Reverse transcription-PCR Method Research Articles

Qualitative detection of E. coli in distributed drinking water using real-time reverse transcription PCR targeting 16S rRNA: Validation and practical experiences

Escherichia coli (E. coli) plays a central role as an indicator for fecal contamination to predict the possible presence of microbial pathogens in drinking water. Current detection methods for E. coli are based on time-consuming culture-based techniques. There is a strong need for methods to detect fecal contamination rapidly in distributed drinking water to prevent outbreaks of waterborne disease and support water utilities to efficiently manage their operations like actions to repair or maintain distribution pipes, to minimize impact on consumers. This study describes the validation and application of a qualitative real time reverse transcription PCR (RT-PCR) method targeting 16S ribosomal RNA (rRNA) for rapid detection of E. coli in distributed drinking water. The RT-PCR assay targets 16S rRNA, a highly abundant RNA in viable cells, enabling robust detection at the required sensitivity of 1 CFU/100 ml. The validation was performed by comparing the RT-PCR method with the culture-based chromogenic reference method (CCA) using the protocol and criteria described in ISO 16,140–2:2016. The validation demonstrated that this RT-PCR method can be used to specifically detect E. coli in a broad range of drinking water samples with at least the same limit of detection as the culture method (Relative Limit Of Detection = 0.75, range 0.43–1.43). The inclusivity study showed that the RT-PCR method was able to detect a broad range of E. coli strains derived from different sources and geographic areas, including pathogenic serotype O157 strains that are not detected with the culture method. The exclusivity study determined that other bacterial genera are not detected with this RT-PCR. However, Escherichia fergusonii was detected and, based on “in silico” analysis, it is expected that also E. albertii and E. marmotae and Shigella species will be detectable using this RT-PCR. An interlaboratory study confirmed that the RT-PCR and culture method have comparable sensitivities when tested by different participants at different laboratories. The application of RT-PCR to confirm the hygienic quality of distributed drinking water after actions to repair or maintain distribution pipes was compared with the culture method on 8076 routine samples, analyzed by the drinking water laboratories in the Netherlands. This comparison study showed a 96.4 % agreement between RT-PCR and culture. In 3.3 % of the samples E. coli was detected with RT-PCR and not with the culture method and in 0.1 % of the samples E. coli was only detected by culture confirming either a higher sensitivity for RT-PCR or the detection of RNA from uncultivable cells. Finally, the application of RT-PCR was highlighted during a contamination event in Belgium where we demonstrate the potency of RT-PCR as a tool to rapidly monitor the spread of microbial contamination and to monitor the effect of measures to remove the contamination This is the first fully validated rapid nucleic based method for detection of E. coli in distributed drinking water. These results demonstrate that this RT-PCR method can be used as a rapid alternative to the culture method to monitor E. coli in distributed drinking water. However, it should be emphasized that nucleic acid based detection methods rely on highly different detection principles (detection of captured nucleic acids present in a sample) than culture base methods (presence of cells cultivable on a selective medium) resulting in occasional different analysis results. Varying treatment and disinfection steps (UV, chlorine, monochloramine, Ozone) or environmental factors (decay) can influence the results and cause differences between RT-PCR and culture methods.

Placental Cannabinoid Receptor Expression in Preterm Birth.

Background: The cannabinoid receptor (CBR) plays a significant role in oogenesis, pregnancy, and childbirth. It might also play a significant role in preterm birth (PTB). The aim of the study was to investigate the association between the expression of the CBR in the placenta and the incidence of PTB. Methods: This prospective, observational, multicentre preliminary study was conducted on placental samples obtained from 109 women. The study included 95 patients hospitalized due to the high risk of PTB. They were divided into two groups: Group 1, where the expression of the CBR1 and CBR1a was analyzed, and Group 2, in which we examined CBR2 expression. The control group, that is, Group 3, consisted of 14 women who delivered at term, and their placentas were tested for the presence of all three receptor types (CBR1, CBR1a, and CBR2). Results: The study used reverse transcription and real-time PCR methods to assess the expression of CBRs in the placental tissues. The expression of the CBR2, CBR1, and CBR1a receptors was significantly lower in the placentas of women after PTB compared to those after term births, p = 0.038, 0.033, and 0.034, respectively. Conclusions: The presence of CBR mRNA in the human placental tissue was confirmed. The decreased expression of CBRs could serve as an indicator in predicting PTB.

Advancing broad bean true mosaic virus detection using conventional RT-PCR and real-time RT-PCR with novel primer set design

Broad bean true mosaic virus (BBTMV) infects broad beans and peas, reducing yield. As BBTMV is transmitted through broad beans, many countries have implemented regulations to prevent the distribution of infected seeds. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect BBTMV. While the PCR-based method is preferred for seed virus detection due to its sensitivity and speed. A BBTMV-specific PCR detection method has not yet been reported. A universal detection method currently exists that utilizes reverse transcription PCR (RT-PCR) for the Comovirus genus, to which BBTMV belongs. However, sequence analysis is required for species identification. To address this limitation, we developed and verified RT-PCR detection methods using newly designed BBTMV-specific primers. RT-PCR and real-time RT-PCR with these primers were approximately 5 × 105–106 times more sensitive than ELISA and 100–1000 times more sensitive than previously reported RT-PCR methods. Using RT-PCR and real-time RT-PCR employing these primers, we could detect BBTMV with same sensitivity when more than 3.0 × 105 copies were present per gram of broad bean seeds. Our newly developed detection methods can test for BBTMV with high sensitivity and speed.

Protective effect of Tao Hong Si Wu Decoction against inflammatory injury caused by intestinal flora disorders in an ischemic stroke mouse model

Background and aimsRecent studies have shown that intestinal flora are involved in the pathological process of ischemic stroke (IS). The potential protective effect of the traditional Chinese prescription, Tao Hong Si Wu Decoction (THSWD), against inflammatory injury after IS and its underlying mechanisms of action were investigated in the current study.MethodsFifty SPF(Specefic pathogen Free) male C57 mice were randomly assigned to sham operation, model, THSWD low-dose (6.5 g/kg), medium-dose (13 g/kg) and high-dose (26 g/kg) groups (10 mice per group). Mouse models of transient middle cerebral artery occlusion were prepared via thread embolism. Neurological function score, hematoxylin-eosin (HE) staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), 16S ribosomal DNA (rDNA) sequencing, quantitative reverse transcription PCR (qRT-PCR) and other methods were employed to elucidate the underlying molecular mechanisms.ResultsNotably, THSWD induced a reduction in the neurological function score (P < 0.01) and neuronal injury in brain tissue, increase in protein expression of Claudin-5 and zonula occludens-1 (ZO-1) in brain tissue(P < 0.01), and decrease in serum lipopolysaccharide (LPS)(P < 0.01), diamine oxidase (DAO)(P < 0.01) and D-lactic acid(P < 0.01, P < 0.05) levels to a significant extent. THSWD also inhibited the levels of tumor necrosis factor-α (TNF-α)(P < 0.01) and interleukin − 1β (IL-1β)(P < 0.01) in brain tissue, and increased alpha and beta diversity in ischemic stroke mice, along with a certain reversal effect on different microflora. Finally, THSWD inhibited the polarization of microglia cells(P < 0.01) and decreased the protein and gene expression of toll-like receptor-4 (TLR-4)(P < 0.01, P < 0.05) and nuclear factor kappa B (NF-κB)(P < 0.01) in brain tissue.ConclusionOur data indicate that THSWD may interfere with inflammatory response in ischemic stroke by regulating intestinal flora and promoting intestinal barrier repair.

Simultaneous detection of human norovirus GI, GII and SARS-CoV-2 by a quantitative one-step triplex RT-qPCR.

There are many similarities in the clinical manifestations of human norovirus and SARS-CoV-2 infections, and nucleic acid detection is the gold standard for diagnosing both diseases. In order to expedite the identification of norovirus and SARS-CoV-2, a quantitative one-step triplex reverse transcription PCR (RT-qPCR) method was designed in this paper. A one-step triplex RT-qPCR assay was developed for simultaneous detection and differentiation of human norovirus GI (NoV-GI), GII (NoV-GII) and SARS-CoV-2 from fecal specimens. The triplex RT-qPCR assay had high detection reproducibility (CV < 1%) and sensitivity. The lower limits of detection (LLOD95) of the triplex RT-qPCR assay for each target site were 128.5-172.8 copies/mL, and LLOD95 of the singleplex RT-qPCR assay were 110.3-142.0 copies/mL. Meanwhile, among the detection of clinical oropharyngeal swabs and fecal specimens, the results of the singleplex and triplex RT-qPCR assay showed high agreement. The triplex RT-qPCR assay for simultaneous detection of NoV-GI, NoV-GII and SARS-CoV-2 from fecal specimens has high clinical application value.

Comparison of RT-PCR and rRT-PCR Methods in Detection of Viral Hemorrhagic Septicemia Virus (VHSV) in Marine Ornamental Fish

Reverse Transcription PCR (RT-PCR) and Real Time Reverse Transcription PCR (rRT-PCR) are methods that can be used to detect VHSV (Viral Hemorrhagic Septicemia Virus) on freshwater and seawater fish. rRT-PCR is more widely used than the RT-PCR method because of several advantages such as requiring shorter time and fewer stages. Both methods have their own advantages and disadvantages, so it is necessary to compare the two methods in detecting the presence of VHSV in marine ornamental fish. The research was carried out with 3 steps: sensitivity test, specificity test, and sample test. The sensitivity test showed that the smallest dilution concentration that could be detected by both methods was the same at 10-3 with 100 copies in the 77bp band. Specificity tests on both methods showed that VHSV primers used are specific. Sample tests in both methods showed the same results in each of the samples tested. The results of sensitivity, specificity and sample tests conducted on RT-PCR and rRT-PCR methods showed the same results (positive) thus both methods can be used to detect the VHSV in marine ornamental fish.

The role of apoptosis and oxidative stress in the pathophysiology of Acanthamoeba spp. infection in the kidneys of hosts with different immunological status

BackgroundAcanthamoeba spp. are opportunistic pathogens that cause inflammation, mostly in the brain, lungs and cornea. Recent reports indicate kidney dysfunction in hosts with systemic acanthamoebiasis. The aim of the study was to analyze the gene expression and protein concentration of NADPH oxidase 2 and 4 (NOX2 and NOX4, respectively) and nuclear erythroid 2-related factor (Nrf2) in the kidneys of hosts with systemic acanthamoebiasis. We also aimed to determine the protein and gene expressions of Bcl2, Bax, caspases 3 and 9.Methods Mice were divided into four groups based on their immunological status and Acanthamoeba sp. infection: A, immunocompetent Acanthamoeba sp.-infected mice; AS, immunosuppressed Acanthamoeba sp.- infected mice; C, immunocompetent uninfected mice; CS, immunosuppressed uninfected mice. NOX2, NOX4 and Nrf2 were analyzed by quantitative reverse transcription PCR (qRT-PCR) and ELISA methods, while pro-apoptotic and anti-apoptotic proteins (Bax and Bcl-2, respectively), Cas9, Cas3 were analyzed by qRT-PCR and western blot methods. ResultsIncreased gene expression and/or protein concentration of NOX2 and NOX4 were found in both immunocompetent and immunosuppressed mice infected with Acanthamoeba sp. (groups A and AS, respectively). Gene expression and/or protein concentration of Nrf2 were higher in group A than in control animals. Compared to control mice, in the AS group the expression of the Nrf2 gene was upregulated while the concentration of Nrf2 protein was decreased. Additionally in A group, higher gene and protein expression of Bcl-2, and lower gene as well as protein expression of Bax, caspases 3 and 9 were noted. In contrast, the AS group showed lower gene and protein expression of Bcl-2, and higher gene as well as protein expression of Bax, caspases 3 and 9.ConclusionsThis study is the first to address the mechanisms occurring in the kidneys of hosts infected with Acanthamoeba sp. The contact of Acanthamoeba sp. with the host cell surface and/or the oxidative burst caused by elevated levels of NOXs lead to an antioxidant response enhanced by the Nrf2 pathway. Acanthamoeba sp. have various strategies concerning apoptosis. In immunocompetent hosts, amoebae inhibit the apoptosis of kidney cells, and in immunosuppressed hosts, they lead to increased apoptosis by the intrinsic pathway and thus to a more severe course of the disease.Graphical

Artificial Pasture Grazing System Attenuates Lipopolysaccharide-Induced Gut Barrier Dysfunction, Liver Inflammation, and Metabolic Syndrome by Activating ALP-Dependent Keap1-Nrf2 Pathway.

Geese can naturally obtain dietary fiber from pasture, which has anti-inflammatory and antioxidant properties. This study aimed to investigate the inhibitory impacts of pasture on ameliorating LPS-ROS-induced gut barrier dysfunction and liver inflammation in geese. Materials and methods. The lipopolysaccharides (LPS), alkaline phosphatase (ALP), reactive oxygen species (ROS), tight junction proteins, antioxidant enzymes, immunoglobulins, and metabolic syndrome were determined using ELISA kits. The Kelch-like-ECH-associated protein 1-Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) and inflammatory cytokines were determined using the quantitative reverse transcription PCR (RT-qPCR) method. The intestinal morphology was examined using the Hematoxylin and Eosin (H&E) staining method in ileal tissues. Results. Pasture significantly influences nutrient absorption (p < 0.001) by ameliorating LPS and ROS-facilitated ileal permeability (p < 0.05) and systemic inflammation (p < 0.01). Herein, the gut permeability was paralleled by liver inflammation, which was significantly mimicked by ALP-dependent Nrf2 (p < 0.0001) and antioxidant enzyme activation (p < 0.05). Indeed, the correlation analysis of host markers signifies the importance of pasture in augmenting geese's health and production by averting gut and liver inflammation. Conclusions. Our results provide new insight into the mechanism of the pasture-induced ALP-dependent Nrf2 signaling pathway in limiting systemic inflammation in geese.

Molecular detection and expression of virulence factor encoding genes of Pseudomonas aeruginosa isolated from clinical samples

Introduction and Aim: Pseudomonas aeruginosa virulence factors genes are a growing concern as they are involved not only in its pathogenicity, but also cause bacterial resistance to multiple classes of antibiotics. Laboratory identification of clinical isolates carrying the virulence genes would be critical in limiting the bacteria's spread and reducing its pathogenicity. The purpose of this study was to investigate a simple and inexpensive real-time PCR test to find the level of expression of Pseudomonas aeruginosa virulence genes before and after treatment with specific concentration of Lactobacillus acidophilus cell-free supernatants (CFSs). Materials and Methods: Between December 2021 and June 2022, 350 clinical samples collected from Baghdad hospitals, Iraq, were tested for the presence of P. aeruginosa. The P. aeruginosa isolated were tested for their antimicrobial susceptibility using the Kirby-Bauer disk diffusion method. P. aeruginosa virulence genes were detected by using the reverse transcription-PCR method. The expression levels of these genes before and after treatment with Lactobacillus acidophilus cell-free supernatants were measured by real-time PCR. Results: Out of 350 samples tested, 60 isolates were positive for the presence of P. aeruginosa. Antibiotic susceptibility tests revealed a high level of antibiotic resistance, while genetic techniques identified the presence of several virulence genes that exhibited variable expression under the influence of Lactobacillus acidophilus supernatants. Conclusion: The study findings showed that L. acidophilus supernatants had an effect on reducing the expression of certain virulence genes of P. aeruginosa, implying that L. acidophilus could be used as an option in treating P. aeruginosa infection.

The genomic and epidemiological investigations of enteric viruses of domestic caprine (Capra hircus) revealed the presence of multiple novel viruses related to known strains of humans and ruminant livestock species.

In this study, the enteric virome of diarrheic caprine was investigated using next-generation sequencing, reverse transcription PCR methods, and bioinformatics analyses. The complete or nearly complete genomes of seven novel viruses including (i) a caprine astrovirus (OQ758025) of family Astroviridae, four picornaviruses in genera (ii) Boosepivirus (OQ758026), (iii) Enterovirus (OQ758028), (iv) Kobuvirus (OQ758029), and (v) an unassigned picornavirus provisionally called as capripivirus (from the term caprine picornavirus) of the family Picornaviridae (OQ758027), as well as (vi) a tusavirus of family Parvoviridae (OL692339.2) and an (vii) unassigned CRESS DNA virus (OQ758030) have been identified. Structural analyses of the internal ribosomal entry sites revealed the presence of unique motifs and suggest the modular exchange of certain elements between co-infecting viruses. Epidemiological investigations and genotyping PCR reactions of identified RNA virus groups were also conducted using multiple "in-house" developed SYBRgreen-based screening quantitative PCR (qPCR) assays and generic capsid primers on additional fecal samples from ruminant livestock species (n = 62 caprine, n = 32 ovine, and n = 94 cattle) of three different age groups from n = 12 geographically distant sampling sites in Hungary. The results of qPCR screening assays as well as K-means cluster and phylogenetic analyses of the determined sequences suggest that diverse members of the above RNA virus groups were found in all age groups, but mostly in <1-year-old ruminants. Members of the analyzed virus groups were found frequently (37.2%) in multiple (even in quintuple) co-infections, while strains related closely to the identified index viruses were detectable only in certain caprine and ovine samples indicating a wide range of viral diversity. IMPORTANCE Compared with other domestic animals, the virome and viral diversity of small ruminants especially in caprine are less studied even of its zoonotic potential. In this study, the enteric virome of caprine was investigated in detail using next-generation sequencing and reverse transcription PCR techniques. The complete or nearly complete genomes of seven novel viruses were determined which show a close phylogenetic relationship to known human and ruminant viruses. The high similarity between the identified caprine tusavirus (family Parvoviridae) and an unassigned CRESS DNA virus with closely related human strains could indicate the (reverse) zoonotic potential of these viruses. Others, like astroviruses (family Astroviridae), enteroviruses, or novel caripiviruses (named after the term caprine picornavirus) of family Picornaviridae found mostly in multiple co-infections in caprine and ovine, could indicate the cross-species transmission capabilities of these viruses between small ruminants.

Effects of sub-inhibitory concentration of antibiotic and heat stress on the expression of type II TA system genes in Brucella spp.

ObjectiveBacteria can react to stress conditions using the Toxin-Antitoxin (TA) system. This study investigated the expression of TA system genes under heat and antibiotic stresses in Brucella spp. MethodsTo determine the effects of sub-inhibitory concentration (sub-MIC) of rifampin on bacterial survival and growth, a colony-forming unit was quantitated, and turbidity was assessed following the treatment of Brucella isolates, with ½ minimum inhibitory concentration (MIC) of antibiotic at different time intervals. Also, Brucella isolates were exposed to heat stress (42 °C) compared to the control (37 °C). Finally, the expression of TA system genes in Brucella isolates was evaluated one hour after treatment using the quantitative reverse transcription PCR (qRT-PCR) method. ResultsOur results showed that the growth of the Brucella isolates reduced in the presence of the sub-MIC of antibiotics compared to the control. The results of the qPCR assay showed that, in the presence of rifampicin the expression of the TA system genes increased and, under the heat stress conditions, the expression of the TA system genes increased compared to controls expect brnT / brnA system. ConclusionAlthough the exact role of the TA system in response to various stresses is not yet fully understood, our study provided information on the effectiveness of the type II TA system under heat and antibiotic stress conditions by examining the gene expression of type II systems in Brucella isolates.

Analysis of the characteristics of viral infections in children with diarrhea in Beijing from 2018 to 2022

Objective: To explore the characteristics of viral infections in children with diarrhea in Beijing from 2018 to 2022. Methods: Real-time PCR and enzyme-linked immunosorbent assay were used to detect viral nucleic acid of Norovirus (NoV), Sappovirus (SaV), Astrovirus (AstV), Enteric Adenovirus (AdV) or antigen of Rotavirus (RV) in 748 stool samples collected from Beijing Capital Institute of Pediatrics from January 2018 to December 2021. Subsequently, the reverse transcription PCR or PCR method was used to amplify the target gene of the positive samples after the initial screening, followed by sequencing, genotyping and evolution analysis, so as to obtain the characteristics of these viruses. Phylogenetic analysis was performed using Mega 6.0. Results: From 2018 to 2021, the overall detection rate of the above five common viruses was 37.6%(281/748)in children under 5 years old in Beijing. NoV, Enteric AdV and RV were still the top three diarrhea-related viruses, followed by AstV and SaV, accounting for 41.6%, 29.2%, 27.8%, 8.9% and 7.5%, respectively. The detection rate of co-infections with two or three diarrhea-related viruses was 4.7% (35/748). From the perspective of annual distribution, the detection rate of Enteric AdV was the highest in 2021, while NoV was predominant in the other 4 years. From the perspective of genetic characteristics, NoV was predominant by GⅡ.4, and after the first detection of GⅡ.4[P16] in 2020, it occupied the first two gene groups together with GⅡ.4[P31]. Although the predominant RV was G9P[8], the rare epidemic strain G8P[8] was first detected in 2021. The predominant genotypes of Enteric AdV and AstV were Ad41 and HAstV-1. SaV was sporadic spread with a low detection rate. Conclusion: Among the diarrhea-related viruses infected children under 5 years of age in Beijing, the predominant strains of NoV and RV have changed and new sub-genotypes have been detected for the first time, while the predominant strains of AstV and Enteric AdV are relatively stable.

Effect of Vitamin D on the HMGB1/RAGE Pathway and Adipokines Levels in Obese Asthmatic Mice.

Compared to common asthma, obese asthma is difficult to control. Previous studies have shown that vitamin D (Vit D) has a therapeutic effect on asthma. Nevertheless, the action mechanism of Vit D for obese asthma are not well known. In this study, we, therefore, induced obesity and established an obese asthma mouse model using ovalbumin (OVA) stimulation and applied treatment with Vit D (100 ng/kg). Accordingly, thirty mice were randomly divided into 5 equal groups of normal control, asthma, obese asthma, asthma+Vit D, and obese asthma+ Vit D. The levels of inflammatory factors and adipokines were measured by the ELISA assay; then the quantitative reverse transcription PCR (qRT-PCR) method was used to evaluate the expression of high mobility group box 1(HMGB1) and receptor for advanced glycation end products [RAGE] genes.T he results showed that OVA sensitization significantly increased airway resistance, the levels of inflammatory cytokines, and HMGB and RAGE expression in asthmatic and obese asthmatic mice, as compared to the control group. Also, these changes in the obese asthmatic group were notably higher than those in the asthmatic one. In addition, the treatment of asthmatic and obese asthmatic mice with Vit D significantly reduced the raw, serum and BALF levels of inflammatory cytokines, as well as the expression of HMGB1 and RAGE mRNA. To conclude, the present study showed that vitamin D might attenuate lung injury by up-regulating HMGB1 and RAGE expression. Our findings, thus, may offer new concepts and approaches for the treatment and prevention of obese asthma.

Effects of sub-inhibitory concentration of antibiotic and heat stress on the expression of type II TA system genes in Brucella spp.

ObjectiveBacteria can react to stress conditions using the Toxin-Antitoxin (TA) system. This study investigated the expression of TA system genes under heat and antibiotic stresses in Brucella spp. MethodsTo determine the effects of sub-inhibitory concentration (sub-MIC) of rifampin on bacterial survival and growth, a colony-forming unit was quantitated, and turbidity was assessed following the treatment of Brucella isolates, with ½ minimum inhibitory concentration (MIC) of antibiotic at different time intervals. Also, Brucella isolates were exposed to heat stress (42 °C) compared to the control (37 °C). Finally, the expression of TA system genes in Brucella isolates was evaluated one hour after treatment using the quantitative reverse transcription PCR (qRT-PCR) method. ResultsOur results showed that the growth of the Brucella isolates reduced in the presence of the sub-MIC of antibiotics compared to the control. The results of the qPCR assay showed that, in the presence of rifampicin the expression of the TA system genes increased and, under the heat stress conditions, the expression of the TA system genes increased compared to controls expect brnT / brnA system. ConclusionAlthough the exact role of the TA system in response to various stresses is not yet fully understood, our study provided information on the effectiveness of the type II TA system under heat and antibiotic stress conditions by examining the gene expression of type II systems in Brucella isolates.

Sensitivitas Pemeriksaan Covid-19: Insulated Isothermal PCR (iiPCR) Dan Reverse Transcription PCR (RT-PCR)

Covid-19 (Corona Virus disease 2019) is a new type of disease caused by a virus from the Corona Virus group, namely SARS-CoV-2. Covid-19 may cause respiratory system disorders, ranging from mild symptoms such as flu to lung infections, such as pneumonia. Laboratory diagnoses for Covid-19 disease generally include hematological (complete blood examinations), and molecular or a combination of serology and molecular. PCR examination that can be carried out using the Insulated Isothermal PCR and Reverse Transcription PCR methods. This research is descriptive observational research. Data were collected at Grha Kedoya Hospital, West Jakarta, North Kedoya. The result showed a sensitivity of iiPCR score 82,6%, which is relatively lower than gold-standard RT-PCR. Our research suggested that RT-PCR is still an effective and sensitive method for Covid-19 examination.

Gene Expression and Methylation Levels of PCSK9 Gene in Iraqi Patients with Coronary Artery Disease

The expression of the Proprotein Convertase Subtilisin/Kexin Type 9 gene (PCSK9) is inextricably related to lipid levels and a risk of atherosclerotic coronary artery disease (ASCAD). The present study aims to measure the quantity of PCSK9 gene expression and the effect of methylation on its expression level taking part in the pathogenesis of acute coronary artery disorder. A current study included 150 subjects from the Iraqi population, 100 ASCAD patients and 50 healthy controls. The concentration of PCSK9 in each serum sample was determined by the ELISA technique, the expression levels of the PCSK9 gene in whole blood were estimated by RT-qPCR – Quantitative Reverse Transcription PCR method, and DNA methylation level detection in PCSK9 gene by using High Resolution Melting Analysis (HRM) technique. The expression level of the PCSK9 gene was 6.06 ± 1.84 in ASCAD patients group compared with a control group 1.00 ±0.19. That indicates the up-regulation of the PCSK9 gene in patient with atherosclerosis. In addition, the PCSK9 concentration was higher in patient group in comparison to the healthy controls group P= &lt; 0.01. However, PCSK9 methylation levels, a highly significant distinction between the two study groups p=0.002. In conclusion, serum PCSK9 levels are associated with coronary artery lesions as shown in receiver operating characteristic (ROC) analysis. Suggests that, serum PCSK9 might be a good indicator of coronary artery disease development.

Molecular Epidemiology of Bovine Enteroviruses and Genome Characterization of Two Novel Bovine Enterovirus Strains in Guangxi, China.

Bovine enterovirus (BEV) is a highly infectious pathogen that may cause respiratory and gastrointestinal disease outbreaks in cattle. This study aimed to investigate the prevalence and genetic characteristics of BEVs in Guangxi Province, China. A total of 1,168 fecal samples from 97 different bovine farms were collected between October 2021 and July 2022 in Guangxi Province, China. BEV was confirmed using a reverse transcription-PCR (RT-PCR) method targeting the 5' untranslated region (UTR), and isolates were genotyped by sequencing their genomes. The nearly complete genome sequences of eight BEV strains showing cytopathic effects in MDBK cells were determined and analyzed. In total, 125 (10.7%) of 1,168 fecal samples were positive for BEV. BEV infection was significantly associated with farming patterns and clinical symptoms (P < 0.05; odds ratio [OR] > 1). Molecular characterization indicated that five BEV strains from this study belonged to EV-E2 and one strain to EV-E4. Two BEV strains (GXNN2204 and GXGL2215) could not be assigned to a known type. Strain GXGL2215 showed the closest genetic relationship with GX1901 (GenBank accession number MN607030; China) in its VP1 (67.5%) and P1 (74.7%) and with NGR2017 (MH719217; Nigeria) in its polyprotein (72.0%). It was also close to the EV-E4 strain GXYL2213 from this study when the complete genome (81.7%) was compared. Strain GXNN2204 showed the closest genetic relationship with Ho12 (LC150008; Japan) in the VP1 (66.5%), P1 (71.6%), and polyprotein (73.2%). Genome sequence analysis suggested that strains GXNN2204 and GXGL2215 originated from the genomic recombination of EV-E4 and EV-F3 and EV-E2 and EV-E4, respectively. This study reports the cocirculation of multiple BEV types and the identification of two novel BEV strains in Guangxi, China, and it will provide further insights into the epidemiology and evolution of BEV in China. IMPORTANCE Bovine enterovirus (BEV) is a pathogen that causes intestinal, respiratory, and reproductive disease infections in cattle. This study reports on the widespread prevalence and biological characteristics of the different BEV types which currently exist in Guangxi Province, China. It also provides a reference for the study of the prevalence of BEV in China.

Development of a novel real-time RT-PCR method using peptide nucleic acid (PNA) probes for detecting and genotyping of viral haemorrhagic septicaemia virus (VHSV)

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases in salmonid and olive flounder farms. The causative agent of VHS is the VHS virus (VHSV), which has been classified into four genotypes (I–IV), based on sequence analysis of the genes encoding for nucleoprotein, glycoprotein, and non-structural viral protein. Among the various diagnostic methods, real-time reverse transcription PCR method based on TaqMan-probe (RT-qPCR) is a stable, rapid, specific, and highly sensitive method for viral gene detection. However, the currently accepted diagnostic method based on RT-qPCR can only detect viral presence and load, and does not provide information about viral genotype. Peptide nucleic acids (PNAs) are artificially synthesized DNA analogues with an uncharged peptide backbone. PNA probes can effectively detect a target gene by amplification and a specific melting temperature signal. It was reported that PNA probes can effectively distinguish between mismatched sequences based on their different melting temperatures in amplified PCR products. The present study reports a novel real-time RT-PCR method for simultaneous detection and genotyping of VHSV using PNA probes. The newly-developed method showed a sensitivity similar to that of the infectious titre by fish cell cultures inoculated with the virus, except for genotype IVa, where viral inoculation in cell culture showed a 10-fold higher sensitivity than the novel method. The melting point analysis to distinguish the four genotypes was performed on 80 VHSV isolates representing all known genotypes, showing that this novel real-time RT-PCR can distinguish between all VHSV genotypes without the need of further sequencing.

Low-density small extracellular vesicles in bovine follicular fluid carrying let-7i target FASLG to inhibit granulosa cells apoptosis

Apoptosis of granulosa cells is a key factor in mammalian follicular atresia. It has a significant impact on oocyte development and maturation. Extracellular vesicles (EVs) are a group of highly heterogeneous population. Previous studies have found that ovarian follicular fluid is rich in EVs. In the present study, the follicular fluid of 3–5 mm follicles from bovine ovaries without corpus luteum was collected, and a subtype of small EVs (sEVs), low-density small EVs (LD-sEVs), was successfully isolated by differential ultracentrifugation combined with iodixanol density gradient centrifugation. LD-sEVs were identified using transmission electron microscope, nanoparticle tracking analysis and Western blot. Flow cytometry, Quantitative reverse transcription PCR (RT-qPCR), Western blot, and other methods were used to detect the effect of LD-sEVs on follicular granulosa cell apoptosis. After that, let-7i, a highly expressed miRNA component in LD-sEVs, was screened and target validation was carried out in granulosa cells. The results showed that LD-sEVs could be taken up by granulosa cells and inhibited the apoptosis. Further research found that let-7i inhibits the apoptosis of granulosa cells by targeting FASLG. It plays an important role in regulating the apoptosis of follicular granulosa cells, which may affect follicular development.

Evaluation of the Effectiveness of Diagnosing a New Coronavirus Infection during the Period of Seasonal Incidence Using the “Hypercube” Mathematical Model

Abstract Background: With the emergence of new strains of coronavirus and the easing of previously imposed anti-COVID restrictions, it is important to respond quickly and ensure sufficient coverage of laboratory tests for people who come into contact with patients with coronavirus infection. Diagnostics by polymerase chain reaction (PCR) remain the most common and accessible method of laboratory confirmation of a new coronavirus infection COVID-19. The method has a huge number of positive aspects, but it is time-consuming. During an epidemiological outbreak, the speed at which a diagnosis is made is often crucial. In addition, the task of reducing material costs for reagents and consumables used in the analysis by PCR method is also relevant. Objective: The objective is to evaluate the effectiveness of the hypercube mathematical model application for diagnosing a new coronavirus infection COVID-19 by the PCR method. Materials and Methods: In the course of the work, 108 respiratory swabs were examined using the reverse transcription-PCR method; the swabs were tested by the classical method and using the hypercube mathematical algorithm. Results: By applying the group mixing method using the ’Hypercube’ mathematical model, when performing molecular genetic analyzes, we were convinced that it is inexpedient to use sample pooling during the seasonal peak incidence of coronavirus infection. Applying the method, the cost of reagents, consumables, and time increases by at least 1.5–2 times since all our studies required conducting additional clarifying tests. At the same time, our results show that the hypercube pooling technique does not affect the analytical sensitivity. Conclusion: The use of the Hypercube mathematical algorithm for SARS-CoV-2 virus testing during an increase in seasonal incidence is a mostly unpromising strategy since it increases the time of PCR analysis and the cost of reagents and consumables, as well as slows down the validation of the result.