits. The qualitative characteristics of olive oil can be adversely affected by various biotic and abiotic factors. Particularly, microbial pathogens, such as mold fungi, can cause the deterioration of the oil and, thus, be a serious risk to consumer health. In this study, the effectiveness of DNA-based methods, i.e., endpoint PCR, Real-Time PCR (RT-PCR), and loop-mediated isothermal amplification (LAMP), all based on the ITS2-28S region, were used to evaluate the fungal contamination of samples of extra virgin olive oil. All the DNA techniques were able to detect, albeit at different levels, fungal infections affecting some of the basic quality parameters of the olive oils analyzed. However, compared to endpoint PCR and/or RT-PCR, the LAMP assay greatly simplified and accelerated the identification of pathogenic mold in the oil samples. This may encourage the olive oil industry to adopt this method in order to offer the consumer an oil with specific health parameters and therefore guarantee the safety and quality of this precious food product.

• Foliar proline combats lead-induced crop stress. • Enhanced wheat crop health with external proline. • Lead stress impacts biomass, chlorophyll, and yield. • Foliar proline reduces reactive oxygen species. • TaGSK1 gene expression upregulation under proline stress. The application of proline as foliar spraying has emerged as a promising solution to combat lead-induced crop stress. This study highlights the effectiveness of external proline in enhancing wheat crop health under lead stress. We reveal how lead stress negatively impacts key factors like biomass, chlorophyll levels, and crop yield, particularly in 100-grain weight. Gas exchange parameters, including stomatal conductance, CO 2 levels, photosynthesis, water-use efficiency, and transpiration, also suffer under lead stress. Significantly, exposure to lead stress triggers the activation of antioxidant enzymes and elevates levels of proline, calcium, and protein. Nonetheless, the application of foliar proline proves highly effective in alleviating these detrimental effects by reducing reactive oxygen species and enhancing proline and protein levels in wheat. Additionally, our findings demonstrate a significant upregulation of TaGSK1 gene expression at different proline concentrations. Compared to controls, TaGSK1 expression increases by 1.7-fold under 50 mM proline stress and an impressive 4.7-fold under 200 mM proline stress in wheat. This finding holds promise in alleviating the adverse effects of lead stress and strengthening crop resilience for a more secure and sustainable future in wheat cultivation amid various stressors. [ABSTRACT FROM AUTHOR]

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Problem: Increased oxidative stress (OS) and inflammatory responses are major underlying factors behind Chlamydia trachomatis‐associated recurrent spontaneous abortion (RSA). miRNAs are known to regulate inflammation and OS and their dysregulation has been associated with compromised pregnancies. Therefore, aim of this study was to investigate the expression/correlation of OS biomarkers, cytokines and miRNAs in C. trachomatis‐associated RSA. Method of Study: Urine and non‐heparinized blood samples were collected from RSA patients with history of >3 consecutive abortions (cases) and non‐pregnant women with history of >2 successful deliveries (controls) attending Department of Obstetrics and Gynaecology, Safdarjung hospital, New Delhi. C. trachomatis detection was done in urine by PCR. miRNA expression was studied by microarray analysis and validated by real time‐PCR. Evaluation of cytokines and antioxidant genes expression were done by real‐time PCR. Level of OS biomarkers 8‐hydroxy guanosine (8‐OHdG) and 8‐isporostane (8‐IP) were measured by ELISA. Results: Fifty circulating miRNAs were differentially expressed in infected patients compared with controls. Of these, four were overexpressed and 46 downregulated. Thirteen differentially expressed circulating miRNAs were selected to validate microarray results. miRs‐8069, ‐3663‐3p showed maximum upregulation/downregulation in infected versus control group. Expression of cytokines (IL‐8, TNF‐α, IFN‐γ), antioxidant genes SOD2 and OS biomarkers (8‐OHdG,8‐IP) were increased while SOD1 was decreased in infected patients. miR‐8069 showed significant positive correlation with cytokines, SOD2, 8‐OHdG and 8‐IP. miR‐3663‐3p showed significant positive correlation with SOD1. Conclusions: Overall results indicate circulating miRNAs are involved in pathogenesis of C. trachomatis‐associated RSA and are potential modulators of cytokine signalling and OS in infected RSA. [ABSTRACT FROM AUTHOR]

The present study aimed at evaluating the potential contribution of Phosphatase and Tensin Homolog (PTEN) and its gene polymorphism (PTEN rs701848 T/C) in relation to Wingless/integrase-1 (Wnt) signaling in childhood epilepsy and the impact of antiepileptic medications on their serum levels. This study included 100 children with epilepsy (50 pharmacoresistant and 50 pharmacoresponsive) and 50 matched controls. All subjects had their genotypes for the PTEN rs701848T/C polymorphism assessed using TaqManTM assays and real-time PCR. By using the sandwich ELISA technique, the blood concentrations of PTEN and Wnt3a were measured. Serum Wnt3a levels in epileptic patients were significantly higher than in the control group, p < 0.001. Children with epilepsy who received oxcarbazepine had considerably lower serum Wnt3a levels than those who didn't, p < 0.001.With an AUC of 0.71, the cutoff value for diagnosing epilepsy as serum Wnt3a > 6.2 ng/mL has a sensitivity of 55% and a specificity of 80%. When compared to controls, epileptic children had considerably more (TT) genotype and less (TC and CC) genotypes, p < 0.05 for all. Epileptic children had significantly higher (T) allele frequency than controls, p = 0.006 with OR (95%CI) = 1.962(1.206–3.192). Pharmacoresistant epileptic children had significantly higher (TT) genotype compared to pharmacoresponsive type (p = 0.020). We originally found a strong association between PTEN rs701848 T/C and childhood epilepsy, in particular pharmacoresistant type. Serum Wnt3a levels increased in epilepsy, but were not significantly different between different alleles of PTEN. In pharmaco-responsive children Wnt3a levels differed significantly between the different PTEN genotypes. Antiepileptics may affect Wnt3a levels. [ABSTRACT FROM AUTHOR]

The two fungal species Trichophyton rubrum and Trichophytonviolaceum are common pathogens on human, infecting keratinized tissue of the outer body parts. Both species are belonging to the “Trichophyton rubrum complex” and share very high similarity in the genome. Secreted proteinases, key factors for keratin degradation, are nearly identical. Contrary, the ecological niches are differing. Trichophytonrubrum preferably infects skin and nails, whereas T. violaceum preferably infects the scalp. We postulate, that differences in the protease expression contribute to differences in ecological preferences. We analyzed the expression profiles of all 22 endoprotease genes, 12 subtilisins (S8A), 5 deuterolysins (M35) and 5 fungalysins (M36), for both species. To compare the influence of the keratin source, we designed experiments with human nail keratin, sheep wool keratin and keratin free cultivation media. Samples were taken at 12 h, 24 h, 48 h and 96 h post incubation in keratin medium. The expression of the proteases is higher in wool-keratin medium compared to human nail medium, with the exception of MEP4 and SUB6. Expression in the keratin-free medium is lowest. The expression profiles of the two species are remarkable different. The expression of MEP1, MEP3, SUB5, SUB11 and SUB12 are higher in T. rubrum compared to T. violaceum. MEP2, NpIIc, NpIIe, SUB1, SUB3, SUB4, SUB7 and SUB8 are higher expressed in T. violaceum compared to T. rubrum. The differences of the protease expression in the two species may expalin the differences in the ecological niches. Further analysis are necessary to verify the hypothesis.Please check and conform the edit made in title.Here I thinke the species of strains shouldnt be capital， and the right expression should be, "Expression Profiles of Protease in Onychomycosis-Related Pathogenic Trichophyton rubrum and Tinea Capitis-Related Pathogenic Trichophyton violaceum"Author names: Please confirm if the author names are presented accurately and in the correct se-quence (given name, middle name/initial, family name). Author 1 Given name: [Jingjing] Last name [Chen], Author 2 Given name: [Yangmin] Last name [Gao], Author 3 Given name: [Shuzhen] Last name [Xiong], Author 4 Given name: [Ping] Last name [Zhan]. Also, kindly confirm the details in the metadata are correct.YesPlease check and confirm the inserted city and country are correctly identified for affiliation 3.Please change the affiliations, Affiliation 2: ²Jiangxi Provincial Clinical Research Center for Skin Diseases, Dermatology Hospital of Jiangxi Province,The Affiliated Dermatology Hospital of Nanchang University, Nanchang, 330200, Jiangxi; Affiliation 3: 3Institute of Clinical Medicine, Jiangxi Provincial People's Hospital, The First Affiliated Hospital of Nanchang Medical College,Nanchang 330001, Jiangxi. Thanks a lot! [ABSTRACT FROM AUTHOR]

Microbial contamination and foodborne infections are a significant global public health concern. For this reason, the detection, monitoring, and characterization of pathogens represent a significant challenge in quality control settings. Standard approaches, such as culture methods and biochemical tests, are known to be very time-consuming and intensive. Conversely, molecular technologies based on the genomic identification of bacteria are quick and low-cost. Listeria monocytogenes is an opportunistic pathogen and a major concern especially in food industries. It is important to understand and implement multiple quality control measures to control Listeria infection risk and prevent the contamination of products. Standardized detection and confirmation tests such as the API Listeria test, MALDI-TOF MS, and PCR analysis are available. The aim of our work is to provide a specific molecular method, designed according to the EN UNI ISO 16140-3:2021, for the specific detection, monitoring, and characterization of Listeria spp. and Listeria monocytogenes contamination. The verification of this new rapid approach by real-time PCR (qPCR) overcomes the limitations of culture-based techniques, meeting all the verification criteria required by ISO guidelines, including implementation and item confirmation. This system offers a powerful approach to the real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection. [ABSTRACT FROM AUTHOR]

Citrus is affected by many diseases, and hence, the movement of citrus propagative materials is highly regulated in the USA. Currently used regulatory pathogen detection methods include biological and laboratory-based technologies, which are time-consuming, expensive, and have many limitations. There is an urgent need to develop alternate, rapid, economical, and reliable testing methods for safe germplasm exchange. Citrus huanglongbing (HLB) has devastated citrus industries leading to an increased need for germplasm exchanges between citrus growing regions for evaluating many potentially valuable hybrids for both HLB resistance and multilocational performance. In the present study, Next-Generation Sequencing (NGS) methods were used to sequence the transcriptomes of 21 test samples, including 15 well-characterized pathogen-positive plants. A workflow was designed in the CLC Genomics Workbench software, v 21.0.5 for bioinformatics analysis of the sequence data for the detection of pathogens. NGS was rapid and found to be a valuable technique for the detection of viral and bacterial pathogens, and for the discovery of new citrus viruses, complementary to the existing array of biological and laboratory assays. Using NGS methods, we detected beet western yellows virus, a newly reported citrus virus, and a variant of the citrus yellow vein-associated virus associated with the "fatal yellows" disease. [ABSTRACT FROM AUTHOR]

Introduction: Lung cancer (LC) is a leading cause of cancer-related mortality worldwide. Approximately 80% of LC cases are of the non-small cell lung cancer (NSCLC) type, and approximately two-thirds of these cases are diagnosed in advanced stages. Only systemic treatment methods can be applied to patients in the advanced stages when there is no chance of surgical treatment. Identification of mutations that cause LC is of vital importance in determining appropriate treatment methods. New noninvasive methods are needed to repeat and monitor these molecular analyses. In this regard, liquid biopsy (LB) is the most promising method. This study aimed to determine the effectiveness of LB in detecting EGFR executive gene mutations that cause LC. Methods: One hundred forty-six patients in stages IIIB and IV diagnosed with non-squamous cell non-small cell LC were included. Liquid biopsy was performed as a routine procedure in cases where no mutation was detected in solid tissue or in cases with progression after targeted therapy. Liquid biopsy samples were also obtained for the second time from 10 patients who showed progression under the applied treatment. Mutation analyses were performed using the Cobas® EGFR Test, a real-time PCR test designed to detect mutations in exons 18, 20, and 21 and changes in exon 19 of the EGFR gene. Results: Mutation positivity in paraffin blocks was 21.9%, whereas it was 32.2% in LB. Solids and LB were compatible in 16 patients. Additionally, while no mutation was found in solid tissue in the evaluation of 27 cases, it was detected in LB. It has been observed that new mutations can be detected not only at the time of diagnosis, but also in LB samples taken during the follow-up period, leading to the determination of targeted therapy. Discussion: The results showed that "liquid biopsy" is a successful and alternative non-invasive method for detecting cancer-causing executive mutations, given the limitations of conventional biopsies. [ABSTRACT FROM AUTHOR]

Background: SARS-CoV-2 as the cause of novel coronavirus disease (COVID-19) is a member of the family Coronaviridea that has generated an emerging global health concern. Controlling and preventing the spread of the disease requires a simple, portable, and rapid diagnostic method. Today, a standard method for detecting SARS-CoV-2 is quantitative real-time reverse transcription PCR, which is time-consuming and needs an advanced device. The aim of this study was to evaluate a faster and more cost-effective field-based testing method at the point of risk. We utilized a one-step RT-LAMP assay and developed, for the first time, a simple and rapid screening detection assay targeting the Envelope (E) gene, using specific primers. Methods: For this, the total RNA was extracted from respiratory samples of COVID-19 infected patients and applied to one-step a RT-LAMP reaction. The LAMP products were visualized using green fluorescence (SYBR Green I). Sensitivity testing was conducted using different concentrations of the designed recombinant plasmid (TA-E) as positive control constructs. Additionally, selectivity testing was performed using the influenza H1N1 genome. Finally, the results were compared using with conventional real time RT-PCR. Results: It was shown that the RT-LAMP assay has a sensitivity of approximately 15 ng for the E gene of SARS-CoV-2 when using extracted total RNA. Additionally, a sensitivity of 112 pg was achieved when using an artificially prepared TA-E plasmid. Accordingly, for the detection of SARS-CoV-2 infection, the RT-LAMP had high sensitivity and specificity and also could be an alternative method for real-time RT-PCR. Conclusion: Overall, this method can be used as a portable, rapid, and easy method for detecting SARS-CoV-2 in the field and clinical laboratories. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis and is a serious threat to global health. The methods can be used to detect and identify the bacteria is quantitative polymerase chain reaction (qPCR). In this method, denaturation and extension temperatures are determining factors of success that needs to be optimized. Objective: This study aims to optimize denaturation and extension temperatures in M. tuberculosis DNA amplification. Methods: The research used quasi-experimental design. The denaturation temperature optimized were 93, 94, 95, 96, and 97°C, and the extension temperature optimized were 58, 59, 60, 61, and 62°C. The test sample was a 1 ml sputum sample isolated from a patient with isoniazid-resistant M. tuberculosis. Optimization was performed using seven test primers, namely S315T, S315N, S315I, S315R, S315G, S315L, and R463B with the katG gene target and data analysis using Ms Excel. Data optimization results were processed with Excel by taking the lowest Ct value. Results: The results showed that the optimization temperatures for denaturation were different for each primer used. Primers S315T, S315R, and S315G, optimal with denaturation temperature of 96°C, primer S315N optimal with 94°C, primers S315I and R463B optimal with 93°C, and for primer S315L optimal with 95°C, with the most widely used temperature is 96°C. The optimal extension temperature was 58°C for primers S315T, S315N, S315I, and R463B, at 60°C for primers S315R and S315G, and at 61°C for primer S315L. Conclusion: The optimal denaturation temperature in this study was 96°C and the optimal extension temperature was 58°C.

Background: In 2019, the first cases of an acute respiratory infectious disease were announced in the city of Wuhan, China. In places where the speed of transmission and the resulting high prevalence and death from this virus is high, it is important to find things that help prevent and reduce the symptoms and complications of the disease, one of these things. Things are serum levels of vitamin D. As a result of this study, serum levels of vitamin D were measured in patients with Covid-19. Methods: From December to March of 1400, 100 samples of people hospitalized in Khurshid laboratory were examined to identify the RNA of the Covid-19 virus by Real Time PCR method and at the same time to check the serum levels of vitamin D. The data was statistically analyzed using SPSS software. Results: The average age of the patients was between 12.45 and 40.8 years. Comparison the two Covid-19 positive and negative groups in terms of symptoms, it was found that the patients with RH positive had more positive PCR percentage and a significant difference was reported between RH positive and Covid-19 (p=0.006). In comparing the relationship between disease symptoms and the rate of PCR positive reports of gastrointestinal symptoms, history of significant disease, cough, fever, a significant difference was reported (p

Introduction: Recurrent Pregnancy Loss and Recurrent Implantation Failure are major limiting factors in the establishment of pregnancy. Recurrent Pregnancy loss is defined as the failure of two or more clinically recognized pregnancies. A considerable proportion of infertile couples undergoing IVF treatment experiencing recurrent implantation failure (RIF) fail repeatedly to implant following at least three IVF cycles. MicroRNAs (miRNAs) can serve as reliable non-invasive diagnostic and prognostic biomarkers for pregnancy-related complications. Therefore, this study aimed to quantify miR-23a-3p expression level in the plasma of patients with idiopathic recurrent pregnancy loss (iRPL) and recurrent implantation failure compared to healthy subjects to evaluate its potential diagnostic value in iRPL and RIF patients. Material & Methods: This study is a cross-sectional descriptive study. A total of 120 plasma samples were obtained from 40 women with a history of at least two consecutive iRPL, 40 women with RIF, and 40 healthy women without a history of miscarriage to evaluate the expression level of the circulating miR-23a-3p by quantitative real-time polymerase chain reaction (qPCR) technique. All participants were recruited from Assisted Reproductive Technology (ART) and Stem Cell Research Center, Tabriz, Iran, September 2019-March 2020. Statistical analysis was performed using SPSS (version 22). Diagnostic efficiency of the circulating miRNAs was determined by Receiver Operatig Characteristic curve analysis using Graphpad Prism version 9.1.1. Findings: Our results showed that the miR-23a-3p expression level in plasma of iRPL patients was lower than those in healthy controls but without a statistically significant difference (P=0.113). We also found that miR-23a-3p plasma level in patients with RIF tended to be lower compared to healthy participants; however, it was not statistically significant (P=0.974). Discussion & Conclusion: The current study provides evidence indicating that downregulation of miR-23a-3p may be associated with iRPL and RIF. Therefore, further research is needed, such as using different geographic regions, large sample sizes, and other techniques (microarray).

The rising migration and travel from and towards endemic areas has brought renewed concerns about many parasitic infections, including neglected tropical diseases, such as schistosomiasis. Although serology is the most widely used method for the screening of schistosomiasis in non-endemic countries, this technique lacks sensitivity, especially to distinguish between past and ongoing infections. More recently, a molecular test based on the detection of Schistosoma cell-free DNA in the serum has been proposed as a diagnostic procedure for parasitosis. To test the performance of a blood PCR assay, this work investigated 102 serum samples collected from migrants coming from endemic areas by using primers specific to genomic regions of S. mansoni and S. haematobium patients. The results were then compared with the detection of specific IgG Abs with serological tests. Molecular analysis detected Schistosoma DNA in 32 patients. Among them, we characterized nine S. haematobium, 20 S. mansoni, and three coinfections. Compared with molecular assay, serological analysis detected specific antibodies against Schistosoma antigens in 52 out of 102 patients. Concordance between the two tests was found in 76 out of 102 patients (74.51%): in particular, both diagnostic tests were positive in 29 patients (28.43%) and negative in 47 (46.08%). The specificity of the molecular test was 94%. Overall, our data suggest that serological diagnosis could be combined with the molecular approach, providing the clinician with the serotyping of the parasite and useful information about the infection as well as the required further diagnostic procedures.

Botulism is dangerous toxic infection caused by a toxin produced by the bacterium Clostridium botulinum. The mortality rate from botulism can reach 70% of all cases of illness in case of untimely initiation of treatment. The pathogenesis of botulism involves the damage to the central nervous system by a toxin produced by C. botulinum. Currently there are seven recognized antigenic types of this toxin. Botulinum toxin is included into the group of biological agents and it is one of the most likely agents to be used in a biological attack. Since botulinum neurotoxin is a complex nucleoprotein complex and the traces of DNA can be detected even in purified toxin preparations, we have elaborated a technique for detecting and identifying DNA of toxigenic strains of Clostridium botulinum types A, B, E, that cause human botulism in most cases. This technique is based on the the detection of residual amounts of this DNA in botulinum toxin using multiplex real-time polymerase chain reaction (PCR) assay with fluorescent hybridization detection. The main obstacle to development of a technique for the detection and identification of DNA of toxigenic strains is the high variability of the genes responsible for the synthesis of botulinum toxin. We have established a region of the gene with the lowest homology in all strains. This requirement is met by a fragment of the bont gene that encodes a light chain of a neurotoxin and is highly conserved in the strains of C. botulinum producing one type of toxin. The paper represents the results of the definition of analytical sensitivity and specific activity of the developed method. The specificity of the determination is 100%, the analytical sensitivity – 1×102 mc./ml. The method can be used to analyze food, samples of clinical materials and environmental samples suspected of being contaminated with toxigenic strains of C. botulinum.

Abstract The aerobic granular biomass technology was optimized for treating nitrate-polluted groundwater based on the biological denitrification processes in order to provide drinking water. Reactors inoculated with granular biomass were operated at progressively lower C/N rate using acetate and methanol to encourage heterotrophic denitrification, in order to meet the recommended requirements described by European Drinking Water Framework Directive. The granulation and long-term stability of granular biomass under low C/N were successful for all stages, demonstrated compactness of granules and absence of filamentous microorganisms. The nitrate removal was similar in methanol- and acetate-fed reactors, occurring in both cases nitrate removal ratios > 80%, and fact allows the selection of one of both depending groundwater polluted case. Also, feeding reactors with 2 C/N ratio showed nitrate removal values of ≥ 95%, treating highly polluted groundwater (100 mg·L−1). The microbial diversity was higher in the methanol-fed reactor with representative phylotypes as Flavobacterium, Cytophagaceae, NS9 marine group, while species richness was higher in the acetate-fed reactor, which was mainly represented by Flavobacterium genus. Statistical analyses revealed the higher resilience of bacterial population on granules fed with acetate, showing more resistance under drop C/N ratio. Oscillating pollution in groundwater during seasonal periods should be treated using acetate as carbon source for denitrification carried out by granular biomass, while stable pollution concentrations over time allow the use of methanol as a carbon source since the greater microbial diversity allows the elimination of other contaminants present in groundwater.

IntroductionLung cancer (LC) is a leading cause of cancer-related mortality worldwide. Approximately 80% of LC cases are of the non-small cell lung cancer (NSCLC) type, and approximately two-thirds of these cases are diagnosed in advanced stages. Only systemic treatment methods can be applied to patients in the advanced stages when there is no chance of surgical treatment. Identification of mutations that cause LC is of vital importance in determining appropriate treatment methods. New noninvasive methods are needed to repeat and monitor these molecular analyses. In this regard, liquid biopsy (LB) is the most promising method. This study aimed to determine the effectiveness of LB in detecting EGFR executive gene mutations that cause LC.MethodsOne hundred forty-six patients in stages IIIB and IV diagnosed with non-squamous cell non-small cell LC were included. Liquid biopsy was performed as a routine procedure in cases where no mutation was detected in solid tissue or in cases with progression after targeted therapy. Liquid biopsy samples were also obtained for the second time from 10 patients who showed progression under the applied treatment. Mutation analyses were performed using the Cobas® EGFR Test, a real-time PCR test designed to detect mutations in exons 18, 20, and 21 and changes in exon 19 of the EGFR gene.ResultsMutation positivity in paraffin blocks was 21.9%, whereas it was 32.2% in LB. Solids and LB were compatible in 16 patients. Additionally, while no mutation was found in solid tissue in the evaluation of 27 cases, it was detected in LB. It has been observed that new mutations can be detected not only at the time of diagnosis, but also in LB samples taken during the follow-up period, leading to the determination of targeted therapy.DiscussionThe results showed that “liquid biopsy” is a successful and alternative non-invasive method for detecting cancer-causing executive mutations, given the limitations of conventional biopsies.

The rising migration and travel from and towards endemic areas has brought renewed concerns about many parasitic infections, including neglected tropical diseases, such as schistosomiasis. Although serology is the most widely used method for the screening of schistosomiasis in non-endemic countries, this technique lacks sensitivity, especially to distinguish between past and ongoing infections. More recently, a molecular test based on the detection of Schistosoma cell-free DNA in the serum has been proposed as a diagnostic procedure for parasitosis. To test the performance of a blood PCR assay, this work investigated 102 serum samples collected from migrants coming from endemic areas by using primers specific to genomic regions of S. mansoni and S. haematobium patients. The results were then compared with the detection of specific IgG Abs with serological tests. Molecular analysis detected Schistosoma DNA in 32 patients. Among them, we characterized nine S. haematobium, 20 S. mansoni, and three coinfections. Compared with molecular assay, serological analysis detected specific antibodies against Schistosoma antigens in 52 out of 102 patients. Concordance between the two tests was found in 76 out of 102 patients (74.51%): in particular, both diagnostic tests were positive in 29 patients (28.43%) and negative in 47 (46.08%). The specificity of the molecular test was 94%. Overall, our data suggest that serological diagnosis could be combined with the molecular approach, providing the clinician with the serotyping of the parasite and useful information about the infection as well as the required further diagnostic procedures. [ABSTRACT FROM AUTHOR]

In the present study, total of 32 ante-mortem (AM) samples (saliva = 18 and corneal smears = 14) from six animal species (cattle = 5; camel = 1; goat = 1; horse = 1; buffalo = 4; dog = 6) and 28 post-mortem (PM) samples of domestic (cattle = 6; camel = 1; goat = 1; buffalo = 5; dog = 7) and wild animals (lion = 4, mongoose = 2; bear = 1; leopard = 1) were examined for rabies diagnosis in Gujarat, India. Direct fluorescent antibody test (dFAT) and reverse transcriptase polymerase chain reaction (RT-PCR) were applied on AM samples, whereas along with dFAT and RT-PCR, histopathological examination, immunohistochemistry (IHC) and real time PCR (qPCR) were used for PM diagnosis. Nucleotide sequencing of full nucleoprotein (N) and glycoprotein (G) genes were carried out upon representative amplicons. In AM examination, 7/18 saliva and 5/14 corneal impressions samples were found positive in dFAT and 8/18 saliva samples were found positive in RT-PCR. In PM examination, 14/28 samples showed positive results in dFAT and IHC with unusual large fluorescent foci in two samples. In histopathology, 11/28 samples showed appreciable lesion and Negri bodies were visible in 6 samples, only. Out of 23 brain samples examined. 12 samples were found positive in N gene RT-PCR and qPCR, and 10 samples in G gene RT-PCR. Phylogenetic analysis of N gene revealed that test isolates (except sample ID: lion-1; lion, Gir) form a close group with sequence ID, KM099393.1 (Mongoose, Hyderabad) and KF660246.1 (Water Buffalo, Hyderabad) which was far from some south Indian and Sri Lankan isolates but similar to Indian isolates from rest of India and neighboring countries. In G gene analysis, the test isolates form a close group with sequence ID, KP019943.1. [ABSTRACT FROM AUTHOR]

Background: The high prevalence of Hepatitis C virus (HCV) infection contributes to morbidity and mortality among patients on maintenance hemodialysis. Nosocomial transmission can be prevented by strict infection control measures and early screening. Present study aims to determine the prevalence of HCV infection in a hemodialysis unit in Central Kerala, India using Real-time PCR and third-generation ELISA. Methods: The present descriptive cross-sectional study conducted from November 2017 to June 2018 included 117 patients with chronic renal failure who underwent maintenance hemodialysis. Sociodemographic data and detailed history regarding risk factors were taken by interview and from medical records. HCV RNA estimation using real-time PCR and antibody detection using third-generation ELISA were performed using blood samples collected from the patients. Results: Most of the patients were males with a mean age of 48years. Prevalence was 9.4% for HCV RNA and 10.3% for antibodies. The performance of the third-generation ELISA was comparable to the PCR method. Duration of hemodialysis, dialyzer reuse, history of blood transfusion, and positive antibody status was found to be significant risk factors for HCV viremia. Interpretation & conclusions: The prevalence of HCV infection is low in our setting. But there is a need to review infection control practices to identify lapses in dialyzer reuse and safe blood transfusion. A robust infection control program including isolation policy along with screening using high performance third generation ELISA kits can reduce the incidence of HCV infection in hemodialysis centers. [ABSTRACT FROM AUTHOR]

The current study describes a new diagnostic method for the rapid and accurate detection of Tilletia indica, the pathogen accountable for causing Karnal bunt (KB) disease in wheat. This method uses quantitative real-time polymerase chain reaction (qPCR) and a primer set derived from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of T. indica to identify the presence of the pathogen. The qPCR assay using this primer set was found highly sensitive, with a limit of detection (LOD) value of 4 pg of T. indica DNA. This level of sensitivity allows for the detection of the pathogen even in cases of different growth stages of wheat, where no visible symptoms of infection on the wheat plants can be seen by naked eyes. The study also validated the qPCR assay on ten different wheat cultivars. Overall, this study presents a valuable molecular tool for rapid, specific and sensitive detection of KB fungus in wheat host. This method has practical applications in disease management, screening of wheat genotypes against KB and can aid in the development of strategies to mitigate the impact of Karnal bunt disease on wheat production. [ABSTRACT FROM AUTHOR]