Real-time Polymerase Chain Reaction Amplification Research Articles

Pupal Exuviae of Culex Pipiens L. (Diptera: Culicidae) Can be Utilised as a Non-Invasive Method of Biotype Differentiation

BackgroundCulex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay.ResultsGenomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55–80%.ConclusionsThis study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.

RNA interference-mediated knockdown of genes involved in sugar transport and metabolism disrupts psyllid Bactericera cockerelli (Order: Hemiptera) gut physiology and results in high mortality.

The causal agent of zebra chip of potato and vein-greening diseases of tomato is "Candidatus Liberibacter solanacearum" (CLso), a fastidious bacterium transmitted by the potato psyllid. In the absence of disease-resistant cultivars, disease management has relied on minimizing vector population size to reduce CLso transmission, which requires frequent insecticide applications. There is growing interest in the use of RNA interference (RNAi) technology to supplant traditional insecticides with biopesticides. This requires knowledge of genes essential for insect livelihood whose knockdown leads to significant mortality or other phenotypes. Such candidate genes can be evaluated by reverse genetics approaches to further corroborate predicted gene function. Here, five potato psyllid genes involved in sugar homeostasis in the potato psyllid gut, α-glucosidase1 (AGLU1), aquaporin2 (AQP2), facilitated trehalose transporter1 (TRET1), Trehalase1 (TRE1), and Trehalase2 (TRE2), were investigated as candidates for effective gene silencing. Potato psyllid dsRNAs were designed to optimize knockdown of gene targets. Third instar PoP nymphs were given a 48-hr ingestion-access period (IAP) on individual or groups of dsRNA in 20% sucrose. Mortality was recorded 0, 3, 5, 7, and 9 days post-IAP. Gene knockdown was analyzed 9 days post-IAP by quantitative real-time reverse-transcriptase polymerase chain reaction amplification. The individual or stacked dsRNA combinations resulted in 20-60% and 20-40% knockdown, respectively, while subsequent psyllid mortality ranged from 20-40% to >60% for single and stacked dsRNA combinations, respectively. Reverse genetics analysis showed that simultaneous knockdown of the five selected candidate genes with predicted functions in pathways involved in sugar-homeostasis, metabolism, and -transport yielded the highest mortality, when compared with single or combinations of targets. Results confirmed the functions afforded by psyllid gut genes responsible for osmotic homeostasis and sugar metabolism/transport are essential for livelihood, identifying them as potentially lucrative RNAi biopesticide targets and highlighted the translational relevance of targeting multiple nodes in a physiological pathway simultaneously.

Evaluating the effect of gargling with hydrogen peroxide and povidone-iodine on salivary viral load of SARS-CoV-2: A pilot randomized clinical trial.

This study evaluates the salivary viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in hospitalized patients and outpatients before and after gargling with 1% hydrogen peroxide and 0.25% povidone-iodine in comparison with normal saline. This clinical trial was conducted on 120 participants with laboratory-confirmed coronavirus disease 2019 (COVID-19) in two groups: outpatients (n = 60) and hospitalized patients (n = 60). In each group, the patients were randomly divided into three subgroups of 20 based on their given mouthwash for gargling (hydrogen peroxide, povidone-iodine, or normal saline). Two saliva samples were taken from each patient: the first one before gargling and the second one 10 minutes after gargling 10 ml of the respected mouthwashes for 30 seconds. The TaqMan real-time polymerase chain reaction (PCR) amplification of SARS-CoV-2 was used to measure the viral load. Saliva samples from 46% of patients were positive for coronavirus before gargling the mouthwashes. The percentage of patients with an initial positive saliva sample was significantly higher in the outpatient group (83.3%) than in the hospitalized group (5.4%) (P = 0.01). According to the findings, gargling any mouthwash similar to saline did not reduce the viral load (P > 0.05). The saliva of COVID-19 patients in the initial stage of the disease was more likely to contain SARS-CoV-2 than the saliva of the hospitalized patients. Gargling hydrogen peroxide or povidone-iodine did not reduce the salivary SARS-CoV-2 viral load.

Comparison of Serological and Molecular Methods for Detection of Spiroplasma citri in Moroccan Citrus-Growing Areas.

Spiroplasma citri, a helical motile, wall-less, and cultivable microorganism of the class Mollicutes, is the agent of the citrus stubborn disease. There is currently a lack of data about the presence of this pathogen in Moroccan citrus orchards. This study aims to validate serological and molecular methods for routine S. citri diagnosis in Moroccan citrus groves. To provide an update on the present status of the outbreak of the pathogen in Moroccan citrus orchards, a survey of S. citri was conducted in the main citrus-growing regions of Morocco. A total of 575 leaf samples were collected from citrus trees with symptoms attributable to S. citri infection. Samples were collected during 2020 and 2021 from 23 citrus orchards. The presence of S. citri was tested in all samples using the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Using this method, 57 samples were found to be infected with S. citri, 41 samples had doubtful results, and the remaining samples were negative. To corroborate the results of the DAS-ELISA test, 148 samples were chosen for additional molecular testing using conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) based on specific primer pairs targeting three different genes (putative adhesion-like gene P58, putative adhesion gene P89, and spiralin gene). Using primers that target the putative adhesion-like gene P58, S. citri was detected by conventional and real-time PCR amplification from plant tissue with differing degrees of specificity. The results allowed us to determine the incidence of S. citri in all Moroccan citrus orchards, with a wide range of positive samples varying from 6.5% to 78%, and to show that molecular tests, particularly real-time PCR assays that target the putative adhesion-like gene P58, are the most sensitive for making an accurate diagnosis of S. citri. Indeed, the real-time PCR with P58-targeting primers yielded positive results from all positive and doubtful ELISA samples as well as some negative samples, with an OD value close to 1.5× times healthy samples, thus demonstrating a high sensibility of this technique.

Validation of the efficacy of air purifiers using molecular techniques.

The importance of air purifiers has increased in recent years, especially with the "coronavirus disease 2019" pandemic. The efficacy of air purifiers is usually determined under laboratory conditions before widespread application. The standard procedure for testing depends on virus cultivation and titration on cell culture. This, however, requires several days to deliver results. The aim of this study was to establish a rapid molecular assay which can differentiate between intact infectious and distorted non-infectious virus particles. Feline Coronavirus was selected as model for screening. First the samples were pretreated with enzymes (universal nuclease and RNase cocktail enzyme mixture) or viability dye (propidium monoazide) to eliminate any free nucleic acids. The ribonucleic acid (RNA) from intact virus was released via magnetic beads-based extraction, then the amount of the RNA was determined using real-time reverse transcription polymerase chain reaction (RT-PCR) or reverse transcription recombinase-aided amplification (RT-RAA). All results were compared to the infectivity assay based on the calculation of the 50% tissue culture infectious dose (TCID50). The nuclease has eliminated 100% of the free Feline Coronavirus RNA, while propidium monoazide underperformed (2.3-fold decrease in free RNA). Both RT-RAA and real-time RT-PCR produced similar results to the infectivity assay on cell culture with limit of detection of 102 TCID50/mL. Two UV-C air purifiers with prosperities of 100% inactivation of the viruses were used to validate the established procedure. Both real-time RT-PCR and RT-RAA were able to differentiate between intact virus particles and free RNA. To conclude, this study revealed a promising rapid method to validate the efficacy of air purifiers by combining enzymatic pretreatment and molecular assays.

Challenges and future solutions for detection of Clostridioides difficile in adults.

There are no established standards for the diagnosis of Clostridioides difficile infection (CDI), even though the importance of this infection in humans is well known. The effectiveness of the commercially available techniques, which are all standardized for use with human feces, is also limited in terms of the accuracy of the tests. Furthermore, the current approach lacks a point-of-care diagnosis with an acceptable range of sensitivity and specificity. This article reviews the challenges and possible future solutions for the detection of CDI in adults. Existing diagnostic methods, such as enzyme-linked immunoassays and microbial culturing for the detection of toxins A and B, appear to work poorly in samples but exhibit great sensitivity for glutamate dehydrogenase. Real-time polymerase chain reaction and nucleic acid amplification tests have been investigated in a few studies on human samples, but so far have shown poor turnaround times. Thus, developing a multiplex point-of-care test assay with high sensitivity and specificity is required as a bedside approach for diagnosing this emerging infection.

Magnetic-bead-based DNA-capture-assisted real-time polymerase chain reaction and recombinase polymerase amplification for the detection of African swine fever virus.

African swine fever (ASF) is a deadly disease in swine caused by African swine fever virus (ASFV). The global spread of ASFV has resulted in significant economic losses worldwide. Improved early detection has been the most important first line of defense for preventing ASF outbreaks and for activating control measures. Despite the availability of rapid amplification methods, nucleic acid extraction from specimens still needs to be performed in a laboratory. To facilitate this step, we exploited the strong affinity of biotin-streptavidin binding by functionalizing streptavidin-coated magnetic beads with biotinylated oligonucleotide capture probes to efficiently capture genotype II ASFV DNA directly from crude clinical samples. The captured DNA is suitable for detection using real-time quantitative PCR (qPCR) and recombinase polymerase amplification (RPA). In this study, ASFV DNA was efficiently captured from swine feces, serum, and tissue samples. Both DNA-capture-assisted qPCR and RPA-based detection methods have a limit of detection (LOD) of 102 copies/µl, which is comparable to those of commercially available kits. In addition, an RPA-SYBR Green I method was developed for the immediate visual detection of ASFV DNA, which is time-saving and efficient for resource-limited field settings. In summary, a rapid, versatile, sequence-specific DNA capture method was developed to efficiently capture ASFV DNA from swine clinical samples and subsequent detection by qPCR and RPA, which has the potential to be used for robust screening and surveillance of ASFV and in point-of-care (POC) diagnostics.

Reference gene validation for the relative quantification of cannabinoid receptor expression in human odontoblasts via quantitative polymerase chain reaction.

ObjectiveThe aim of this study was to identify and validate the reference genes in cultured human odontoblasts to quantify their cannabinoid receptor transcripts.MethodsThe most stably transcribed genes in cultured human odontoblast cells were identified using the RefGenes tool and were selected for real-time polymerase chain reaction (PCR) amplification. Human odontoblast cells were differentiated from mesenchymal stem cells using a transforming growth factor-β-supplemented differentiation medium, and total RNA was purified. Reverse transcription-quantitative PCR and relative quantification analyses were performed using the Schefe's method. The relative expression dataset was analyzed to select the most stable genes.ResultsThe analysis showed that the transcripts of cholinergic receptor nicotinic beta 2 subunit, LIM homeobox transcription factor 1 beta, and family with sequence similarity 223 member B presented the lowest standard deviation (SD) in expression (SD: 0.2, 0.17, and 0.16, respectively). These genes showed similar expression levels as the target genes (cannabinoid receptors). Significant differences were found in the relative expression levels of cannabinoid receptors using the selected genes compared to those calculated using beta actin transcripts as references (p < 0.05).ConclusionsThe strategy reported here for searching and verifying new reference genes will aid in the accurate and reliable expression of cannabinoid receptors in human odontoblast cells.

Identification and molecular characterization of simian endogenous retrovirus in Macaca fascicularis and Macaca nemestrina from captive breeding facilities in Bogor, Indonesia.

Background and Aim:Endogenous retroviruses (ERVs) found in all vertebrates, including non-human primates (NHPs), are known to be genetically inherited. Thus, recent studies have explored ERVs for human immunodeficiency virus vaccine development using human ERV (HERV) due to the hypervariability of exogenous retroviruses which cause conventional vaccines to be ineffective. HERV was also found to be able to induce an immune response in cancer patients. This study aimed to identify and molecularly characterize ERVs from Indonesian NHPs: Macaca fascicularis and Macaca nemestrina. Then, we described the phylogenetic relationship of these isolates with those of the simian ERVs (SERVs) characterized in other species and countries.Materials and Methods:First, 5 mL of whole blood samples was taken from 131 long-tailed macaques and 58 pig-tailed macaques in captive breeding facilities at Bogor, Indonesia, for DNA extraction. Next, the DNA samples were screened using the SYBR Green real-time polymerase chain reaction (PCR) technique with specific primers for env (simian retroviruses [SRV]1-5 7585U19 and SRV1-5 7695L21). Positive SERV results were those with cycle threshold (CT) values < 24 (CT < 24) and melting temperature (TM) ranges of 80°C–82°C. Then, whole-genome nucleotide sequences from two pig-tailed macaques samples detected as positive SERV were generated using a nucleic acid sequencing technique which utilized the walking primer method. Subsequently, the sequences were analyzed using bioinformatics programs, such as 4Peaks, Clustal Omega, and BLAST (NCBI). Subsequently, a phylogenetic tree was constructed using the neighbor-joining method in MEGA X.Results:SYBR Green real-time PCR amplification results indicated that SERV (Mn B1 and Mn B140910)-positive samples had CT values of 22.37–22.54 and TM of 82°C. Moreover, whole-genome sequences resulted in 7991 nucleotide sequences, comprising long terminal repeat, gag, pro, pol, and env genes identical between the sequenced samples. Furthermore, the phylogenetic tree results indicated that both samples from M. nemestrina had 99%–100% nucleotide identities to the Mn 92227 sample identified at the National Primate Center University of Washington (NaPRC UW) which was imported from Indonesia in 1998, confirmed as a novel SERV strain. The phylogenetic tree results also indicated that although SERV whole-genome nucleotide and env amino acid sequences were clustered with SRV-2 (identity values of 82% and 79%, respectively), they had a 99%–100% nucleotide identity to Mn 92227. Meanwhile, the gag, pro, and pol amino acids were clustered with SRV-1, SRV-3, SRV-4, SRV-5, SRV-8, and SERV/1997, with 82% and 88% identity values.Conclusion:Based on the SYBR Green real-time PCR profiles generated, similarities with Mn 92227 were observed. Subsequent phylogenetic analysis confirmed that both samples (Mn B1 and Mn B140919) from pig-tailed macaques in the country of origin were novel SERV strains at NaPRC UW. Therefore, it could be used in biomedical research on ERVs.

Comparison of Simple RNA Extraction Methods for Molecular Diagnosis of Hepatitis C Virus in Plasma

Nucleic acid extraction from biological samples is an important step for hepatitis C virus (HCV) diagnosis. However, such extractions are mostly based on silica-based column methodologies, which may limit their application for on-site diagnosis. A simple, rapid, and field-deployable method for RNA extraction is still needed. In this study, we evaluated the efficacy of four simple RNA extraction methods for the detection of HCV in plasma samples: a silica-membrane-based method, a magnetic-beads-based method, boiling with diethyl pyrocarbonate (DEPC)-treated distilled water, and using a commercial lysis buffer. HCV RNA was detected using both real-time reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Using real-time RT-PCR, extracted RNA from the silica-membrane-based and magnetic-beads-based methods had a 100% detection rate for RNA extraction from plasma. Using RT-LAMP, extracted RNA from the silica-membrane-based method showed a 66% detection rate, while the magnetic-beads-based method had a 62% detection rate. In summary, magnetic-beads-based extraction can be used as an alternative RNA extraction method for on-site HCV detection. Boiling with DEPC-treated distilled water was not appropriate for low HCV load samples, and boiling with a lysis buffer was not recommended.

Molecular Diagnostic Methods for the Detection of Leptospirosis

Leptospirosis is a widespread infectious disease caused by the spirochete Leptospira. The clinical features of leptospirosis are fever, headache, vomiting, jaundice, and the acute form of the disease is commonly called Weil’s disease. The microscopic agglutination test (MAT) is a gold standard method used to detect leptospirosis. However, it requires 14 days of time and skilled personnel to detect leptospirosis. Various molecular methods were developed for the rapid detection process, including polymerase chain reaction (PCR), multiplex PCR, nested PCR, real-time PCR, and Loop-mediated isothermal amplification (LAMP). Other immuno-based biosensor kits are readily available for the diagnosis of leptospirosis. Though these methods claim to be highly sensitive and specific, each method has its drawbacks. This review discusses the different molecular diagnostic techniques applied for the diagnosis of leptospirosis; elaborating on each method’s sensitivity, specificity, and detection time and the different samples of water, blood, and urine used.

Algorithm development and diagnostic accuracy testing for non-invasive foetal RHD genotyping: an Indian experience.

The discovery of the cell-free foetal DNA (cffDNA) circulating in the maternal plasma enabled prediction of foetal RHD thus eliminating the risks associated with invasive procedures. Non-invasive foetal RHD genotyping has now become the standard approach in developed countries for management of alloimmunised women and is also used for targeted antenatal prophylaxis in non-alloimmunised women. cffDNA was extracted from the plasma of 217 RhD negative pregnant women at a gestational age of 10-34 weeks. The foetal RHD genotype was determined by real-time polymerase chain reaction (real-time PCR) amplification of exons 4, 5 and 10 in duplicates. After an initial 54 samples, foetal typing was carried out with RHD exons 5 and 10 for the remaining samples. CCR5, SRY and RASSF1A genes were used as controls. Results were compared with cord blood serological typing at birth. Out of the 217 women, 193 were non-immunised and 24 were alloimmunised. A conclusive diagnosis was obtained in 203 samples. Diagnosis was inconclusive in 14 samples; of these, foetal RHD genotype could be resolved in six samples after maternal and paternal RHD genotyping. A 100% diagnostic accuracy, sensitivity and specificity were demonstrated in 209 women who had had a conclusive result. When the inconclusive samples were included, diagnostic accuracy and sensitivity were more than 95% and specificity was 78.95%. Anti-D is still the leading cause of haemolytic disease of the foetus and the newborn in India. There is, therefore, a need to establish and develop an algorithm for antenatal RhD negative women in India. The positive results of non-invasive foetal RHD genotyping, from the start of the 10th week of gestation using two RHD exons giving 100% diagnostic accuracy, show promise for routine diagnostic use to the benefit of the antenatal RhD negative Indian population.

Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing

The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.

Molecular Detection of Wheat Blast Pathogen in Seeds.

Pyricularia oryzae is a fungal plant pathogen causing blast disease in several species of the Poaceae family. It encompasses several genetic lineages, including one that is pathogenic on wheat and belongs to the Triticum lineage of P. oryzae. The fungus spreads at short distances by its airborne and rain-splash dispersed spores, and at longer distances via cryptically infected wheat seeds, through trade. Here, we describe a practical method to detect P. oryzae Triticum lineage in wheat seeds, after a biological enrichment step, with various options for molecular testing involving several DNA-based technologies: polymerase chain reaction (PCR), real-time PCR, and loop-mediated isothermal amplification (LAMP). The array of available molecular assays is presented in this protocol, each of them targeting specific regions of the P. oryzae Triticum lineage and offering different levels in terms of sensitivity and specificity.

Latent potential of current plant diagnostics for detection of sugarcane diseases

This review evaluates the usability of commonly used techniques for sugarcane sample preparation and pathogen detection, emphasising on-site diagnostics, and the associated challenges. A critical analysis of state-of-the-art methods to overcome these challenges is also presented. • The importance of on-site diagnostics for early detection sugarcane diseases causative organisms is described. • Commonly used techniques for sugarcane sample preparation and pathogen detection are described. • Currently available portable devices for on-site diagnostics are critically evaluated. • The biological and technical challenges of on-site detection are addressed. • A critical analysis of state-of-the-art methods to overcome these challenges is presented. Cropping industries are impacted by multiple biotic constraints that result in significant economic and productivity losses due to pests and diseases, and disease management costs both pre- and post-harvest. Bacterial, fungal, and viral pathogens are estimated to cause 16% of global crop losses pre-harvest, and thus accurate and informed disease management is required to ensure optimal yields. Significant economic risks are posed by several major diseases, which require host management approaches and accurate, sensitive, and rapid detection of the causative organisms to inform plant disease management. Over the last decade, many methods have been developed for detecting disease-causing organisms using enzyme-linked immunosorbent assays, real-time polymerase chain reaction, microarray, and loop-mediated isothermal amplification based tools. Most of these methods are highly selective and specific but unsuitable for on-site application. These methods are time consuming and require complex instrumentation. Sugarcane is a major cash crop in Australia with economic activity of over a billion dollars annually and many billions more globally, and thus there is a need to develop rapid on-site diagnostic tools with less complex assays for the early detection of sugarcane pathogens. This review considers the usability of commonly used sugarcane sample preparation and pathogen detection techniques, with a particular focus on on-site diagnostics and the associated challenges. In addition, the state-of-the-art methods for overcoming these challenges are critically analyzed.

Ultra-rapid real-time microfluidic RT-PCR instrument for nucleic acid analysis.

The polymerase chain reaction (PCR) is paramount in nucleic acid amplification testing, and for many assays, the use of PCR or qPCR is considered the 'gold standard'. While instrumentation for executing PCR has advanced over the last two decades, a growing interest in point-of-need testing has highlighted the deficit that exists for 'rapid PCR' systems. Here, we describe a field-forward prototype instrument capable of ultra-fast thermal cycling for real-time PCR amplification of DNA and RNA. The custom-designed, injection-molded microfluidic chips interface with a novel mechatronic system to complete 40 cycles of real-time PCR in under 10 minutes, an 84% reduction in time compared to a standard 50 minute assay. Such rapid amplification is enabled by two thermoelectric Peltiers capable of efficiently heating and cooling the sample at 12 and 10 °C s-1, respectively. Judicious selection and strategic placement of the thermal cyclers and fluorescence detector relative to the microchip enable synchronized thermal cycling and fluorescence monitoring, further reducing time-to-result. Robust amplification and detection of DNA and RNA targets empowers laboratories to achieve rapid, actionable information in endless applications.

Association of GSTM1 Deletion With Progression of CKD in Children: Findings From the Chronic Kidney Disease in Children (CKiD) Study

Loss of function of the product of the GSTM1 gene has been implicated in rapid progression of adult chronic kidney disease (CKD). Its role in pediatric CKD has not been previously described. Secondary analysis of a prospective observational cohort examining the association between deletions in GSTM1 and progression of CKD. We used data and samples from the prospective Chronic Kidney Disease in Children (CKiD) cohort aged 1-16 years at enrollment with CKD. We defined the exposure as fewer than 2 GSTM1 alleles on real-time polymerase chain reaction amplification. The primary outcome was a composite of 50% decrease in estimated glomerular filtration rate (eGFR) or start of kidney replacement therapy. Secondary outcomes included remission of proteinuria in children with glomerular disease and cardiovascular complications. The primary analysis was by Cox proportional hazards model. Analysis was adjusted for age, sex, race, ethnicity, body mass index category, diagnosis category, and eGFR. The analysis included 674 children. Their mean age at most recent visit was 11.9 years; 61% were male, and 20% were Black. There were 241 occurrences of the primary outcome at the time of analysis. After adjustment for baseline characteristics, the risk of progression of CKD for exposed children was 1.94 (95% CI, 1.27-2.97). The effect size was similar with either 1 or 2 deletions (autosomal dominant inheritance). The relationships between number of functional GSTM1 alleles and prespecified secondary outcomes were not statistically significant after adjustment. Missing data, especially for secondary outcomes, and relatively small sample size compared to genetic studies in adults. GSTM1 deletion is associated with more rapid progression of pediatric CKD after adjustment in this large prospective cohort. No statistically significant associations were seen with secondary outcomes. If replicated, these findings may inform development of interventions for CKD in children.

Evaluating the expression of key genes involved in resistance to oxidative stress in ALL patients

Background: Leukemia accounts for about 8% of all cancers and causes approximately 7% of mortalities due to malignancies. Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and rare in older subjects. The aim of this study was to evaluate the expression of oxidative stress resistance genes including Catalase, manganese superoxide dismutase (MnSOD), Forkhead Box O3 (Foxo3a), and sirtuin-1 (SIRT1) in ALL patients that may be applied for therapeutic purposes in the future.&#x0D; Materials and Methods: In this observational case-control study, blood samples were drawn from 60 newly diagnosed ALL patients and 10 healthy individuals as a control group. After RNA extraction and cDNA synthesis, real-time polymerase chain reaction (RT-PCR) amplification was performed using specific primers for evaluating the expression of Catalase, MnSOD, Foxo3a, and SIRT1 genes.&#x0D; Results: The expression of all studied genes were significantly higher in ALL patients than in the control group; catalase gene, FOX gene, MnSOD gene, and SIRT1 gene were expressed 4 times (p =0.04), 4.5 times (p =0.001), 2.2 times (p =0.05) and 4.8 (p =0.01) times higher than healthy individuals in the control group respectively. However, no significant relationship between their expression and the stage of the disease and blast percentage was demonstrated (P&gt;0.05).&#x0D; Conclusion: According to these results, the authors believe that the pathways involved in oxidative stress may be one of the most important causes of ALL disease's development and progression. In this regard, targeting the critical genes of these pathways can be considered a potential treatment with fewer side effects.

Robustness of digital PCR and real‐time PCR against inhibitors in transgene detection for gene doping control in equestrian sports

Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time PCR against gene doping. Digital PCR amplification was significantly inhibited by high concentrations of proteinase K (more than 0.1μg/μl), ethylenediaminetetraacetic acid (more than 5nmol/μl), and heparin (more than 0.05 unit/μl) but not by ethanol or genomic DNA. In addition, phenol affected droplet formation in the digital PCR amplification process. Real-time PCR amplification was inhibited by high concentrations of phenol (more than 1% v/v), proteinase K (more than 0.001 μg/μl), ethylenediaminetetraacetic acid (more than 1nmol/μl), heparin (more than 0.005 unit/μl), and genomic DNA (more than 51.9ng/μl) but not by ethanol. Although both PCR systems were inhibited by nearly the same substances, digital PCR was more robust than real-time PCR against the inhibitors. We believe that our findings are important for the development of better methods for transgene detection and prevention of false negative results in gene doping testing.

Immunological aspects of SARS-CoV-2 coronavirus damage

In 2020 the whole world was faced with an epidemiological outbreak caused by a new coronavirus SARS-CoV-2. The information available to date suggests that the newly isolated SARS-CoV-2 coronavirus should be assigned to superantigens, the main manifestations of which, as it is known, are suppression of nonspecific resistance factors and suppression of innate immunity mechanisms associated with the formation of a systemic inflammatory response in the form of cytokine storm and pathological activation of phagocytes in the lung tissue with its alteration and subsequent fibrosis. In this case, it is quite difficult and sometimes even impossible to observe the formation of fully-fledged specific immune answer on the effect of such antigens. This, along with the high infectious nature of the disease and the associated mortality, requires special attention to the underlying immunopatomechanism(s). Perhaps that is why little information has been obtained regarding the immunogenic properties of the newly isolated SARS-CoV-2 coronavirus so far, as well as, most importantly, about the structures of the virus itself responsible for the formation of specific immunity to it. The latter will serve as the basis for patient management and vaccine development. Nevertheless, a certain point of view on this issue is already beginning to form, as tools for detecting specific antibodies are being actively developed, as well as modern diagnostic tests for coronavirus, which include real-time polymerase chain reaction, real-time reverse transcription polymerase chain reaction and isothermal amplification mediated by reverse transcription. The presented analysis makes it possible to expand the understanding of the issue concerning the immunopathogenesis of COVID-19, the mechanisms of the onset and development of the disease in a living organism, the formation of an immune response to the new coronavirus, and also to determine the therapeutic tactics of managing patients with severe coronavirus infection. Elucidating the mechanisms of the emergence and development of a new coronavirus infection can help scientists, general practitioners, clinicians, and laboratory physicians respond correctly to the COVID-19 pandemic.