Fluorescent Polymerase Chain Reaction Research Articles

Molecular Epidemiology of Clostridioides difficile Colonization in Families With Infants.

Community-associated Clostridioides difficile infection is a major public health hazard to adults and older children. Infants frequently excrete toxigenic C difficile asymptomatically in their stool, but their importance as a community reservoir of C difficile is uncertain. Families of healthy infants were recruited at the baby's 4-month well child visit and were followed longitudinally until the baby was approximately 9 months old. Babies and mothers submitted stool or rectal swabs every 2 weeks that were cultivated for C difficile; fathers' participation was encouraged but not required. Clostridioides difficile isolates were strain-typed by fluorescent polymerase chain reaction ribotyping and by core genome multilocus sequence typing, and the number of families in whom the same strain was cultivated from >1 family member ("strain sharing") was assessed. Thirty families were enrolled, including 33 infants (3 sets of twins) and 30 mothers; 19 fathers also participated. Clostridioides difficile was identified in 28 of these 30 families over the course of the study, and strain sharing was identified in 17 of these 28. In 3 families, 2 separate strains were shared. The infant was involved in 17 of 20 instances of strain sharing, and in 13 of these, the baby was identified first, with or without a concomitantly excreting adult. Excretion of shared strains usually was persistent. Clostridioides difficile strain sharing was frequent in healthy families caring for an infant, increasing the likelihood that asymptomatically excreting babies and their families represent a reservoir of the organism in the community.

Regulating effect of Qifu Yin on intestinal microbiota in mice with memory impairment induced by scopolamine hydrobromide

Ethnopharmacological relevanceQifu Yin (QFY) originates from “Jingyue Quanshu · Volume 51 · New Fang Bazhen · Buzhen” a work by Zhang Jingyue, a distinguished Chinese medical practitioner from the Ming Dynasty. QFY is composed of Ginseng Radix et Rhizoma, Rehmanniae Radix Praeparata, Angelicae Sinensis Radix, Atractylodis Macrocephalae Rhizoma, Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle, Ziziphi Spinosae Semen, and Polygalae Radix. QFY is frequently employed to address memory loss and cognitive impairment stemming from vascular dementia, Alzheimer's disease (AD), and related conditions. Our findings indicate that QFY can mitigate nerve cell damage. Moreover, the study explores the impact of QFY on the calcium ion pathway and sphingolipid metabolism in mice with myocardial infarction, presenting a novel perspective on QFY's mechanism in ameliorating myocardial infarction through lipidomics. While this research provides an experimental foundation for the clinical application of QFY, a comprehensive and in-depth analysis of its improvement mechanism remains imperative. Aim of the studyTo clarify the regulatory mechanism of QFY on intestinal microecology in mice with memory impairment (MI). Material and methodsThe memory impairment mouse model was established by intraperitoneal injection of scopolamine hydrobromide. Kunming (KM) mice were randomly divided into blank group, Ginkgo tablet group (0.276 g/kg), QFY high, medium and low dose groups (17.2 g/kg, 8.6 g/kg, 4.3 g/kg). The effect on memory ability was evaluated by open field and step-down behavioral experiments. The morphological changes of nerve cells in the hippocampus of mice were observed by pathological method. The contents of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GSH-Px) in the brain tissue of mice were detected. The expression levels of CREB, Brain-Derived Neurotrophic Factor (BDNF) and Recombinant Amyloid Precursor Protein (APP) in the hippocampus of mice were determined using immunohistochemistry. The expression of N-methyl-D-aspartate receptor (NMDAR) and cAMP response element binding protein (CREB) related factors in the serum of mice was analyzed by ELISA. The levels of apoptosis signal-regulating kinase-1 (ASK1) and c-Jun N-terminal kinase (JNK) mRNA in the hippocampus were detected by quantitative real-time fluorescence polymerase chain reaction (qPCR). The intestinal feces of mice were collected, and the 16 S rDNA technology was used to detect the changes in intestinal microbiota microecological structure of feces in each group. ResultsBehavioral experiments showed that the high-dose QFY group exhibited a significant increase in exercise time (P<0.05) and a decrease in diagonal time (P<0.05) compared to the model group. The medium-dose group of QFY showed a reduction in diagonal time (P<0.05). Additionally, the latency time significantly increased in the medium and high-dose groups of QFY (P<0.01). The number of errors in the low, medium and high dose groups was significantly decreased (P<0.05, P<0.01, P<0.01). The nerve cells in the CA1 and CA3 regions of QFY-treated mice demonstrated close arrangement and clear structure. Furthermore, the content of SOD significantly increased (P<0.01) and the content of MDA significantly decreased (P<0.05) in the low and high-dose QFY groups. The content of CAT in the medium-dose group significantly increased (P < 0.05). Immunohistochemical analysis showed a significant reduction in the number of APP expression particles in the CA1 and CA3 regions of all QFY groups. Moreover, BDNF expression significantly increased in the medium and high-dose groups, while CREB expression significantly increased in the low and medium-dose groups of QFY within the CA1 and CA3 regions. Serum analysis revealed significant increases in CREB content in the low, medium, and high dose groups of QFY (P<0.01, P<0.05, P<0.05), and decreases in NMDAR content across all QFY dose groups (P<0.01). PCR analysis showed a significant decrease in the contents of ASK1 and JNK in the medium-dose group (P<0.01). Microecological analysis of intestinal microbiota demonstrated a significant restoration trend in the relative abundance of Fusobacteria, Planctomycetes, and Verrucomicrobia (P<0.01 or P<0.05) at the phylum level in the QFY groups. At the genus level, Akkermansia, Paramuribaculum, Herminiimonas, Erysipelatoclostridium and other genera in the QFY groups showed a significant trend of relative abundance restoration (P<0.01 or P<0.05). ConclusionQFY can improve the memory of MI animals induced by scopolamine hydrobromide by restoring the homeostasis of intestinal microbiota and regulating related indexes in serum and brain tissue.

Time-resolved fluorescent microsphere-based lateral flow assay combined with polymerase chain reaction for quantitatively detecting beef to assess the authenticity of beef products

Ensuring the authenticity of meat products is crucial for food safety. In this study, we proposed a method employing a combination of time-resolved fluorescent microsphere-based lateral flow assay and polymerase chain reaction (TRFM-LFA-PCR) for quantitatively detecting beef content to assess the authenticity of beef products, without the need of knowing the types of adulterants. The PCR amplicons (45 min) were detected by TRFM-LFA and the results can be observed within 20 min. This approach had demonstrated the ability to detect beef at concentrations as low as 0.005%. Furthermore, a calibration curve was established between the T/C ratio and the logarithmic value of beef content (0.1%–100%), with an R2 value of 0.99. The TRFM-LFA-PCR method was applied to detect adulterated samples, with recoveries between 82.91% and 116.29% and relative standard deviation ranging from 2.90% to 13.72%. Consequently, TRFM-LFA-PCR shows great potential for detection of beef adulteration without the need of knowing the types of adulterants.

Response of Rhododendron simsii and Rhododendron delavayi Superoxide Dismutase Family Genes to High-Temperature Stress

Superoxide dismutases (SODs) are the first line of defense in the antioxidant defense system, and they play an essential role in various adversity stress adaptations in Rhododendron. In this study, 9 Rhododendron simsii SODs (RsSODs) and 11 Rhododendron delavayi SODs (RdSODs) genes were identified in the genomes of R. simsii and R. delavayi. Phylogenetic relationship analysis classified SOD proteins from two Rhododendron species and other related species into three subfamilies. The results of gene structure and conserved motif analysis show that SOD proteins are strongly evolutionarily conserved, and SODs of the same subfamily have similar motif distributions, positions, and lengths. Twenty-two light-responsive elements, eight phytohormone regulatory elements, five adversity stress-related elements, and three growth and development regulatory elements were detected in the RsSOD and RdSOD promoters. Quantitative real-time fluorescence polymerase chain reaction analysis showed that among the 20 candidate genes, except for RdCSD5, the other SODs were expressed in at least one of four tissues, and all of these gene family members had high expression levels in the leaves. We then investigated the response of the RsSOD and RdSOD gene families to high-temperature stress in combination with the following specific stressors: abscisic acid, ethephon, and hydrogen peroxide treatments, followed by high-temperature stress. Different degrees of upregulated expression of the detected SOD gene family members were found for exogenous reagent treatments and different times of high-temperature stress. Thus, we provide a basis for the further functional characterization of SOD genes in R. simsii and R. delavayi in the future.

CircSLC25A16 facilitates the development of non-small-cell lung cancer through the miR-335-5p/CISD2 axis.

Non-small-cell lung cancer (NSCLC) is a common malignancy with high morbidity and mortality. Circular RNAs are widely involved in NSCLC progression. However, the mechanism of circSLC25A16 in NSCLC has not been reported. The expressions of circSLC25A16, microRNA-335-5p (miR-335-5p), and CDGSH iron-sulfur domain-containing protein 2 (CISD2) were monitored by quantitative real-time fluorescence polymerase chain reaction. Western blot was also carried out to measure the protein levels of CISD2, hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA). For functional analysis, cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine, flow cytometry, transwell, and wound healing assays were utilized to examine cell proliferation, apoptosis, and migration. Glucose uptake and lactate production were detected using commercial kits. The relationship between miR-335-5p and circSLC25A16 or CISD2 was verified by dual-luciferase reporter and RNA immunoprecipitation assays. Furthermore, tumor xenograft was established to explore the function of circSLC25A16 in vivo. CircSLC25A16 and CISD2 were overexpressed in NSCLC, but miR-335-5p was downregulated. CircSLC25A16 acted as a miR-335-5p sponge, and silencing of circSLC25A16 arrested cell proliferation, migration, and glycolysis, and promoted apoptosis, but these impacts were resumed by miR-335-5p inhibition. CISD2 was a miR-335-5p target, and overexpression of CISD2 abolished the suppressive function of miR-335-5p mimic on the malignant behavior of NSCLC cells. CircSLC25A16 could adsorb miR-335-5p to mediate CISD2 expression. Additionally, silencing circSLC25A16 restrained the growth of NSCLC tumor xenograft in vivo. CircSLC25A16 facilitated NSCLC progression via the miR-335-5p/CISD2 axis, implying that circSLC25A16 may serve as a novel biomarker for NSCLC treatment.

Causative role of a novel intronic indel variant in FBN1 and maternal germinal mosaicism in Marfan syndrome

BackgroundMarfan syndrome (MFS) is an autosomal dominant connective tissue disease with wide clinical heterogeneity, and mainly caused by pathogenic variants in fibrillin-1 (FBN1).MethodsA Chinese 4-generation MFS pedigree with 16 family members was recruited and exome sequencing (ES) was performed in the proband. Transcript analysis (patient RNA and minigene assays) and in silico structural analysis were used to determine the pathogenicity of the variant. In addition, germline mosaicism in family member (Ι:1) was assessed using quantitative fluorescent polymerase chain reaction (QF-PCR) and short tandem repeat PCR (STR) analyses.ResultsTwo cis-compound benign intronic variants of FBN1 (c.3464–4 A > G and c.3464-5G > A) were identified in the proband by ES. As a compound variant, c.3464-5_3464-4delGAinsAG was found to be pathogenic and co-segregated with MFS. RNA studies indicated that aberrant transcripts were found only in patients and mutant-type clones. The variant c.3464-5_3464-4delGAinsAG caused erroneous integration of a 3 bp sequence into intron 28 and resulted in the insertion of one amino acid in the protein sequence (p.Ile1154_Asp1155insAla). Structural analyses suggested that p.Ile1154_Asp1155insAla affected the protein’s secondary structure by interfering with one disulfide bond between Cys1140 and Cys1153 and causing the extension of an anti-parallel β sheet in the calcium-binding epidermal growth factor-like (cbEGF)13 domain. In addition, the asymptomatic family member Ι:1 was deduced to be a gonadal mosaic as assessed by inconsistent results of sequencing and STR analysis.ConclusionsTo our knowledge, FBN1 c.3464-5_3464-4delGAinsAG is the first identified pathogenic intronic indel variant affecting non-canonical splice sites in this gene. Our study reinforces the importance of assessing the pathogenic role of intronic variants at the mRNA level, with structural analysis, and the occurrence of mosaicism.

Prognostic value of BRCA1 promoter methylation for patients with epithelial ovarian cancer

ObjectiveBRCA1 promoter methylation (BRCA1pm) is suspected to alter prognosis of patients with epithelial ovarian cancer (EOC). We aimed to evaluate the prognostic impact of this epigenetic modification. MethodsWe conducted a retrospective, monocentric study from 11/2006 to 08/2018. Patients with EOC and available status concerning somatic BRCA1/2 mutation and BRCA1pm were included. Three groups were defined: patients without BRCA1/2 mutation or BRCA1pm, patients with BRCA1/2 mutation and patients with BRCA1pm. BRCA1/2 mutations were analyzed in current care settings by next-generation sequencing (NGS). BRCA1pm analysis was assessed and quantified from bisulfite converted DNAs using fluorescent methylation specific polymerase chain reaction (PCR) and fragment analysis. All patients signed a consent form and the study was authorized by a Personal Protection Committee. Descriptive statistics were used to describe groups. Multivariate analysis was performed using the logistic regression model and including the variables that could be known at the time of diagnosis and that were significant at univariate analysis. Survival was compared between the groups. Kaplan-Mayer curves were used to express the differences in survival that were compared using log rank tests. Results145 patients were included: 95 (65.5 %) patients without BRCA1/2 mutation or BRCA1pm, 32 (22.1 %) patients with BRCA1/2 mutation, 18 (12.4 %) patients with BRCA1pm. Median survival was decreased in patients with BRCA1pm. Comparison of survival revealed a significant difference in overall survival (p = 0.0078) with a worse prognosis for patients with a BRCA1pm. ConclusionBRCA1pm in patients with EOC is an independent factor associated with a decreased overall survival. SynopsisBRCA1 promotor methylation in patients with epithelial ovarian cancer is an independent factor associated with a decreased overall survival.

The effects of exosomes originating from different cell sources on the differentiation of bone marrow mesenchymal stem cells into schwann cells

BackgroundBone marrow mesenchymal stem cells (BMSCs) can differentiate into Schwann cells (SCs) during peripheral nerve injury; in our previous research, we showed that SC-derived exosomes (SC-exos) played a direct induction role while fibroblast-derived exosomes (Fb-exos) had no obvious induction role. The induction role of neural stem cell (NSC)-derived exosomes (NSC-exos) has also been widely confirmed. However, no studies have compared the induction effects of these three types of cells at the same time. Therefore, by investigating the effect of these three cell-derived exosomes upon the induction of BMSCs to differentiate into SCs, this study explored the role of different exosomes in promoting the differentiation of stem cells into SCs cells, and conducted a comparison between the two groups by RNA sequencing to further narrow the range of target genes and related gene pathways in order to study their related mechanisms.Materials and methodsWe extracted exosomes from SCs, fibroblasts (Fb) and neural stem cells (NSC) and then investigated the ability of these exosomes to induce differentiation into BMSCs under different culture conditions. The expression levels of key proteins and gene markers were detected in induced cells by fluorescence immunoassays, western blotting and polymerase chain reaction (PCR); then, we statistically compared the relative induction effects under different conditions. Finally, we analyzed the three types of exosomes by RNA-seq to predict target genes and related gene pathways.ResultsBMSCs were cultured by three media: conventional (no induction), pre-induction or pre-induction + original induction medium (ODM) with exosomes of the same cell origin under different culture conditions. When adding the three different types of exosomes separately, the overall induction of BMSCs to differentiate into SCs was significantly increased (P < 0.05). The induction ability was ranked as follows: pre-induction + ODM + exosome group > pre-induction + exosome group > non-induction + exosome group. Using exosomes from different cell sources under the same culture conditions, we observed the following trends under the three culture conditions: RSC96-exos group ≥ NSC-exos group > Fb-exos group. The overall ability to induce BMSCs into SCs was significantly greater in the RSC96-exos group and the NSC-exos group. Although there was no significant difference in induction efficiency when comparing these two groups, the overall induction ability of the RSC96-exos group was slightly higher than that of the NSC-exos group. By combining the differentiation induction results with the RNA-seq data, the three types of exosomes were divided into three comparative groups: RSC vs. NSC, RSC vs. Fb and NSC vs. Fb. We identified 203 differentially expressed mRNA target genes in these three groups. Two differentially expressed genes were upregulated simultaneously, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). We did not identify any co-upregulated target genes for the miRNAs, but did identify one target gene of the lncRNAs, namely ENSRNOG00000065005. Analysis identified 90 GO terms related to nerves and axons in the mRNAs; in addition, KEGG enrichment and GASA analysis identified 13 common differential expression pathways in the three groups.ConclusionsOur analysis found that pre-induction + ODM + RSC96/NSC-exos culture conditions were most conducive with regards to induction and differentiation. RSC96-exos and NSC-exos exhibited significantly greater differentiation efficiency of BMSCs into SCs. Although there was no statistical difference, the data indicated a trend for RSC96-exos to be advantageous We identified 203 differentially expressed mRNAs between the three groups and two differentially expressed target mRNAs were upregulated, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). 90 GO terms were related to nerves and axons. Finally, we identified 13 common differentially expressed pathways across our three types of exosomes. It is hoped that the efficiency of BMSCs induction differentiation into SCs can be improved, bringing hope to patients and more options for clinical treatment.

Analysis of the epidemiological situation of influenza in Guangzhou under the prevention and control of COVID-19 in June 2022.

Under the prevention and control measures of COVID-19, the epidemiological situation of respiratory pathogens is not well known. Understanding the patterns of respiratory pathogens epidemiology under the prevention and control measures of COVID-19 is important to guide resource allocation for existing and future treatment and prevention strategies. In total, 659 fever outpatients nasopharyngeal swabs were collected at fever illness onset during June in 2022 at the First Hospital of Guangzhou Medical University. Swabs were tested by real-time fluorescent single-tube multiplex polymerase chain reaction (PCR) for 12 respiratory pathogens. Moreover, 108 of the 659 swabs were tested for influenza virus antigen. At least one pathogen was detected in 477 (72.38%) of 659 fever outpatients with multiple pathogens identified in 25 (3.79%). The highest multiple infectious pattern is parainfluenza virus in combination with influenza (five cases). Influenza A virus (IFA), human rhinovirus (HRV), and parainfluenza virus are the three leading virus pathogens with proportions of 64.64%, 5.01%, and 2.88%. School-age children and adult groups have the highest pathogens positivity rate of 81.28% and 83.87%. A high proportion of adolescents and adults has respiratory pathogens detected during fever illnesses during June in 2022 under the prevention and control of COVID-19. These data indicate that diagnosis, prevention, and control of respiratory tract infection should be paid attention under the prevention and control of COVID-19.

Current Diagnostic Approaches in the Genetic Diagnosis of Disorders of Sex Development.

Disorders of sex development (DSD) are a clinically and genetically highly heterogeneous group of congenital disorders. The most accurate and rapid diagnosis may be possible with a complementary multidisciplinary diagnostic approach, including comprehensive clinical, hormonal, and genetic investigations. Rapid and accurate diagnosis of DSD requires urgency in terms of gender selection and management of the case. Despite the genetic tests performed in current daily practice, the genetic cause is still not elucidated in a significant proportion of cases. Karyotype analysis can be used as a standard for sex chromosome identification. In addition, quantitative fluorescent polymerase chain reaction (QF-PCR) or fluorescence in situ hybridization (FISH) analysis can be used for faster and more cost-effective detection of the sex chromosome and SRY gene. Multiplex ligation-dependent probe amplification (MLPA), single-gene sequence analysis, next-generation sequence analysis (NGSA), targeted NGSA, whole-exome sequencing (WES), and whole-genome sequencing (WGS) analyses can be performed according to preliminary diagnoses. Microarray analysis (array comparative genomic hybridization (aCGH) and single nucleotide polymorphism array (SNPa)) should be performed in cases with syndromic findings and if no pathology is detected with other tests. In DSD cases, the use of optical genome mapping and techniques, which will probably be in daily practice in near future, may be considered. In conclusion, the clinical and genetic diagnosis of DSD is difficult, and molecular genetic diagnosis is often not available. This has psychosocial and health implications for patients and their families. New genetic techniques, especially those targeting the whole genome, may provide a better understanding of DSD through the identification of little-known genetic causes. This review focuses on conventional genetic and next-generation genetic techniques used in the genetic diagnosis of DSD, as well as possible genetic diagnostic techniques and approaches that may be used in routine practice in near future.

The Rapid Evaluation of Down Syndrome With Quantitative Fluorescence Polymerase Chain Reaction (QF-PCR): A Pilot Study Among the Population in Eastern Uttar Pradesh, India.

Background and objective Down syndrome (DS) is characterized by the presence of an additional chromosome; it is a typical chromosomal disorder causing intellectual disability in individuals. The diagnostic process for DS often involves conventional karyotyping, which can be time-consuming. Trisomy 21 and other chromosomal abnormalities may now be quickly and accurately diagnosed using quantitative fluorescence polymerase chain reaction (QF-PCR). In light of this, this study aimed to investigate chromosomal abnormalities in DS using conventional karyotyping and QF-PCR among thepopulation in easternUttar Pradesh, India. Methods Blood samples from 40individuals with clinically diagnosed DS were collected. Conventional karyotyping involved standard cytogenetic techniques, while QF-PCR utilized DNA extraction and analysis with chromosome-specific short tandem repeat (STR) markers. Results Various distinct physical characteristics were observed in the DS individuals, such as mongoloid slant and low-set ears. Karyotyping and QF-PCR analyses revealed different chromosomal configurations associated with DS trisomy 21, with additional chromosomal abnormalities found in some individuals, including partial monosomy 18 and mosaic trisomy 21. However, in a few cases, neither karyotyping nor QF-PCR revealed any abnormalities. Conclusions The study demonstrated that QF-PCR is a reliable and rapid method for diagnosing DS, providing results within 24 hours. This approach allows for the simultaneous diagnosis of a large number of samples and reduces the time required to obtain results. In the diagnostic procedure for DS, we believe QF-PCR will prove to be a usefultool. Furthermore, therapeutic interventions based on their clinical traits and molecular karyotyping can enhance the quality of life of people with DS.

Correlation of DNA load, genotyping, and clinical phenotype of Mycoplasma pneumoniae infection in children.

This study aimed to investigate the correlation between Mycoplasma pneumoniae (MP)-DNA load in the bronchoalveolar lavage fluid (BALF) of children with MP pneumonia (MPP) and its subtypes, relevant laboratory data, imaging, extrapulmonary complications in infected children, and its clinical significance in evaluating the disease. Children hospitalized with MPP at Tianjin Children's Hospital between December 2017 and December 2020 were selected for the study, excluding those with mixed viral, bacterial, and fungal infections. Children were divided into low- and high-load groups according to the MP DNA load in BALF using real-time quantitative fluorescence polymerase chain reaction (PCR). After a successful MP culture, positive specimens were subjected to PCR-Restriction fragment length polymorphism and Multiple-locus variable number tandem repeat analysis typing. Basic data, clinical information, laboratory data, and radiological results were collected from all children included in the study. The PI-I type dominated the different load groups. Children in the low-load group had more wheezing and shortness of breath; however, children in the high-load group had a higher length of hospitalization, maximum fever temperature, higher chills/chilliness, incidence of abdominal pain, and higher C-reactive protein (CRP), procalcitonin (PCT) and aspartate aminotransferase (AST) levels. Children in the high-load group were more likely to have imaging changes such as pleural effusion, and the incidence of respiratory infections and extrapulmonary complications was higher than that of those in the low-load group. We applied Spearman's correlation analysis to clarify the relationship between MP DNA load and the clinical severity of MPP. We found that MP DNA load was positively correlated with length of hospitalization, maximum fever temperature, CRP, PCT, Interleukin-6 (IL-6), and AST levels, and negatively correlated with fever and cough durations, white blood cell count (WBC), and proportion of monocytes (MONO). The degree of correlation was as follows: length of hospitalization > IL-6 > cough duration > AST > fever duration > PCT > WBC > proportion of MONO > maximum fever temperature > CRP levels. MP DNA load was not correlated with MP typing but was significantly correlated with the children's clinical phenotype. Therefore, the MP DNA load helps in the early diagnosis of infection and can better predict disease regression.

Molecular characterization of Marek's Disease virus reveals reticuloendotheliosis virus-long terminal repeat integration in the genome of the field isolates in Egypt

The highly contagious, immunosuppressive, and cancer-causing Marek's disease virus (MDV) infects chickens. The financial costs of Marek's disease (MD) are significant for the chicken industry. In this study, a total of 180 samples from chicken farms suspected to be MDV-infected were collected. The chickens were sampled during the period between the months of October 2016 and February 2018 at Dakahlia and Damietta Governorates, Egypt. A total of 36 pooled samples were created. The prepared samples were inoculated into embryonated chicken eggs (ECEs). Indirect fluorescent antibody technique (IFAT) and ICP4 gene-based polymerase chain reaction (PCR) were used for MDV identification. For the genetic characterization of the identified virus, The ICP4 gene sequence was identified and compared with the sequences available from various regions of the world. Furthermore, the genomes of all detected MDVs were screened for the long terminal repeat (LTR) region of reticuloendotheliosis (REV) in their genomes. The results showed that 31 out of 36 pooled samples (86.1%) inoculated into ECEs displayed the characteristic pock lesions. By using IFAT and PCR to identify MDV in ECEs, positive results were found in 27 samples (75%). The Egyptian virus is thought to be genetically closely related to MDVs circulating in Ethiopia, China, and India. REV-LTR was amplified from 6 out of 27 field isolates genomes (22.2 %) while MDV vaccine strains were free from REV-LTR insertion. The integrated REV-LTRs depicted a close genetic relationship with those integrated in fowl poxvirus (FWPV) circulating in Egypt as well as those integrated in FWPVs and MDVs from China, USA, South Africa, and Australia. To the best of our knowledge, this investigation represents the first identification and characterization of REV-LTR insertions in Egyptian MDV field isolates. Given the findings above, additional research in the future seems crucial to determine how the REV-LTR insertions affect MDV pathogenesis, virulence, and insufficient vaccination protection.

Post-mortem rapid aneuploidy testing for holoprosencephaly.

Abortion and fetal death are common in fetuses with holoprosencephaly, so genetic examinations often have to be made in a post-mortem setting. The efficiency of the conventional karyotyping using cultured fibroblasts in these situations is limited due to frequent culture failure. In the current study, archived cases of holoprosencephaly, where post-mortem genetic evaluation was requested and sufficient frozen material was available, were reevaluated using the quantitative fluorescence polymerase chain reaction (QF-PCR) technique. Testing for aneuploidies of chromosomes 13, 15, 16, 18, 21, 22, X, and Y with the QF-PCR technique was carried out on DNA isolated from archived frozen chorionic villi in seven cases of holoprosencephaly. QF-PCR was successful in all seven cases. Two cases of trisomy 13, two cases of triploidy, and one case of trisomy 18 was found meaning a 71% diagnostic yield. The success rate of QF-PCR (100%, 7/7) was superior compared to conventional karyotyping (43%, 3/7). Rapid aneuploidy testing using the QF-PCR technique is a simple, reliable, time- and cost-effective method sufficient to conclude the etiologic investigation in the majority of holoprosencephaly cases post-mortem.

Changes in &lt;i&gt;DNMT1&lt;/i&gt; expression as a marker of epigenetic regulation disturbanses in multiple sclerosis patients

BACKGROUND: Multiple sclerosis is a chronic neurodegenerative autoimmune disease characterized by the presence of foci of inflammation and demyelination in the central nervous system. The initiation of pathological processes in multiple sclerosis is caused by a complex interaction of genetic factors, unfavorable environmental factors and epigenetic influences. Progressive neurological symptoms caused by axonal conduction disorders, axonal death and neurodestruction lead to a significant decreased patients’ quality of life and disability. The search for a new markers to improve diagnostic and therapeutic methods, including taking into account the genetic background and epigenetic interactions, is an urgent task.&#x0D; AIM: The work was aimed to study the changes in DNMT1 mRNA expression in multiple sclerosis patients with different disease duration, to analyze methylation of DNMT1 promoter, and compare the changes in the level of DNMT1 expression with the homocysteine content in the blood, and the presence of polymorphic variants in genes coding the key folate cycle enzymes.&#x0D; MATERIALS AND METHODS: The level of DNMT1 mRNA expression in peripheral mononuclear blood cells was assessed by reversed transcription followed by polymerase chain reaction. Fluorescent polymerase chain reaction followed by methyl-sensitive analysis of high-resolution melting curves was used to analyze methylation of the DNMT1 promoter. The content of homocysteine in the blood was determined by chemiluminescence immunoassay. The real-time polymerase chain reaction was used for genotyping by polymorphism of folate cycle genes; the fluorescent probes with the LNA modifications were used to discriminate alleles.&#x0D; RESULTS: It has been shown that in multiple sclerosis patients, including those at the onset of the disease, the level of DNMT1 mRNA expression is significantly lower than in the control group. No relationship was found between the decrease in DNMT1 expression and the level of promoter methylation. Strong positive relationship between the level of DNMT1 mRNA expression and homocysteine content in patients with multiple sclerosis and the combined effects of the genotypes of MTR A2756G and MTHFR C677T polymorphism on the expression of DNMT1 have been shown. These findings suggest that genetically determined features of folate metabolism may contribute to the disruption of epigenetic regulation in multiple sclerosis.&#x0D; CONCLUSIONS: The obtained results indicate the promise of research aimed to identifying the factors causing epigenetic changes in multiple sclerosis. Studying the mechanisms of the folate cycle genes polymorphic variants contribution to the pathogenesis of multiple sclerosis could be one of the possible ways to improve diagnostic and therapeutic approaches.

Accurate and reliable surface-enhanced Raman spectroscopy assay for early detection of SARS-CoV-2 RNA with exceptional sensitivity

This research proposes a highly sensitive and simple surface-enhanced Raman spectroscopy (SERS) assay for the detection of SARS-CoV-2 RNA using suitably designed probes specific for RdRp and N viral genes attached to a Raman marker. The sensitivity of the assay was optimized through precise adjustments to the conditions of immobilization and hybridization processes of the target RNA, including modifications to factors such as time and temperature. The assay achieved a remarkable sensitivity down to 58.39 copies/mL, comparable to or lower than the sensitivities reported for commercial fluorescent polymerase chain reaction (PCR) based methods. It has good selectivity in discriminating SARS-CoV-2 RNA against other respiratory viruses, respiratory syncytial virus (RSV), and influenza A virus. The reliability of the assay was validated by testing 24 clinical samples, including 12 positive samples with varying cycle threshold (Ct) values and 12 negative samples previously tested using real-time PCR. The assay consistently predicted true results that were in line with the PCR results for all samples. Furthermore, the assay demonstrated a notable limit of detection (LOD) of Ct (38 for RdRp gene and 37.5 for N-gene), indicating its capability to detect low concentrations of the target analyte and potentially facilitating early detection of the pathogen.

Copy number variation sequencing for the products of conception: What is the optimal testing strategy

BackgroundCopy number variation sequencing (CNV-seq) is crucial in prenatal diagnosis, but its limitations in detecting polyploidy, maternal cell contamination (MCC), and uniparental disomy (UPD) restrict its application in the analysis of products of conception (POCs). This study aimed to investigate an optimal genetic testing strategy for POCs in the era of CNV-seq. MethodsCNV-seq and quantitative fluorescent polymerase chain reaction (QF-PCR) were performed in all 4,211 spontaneous miscarriage cases. Different testing strategies were compared and the optimal testing strategies were proposed. ResultsOf the 4,211 cases, 2561 (60.82%) exhibited clinically significant chromosomal abnormalities. CNV-seq alone, without QF-PCR, might misdiagnose 311 (7.39%) cases, including 278 polyploidy, 13 UPD, and 20 MCC. In 20 MCC cases identified by QF-PCR, CNV-seq successfully pinpointed the cause of miscarriage in 13 cases. Furthermore, in cases where QF-PCR suggested polyploidy, CNV-seq improved the diagnostic accuracy in 54 (1.28%) hypo/hypertriploidy cases. After comparing four different strategies, the sequential approach (initiating with CNV-seq followed by QF-PCR if necessary) emerged as advantageous, reducing approximately 70% of the cost associated with QF-PCR while maintaining result accuracy. ConclusionsWe propose an initial CNV-seq followed by QF-PCR if needed—an efficient and cost-effective strategy for the genetic analysis of POCs.

Characteristics of reproductive tract infections caused by common pathogens among the outpatients of reproductive medicine center in Putian: retrospective study

BackgroundThis study aims to explore the infection and age distribution of Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Herpes simplex virus type II (HSV II) among the outpatients of Reproductive Medicine Center in Putian, Fujian Province to provide a clinical basis for the early diagnosis and treatment of various reproductive tract diseases and infertility in this region.MethodsA total of 1736 samples of secretions and exfoliated cervical cells were collected from the outpatients of the Reproductive Medicine Center of the Affiliated Hospital of Putian University from December 2021 to April 2023. The infections of UU, CT, NG and HSVII were detected by real-time fluorescence polymerase chain reaction (PCR), and the infection statuses of the patients with different genders, ages and diagnoses were analysed.ResultsAmong the 1736 patients, 611 were male and 1125 were female. The male patients had higher UU infection rate but lower HSV II infection rate than the female patients. No significant difference in CT and NG infection rates was observed between the genders. The CT infection rate gradually decreased with the increase in the age. The difference in UU, NG and HSV II infection rates among the different age groups was not statistically significant. For UU infection, the male infertile patients had the highest rate of 37.72% (172/456). Meanwhile, the differences in CT, NG and HSV II infection rates among the different diagnosis groups were not statistically significant. Among the male and female infertile patients, the CT infection rate was the highest in the 21–25 years of age group at 11.11% (2/18) and 9.47% (9/95), respectively. No statistically significant difference in UU, CT, NG and HSV II infection rates was observed among the different age groups of patients diagnosed in relation to the family planning guidance and between the male and female patients with other diagnoses results.ConclusionsThis study showed that UU was the most frequently identified pathogen in infertile men in Putian, Fujian Province. The CT infection rate was the highest in people under 20 years old, and the infection showed a tendency toward young individuals. Therefore, the publicity of sexual health knowledge must be strengthened, and the prevention and treatment of venereal diseases among young and middle-aged people must be improved. Moreover, the pathogen infection is related to infertility to a certain extent, which is conducive to clinical diagnosis and treatment.

Blood Chimerism in Fertile Female with 46, XY Karyotype Report

Introduction: The mammalian Y chromosome is a vital sex-determining factor in mammals by encoding gonadal genes. However, there are some discordant conditions with the XY female chromosome, which can lead to their inclusion in the disorders of sex development group.&#x0D; Case Report: The person under study is a 22-year-old woman for whom written consent was obtained, and all ethical procedures were followed. Herein, we report an extraordinarily fertile adolescent phenotypic mother with congenital XY abnormality who reared as female, spontaneous puberty with regular menstruation and two gestations. This condition was initially diagnosed using the karyotype technique and finally confirmed with the specialized Fluorescence in-situ hybridization technique. Finally, the quantitative fluorescent polymerase chain reaction technique was used for more certainty. Usually, the male genotype cannot be fertile; since this case is unique, various reasons are involved in its occurrence. One of these cases can be a bone marrow or blood transplant, but according to the study, the possible development of this disorder can be caused by the fetal period.&#x0D; Conclusion: As the prior clinical history of the case report revealed that her twin brother died during her mother’s expectancies, it can be assumed that the blood supply between the two fetuses has been shared unevenly or that feto-fetal transfusion has occurred. This incident has caused her to become a chimera with XY karyotype.

The Genetic Approach to Stillbirth: A Systematic Review

ABSTRACT Stillbirth represents a significant burden to individuals and families and has a great variety of causes, but much is still unknown about the underlying etiologies. The overall contribution of genetic abnormalities to stillbirth is not well known; this study was designed to evaluate known genetic causes of unexplained stillbirths, as well as understand the current position and future direction for using genetic and genomic testing in this field. This study was a systematic review of literature published between 1953 and 2020 on genetic testing and stillbirth. Inclusion criteria for studies were human cases, details and specific data on cohorts that included proportion of unexplained fetal deaths, and data on genetic testing and the subsequent results. Exclusion criteria were pregnancy loss before 22 weeks of gestation, focus on known causes of stillbirth, focus on fetal pathology or abnormalities, investigating neonatal death, genetic testing in populations with congenital anomalies or suspected specific syndromes, and those without details on genetic testing. The final review included 9 studies, 1 comparing karyotyping and chromosomal microarray analysis (CMA); 1 comparing karyotyping, CMA, and quantitative fluorescence–polymerase chain reaction; 2 with CMA only; and 5 with exome sequencing data. The studies that examined only CMA found 1 case of copy number variants (CNVs) in 24 cases and 31 CNVs in 96 cases. The largest study included here analyzed 532 stillbirths and compared CMA and karyotyping. Chromosomal microarray analysis showed a higher rate of detection of CNVs, increasing from 5.8% to 8.3% (P = 0.007) when compared with karyotype analysis. One challenge presented by this topic is that some disorders that could contribute to unexplained stillbirth are typically diagnosed when symptoms appear later in life and so are not usually associated with stillbirth or the risk of stillbirth. One example of this could be cardiac rhythm anomalies or other rare genetic variants that lead to stillbirth when appearing in a particular variation. One study included a cohort of unexplained stillbirth after postmortem examination that excluded obvious causes. Genetic analysis showed that several cases carried a variant that was functionally disruptive that may have contributed to stillbirth, but several additional cases that were functionally normal. Genetic variations relating to unexplained stillbirth are a broad and largely unexplored topic. Genetics play a vital role in clinical exploration and treatment of many disorders and in diagnosing causes of fetal mortality and morbidity. This review shows that genetic testing for stillbirth varies greatly according to test availability and guidelines of use, with some tests showing greater diagnostic yield than others. Although these studies incorporated large cohorts of unexplained stillbirths, genetic analysis identified potential causes for only a small percentage. Future research is needed to explain potential genetic causes for stillbirth.