ABI Prism Research Articles

Individual Identification and Assessment of Genetic Diversity Using Microsatellite Markers in Racing Pigeons Raised in Turkiye

The implementation of a swift and economical molecular genetic approach, ensuring both efficacy and cost-effectiveness and facilitating population certification, is of utmost significance for breeders and the conservation of Turkiye's native pigeon biodiversity. In this study, we aimed to examine the genetic structure of racing pigeons (Columba livia domestica) raised in Turkiye using a genetic marker panel consisting of eight short tandem repeat (STR) loci. For this purpose, DNA was isolated from the shed feathers of 216 pigeons. Genomic DNA was amplified using the multiplex allele-specific PCR and subsequent capillary electrophoresis with ABI PRISM 3130XL Genetic Analyzer. Next, PCR products were analyzed in the GeneMapper Software program (Applied Biosystems). For parent testing, paternity index (PI), combined paternity index (CPI), and cumulative probability of paternity (CPP) were calculated. Furthermore, population genetic diversity was evaluated using heterozygosity (He), polymorphism information content (PIC), and Hardy–Weinberg equilibrium (HWE) testing. Results revealed that the total number of alleles is 81 and the number of alleles per locus varies between 4 and 19. The similarity rate between parent and offspring was calculated as 99.99% and above. Since no pedigree information was given when the samples were analyzed, obtaining this similarity ratio demonstrates the reliability of the panel. He values range from 0.362 to 0.919, and the PIC values range from 0.295 to 0.909. Loci PG-1, PG-2, and PG-3 show significant genetic diversity, with moderate to high PIC values reflecting varied allele frequencies in the population. Consequently, the set of seven STR markers (+ one sex marker) can be applied to identify and confirm parentage on a regular basis, thereby facilitating efficient breeding programs and ensuring genetic diversity conservation. This panel enables efficient pedigree analysis and gender determination, optimizing cost-effectiveness. The methodology presented in this study is ideal for pedigree analysis and breed certification in the Turkish pigeon breeding industry. Consequently, we affirm that the study data carries considerable national importance.

Hyaline fibromatosis syndrome: a rare, yet recognizable syndrome.

Hyaline fibromatosis syndrome is a rare autosomal recessive disorder caused by ANTXR2 pathogenic variants. The disorder is characterized by the deposition of amorphous hyaline material in connective tissues. The hallmarks of the disease are joint contractures, generalized skin stiffness, hyperpigmented papules over extensor surfaces of joints, fleshy perianal masses, severe diarrhea, and gingival hypertrophy. The severity of the disease varies and prognosis is poor. No specific treatment is yet available. Most patients with the severe form of the condition pass away before the second year of age. In this study, we describe the clinical and molecular findings of a cohort of seven hyaline fibromatosis syndrome patients who were diagnosed and followed up at a single tertiary reference center in Turkey. Genomic DNA was extracted by standard salting out method from peripheric blood samples of three patients. In one patient DNA extraction was performed on pathology slides since peripheric blood DNA was not available. All coding exons of the ANTXR2 were amplified and sequenced on ABI Prism 3500 Genetic Analyser. Sanger sequencing was performed in 3 patients and homozygous c.945T>G p.(Cys315Trp), c.1073dup p.(Ala359CysfsTer13), and c.1074del p.(Ala359HisfsTer50) variants were identified in ANTXR2. All patients passed away before the age of five years. HFS is a rare, progressive disorder with a broad phenotypic spectrum. HFS can be recognized easily with distinctive clinical features. Nevertheless, it has poor prognosis with increased mortality due to severe clinical decompensation.

First report of Tunisian patients with CDKL5-related encephalopathy.

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) are associated with a wide spectrum of clinical presentations. Early-onset epileptic encephalopathy (EOEE) is the most recognized phenotype. Here we describe phenotypic features in eight Tunisian patients with CDKL5-related encephalopathy. We included all cases with clinical features consistent with CDKL5-related encephalopathy: infantile epileptic spasm, acquired microcephaly, movement disorders and visual impairment. We collected data about seizure types, electroencephalogram, magnetic resonance imaging, and metabolic analysis. The diagnosis of CDKL5 mutation was made thanks to Sanger sequencing with an ABI PRISM 3100-Avant automated DNA sequencer using a Big Dye Terminator Cycle Sequencing Reaction Kit v1.1. and Next Generation Sequencing (NGS) since the development of a gene panel responsible for DEE within the framework of "Strengthening the Sfax University Expertise for diagnosis and management of epileptic encephalopathies". We collected four boys and four girls aged meanly 6 years old with confirmed mutation on CDKL5 gene. Overall, we identified five de novo CDKL5 mutations including three Frame-shift mutations, one missense mutation, and a splicing variant. The mean age at first seizure onset was 4 months. The first seizure type was infantile epileptic spasm (4/8) followed by tonic (2/8) and myoclonic seizures (2/8). Out of eight cases, four exhibited two stages epileptic course while epilepsy in three other patients progressed on three stages. Regarding development, most cases (6/8) had psychomotor retardation from the start whilst the two others showed psychomotor regression with the onset of seizures. Additional clinical features included visual impairment (7/8), tone abnormalities (7/8), stereotypies (7/8), and acquired microcephaly (6/8). Our present report delineates an unusual phenotype of CDKL5-related encephalopathy with male gender predominance and delayed onset epilepsy. It interestingly described new phenotypic features and uncommon benign developmental profiles in boys, different patterns of CDKL5-epilepsy, neuroimaging findings, and CDKL5 mutational spectrum.

Role of IL-6, IL-10 and TNFα Gene Variants in Preterm Birth.

Background: The association of gene variants for interleukin 6 (IL-6) (rs1800796), interleukin 10 (IL-10) (rs1800896) and tumor necrosis factorα (TNFα (rs1800629) with the occurrence of spontaneous preterm birth (PTB) was investigated to determine whether these genetic variants are a risk factor. Methods: A total of 199 blood samples from pregnant women who had given birth prematurely and 200 control blood samples were analyzed to determine single nucleotide polymorphisms (SNPs) of genes for IL-6 (rs1800796), IL-10 (rs1800896) and TNFα (rs1800629). The control samples were samples from pregnant women with term delivery. The isolation of DNA was performed on mini-spin columns according to the manufacturer's protocol. The quality and purity of the isolated DNA were tested using a Qubit 3 fluorometer. Genotyping was performed with an ABI PRISM 7500 SDS using TaqMan SNP genotyping assays. The genotypes obtained were analyzed using the 7500 Software v2.3 package. Results: Carriers of the A/A genotype for the rs1800629 SNP of the TNFα gene have a 4.81 times greater chance of late-onset PTB compared to carriers of the G/G and A/G genotypes in the recessive inheritance model. The presence of the G/G genotype in the recessive inheritance model compared with the G/A and A/A genotypes for the rs1800896 SNP of the IL-10 gene represents a potentially protective factor, with mothers in the term-birth group having an almost 2-fold lower odds of PTB in general and an almost 10-fold lower odds of early PTB. On the other hand, carriers of the A/G genotype of rs1800896 have a 1.54-fold higher chance of preterm birth in general and a 1.6-fold higher chance of late preterm birth in the superdominant inheritance model compared to the A/A and G/G genotypes in the group of mothers with PTB. In this study, no association was found between PTB and the rs1800796 SNP of the IL-6 gene. Conclusions: rs1800629 in mothers was associated with PTB. rs1800896 shows a potentially protective effect for the occurrence of PTB in this study. No association was found between PTB and rs1800796.

Epstein–Barr virus (Orthoherpesviridae: <i>Lymphocryptovirus</i>) among Russian ethnic groups: Prevalence of EBV types (EBV-1 and EBV-2), <i>LMP1</i> gene variants and malignancies

Introduction. The discovery of two EBV types (EBV-1 and EBV-2) has stimulated the study of their prevalence in populations and association with malignancies.
 Objective. To study the prevalence of EBV-1 and EBV-2 types among ethnic groups in Russia, to analyze PCR products of the LMP1 gene in virus isolates, and to evaluate the contribution of EBV types to the incidence of malignant neoplasms.
 Materials and methods. EBV isolates from oral lavages of the Republics Adygea, Kalmykia, Tatarstan and the Moscow Region (MR) representatives were studied by nested PCR for the belonging to EBV-1 and EBV-2 types. LMP1 amplicons obtained by real-time PCR from viral isolates DNA were classified and sequenced on an automatic DNA sequencer ABI PRISM 3100-Avant (USA). The sequencing results were analyzed using Chromas 230 and Vector NT programs (Invitrogen, USA). The reliability of the obtained data was assessed using statistical packages Statistica for Windows, 10.0.
 Results. The prevalence rates of EBV-1 and EBV-2 in representatives of four ethnic groups were compared with the incidence rates of some tumors in the population of three Republics and MR.
 The dominant persistence of the transforming in vitro EBV-1 type in representatives of the Republic of Tatarstan and MR correlated with a high incidence of gastric cancer and lymphomas in the population of these territories. On the contrary, predominant infection of the non-transforming in vitro EBV-2 type and both types of the virus in approximately the same percentage of representatives of Adygea and Kalmykia, respectively, correlated with a lower level incidence of above tumors in populations of these Republics. The differences between the incidence rates of neoplasms in the compared ethnic populations were statistically insignificant (p 0.05). LMP1 variants of viral isolates did not reflect either the level of EBV persistence types or the incidence of tumors.
 Conclusion. Infection of ethnic groups with EBV-1 and EBV-2 may vary significantly under the influence of various factors. The predominance of the in vitro transforming EBV-1 type in the population did not increase the incidence of tumors due to cases associated with the dominant virus type.

Genetic diversity of HPV35 in Chad and the Central African Republic, two landlocked countries of Central Africa: A cross-sectional study.

Human Papillomavirus (HPV)-35 accounts for up 10% of cervical cancers in Sub-Saharan Africa. We herein assessed the genetic diversity of HPV35 in HIV-negative women from Chad (identified as #CHAD) and HIV-infected men having sex with men (MSM) in the Central African Republic (CAR), identified as #CAR. Ten HPV35 DNA from self-collected genital secretions (n = 5) and anal margin samples (n = 5) obtained from women and MSM, respectively, were sequenced using the ABI PRISM® BigDye Sequencing technology. All but one HPV35 strains belonged to the A2 sublineage, and only #CAR5 belonged to A1. HPV35 from #CAR had higher L1 variability compared to #CHAD (mean number of mutations: 16 versus 6). L1 of #CAR5 showed a significant variability (2.29%), suggesting a possible intra-type divergence from HPV35H. Three (BC, DE, and EF) out of the 5 capsid loops domains remained totally conserved, while FG- and HI- loops of #CAR exhibited amino acid variations. #CAR5 also showed the highest LCR variability with a 16bp insertion at binding sites of the YY1. HPV35 from #CHAD exhibited the highest variability in E2 gene (P<0.05). E6 and E7 oncoproteins remained well conserved. There is a relative maintenance of a well conserved HPV35 A2 sublineage within heterosexual women in Chad and MSM with HIV in the Central African Republic.

DNA Extraction from Blood Stored on FTA Cards

Informed consent was obtained from 238 unrelated Libyan individuals (Benghazi region). DNA was extracted from blood stains collected on FTA1 cards About 1.2 mm FTA1 disc and 1 ng of DNA purified from buccal swabs was used products were separated and detected using the ABI Prism 310xl Genetic Analyzer (Applied Biosystems) according to the manufacturer’s recommended protocol. The data were analysed using GeneMapper ID v3.2 (Applied Biosystems). Quality control - The laboratory has participated in the Y-STR Haplotyping Quality Assurance Exercise (Certified at 2010-5-20). We conclude that amplification of DNA stored on FTA cards can be used safely to analyse DNA profiles.

Detection of a status of microsatellite instability in tumors of patients with endometrioid adenocarcinoma of the ovaries and/or of uterine corpus

Introduction. Multiple primary malignant neoplasms of female reproductive organs are a rare pathology. However, over the past decades, there has been an upsurge of interest in the study of this phenomenon in oncology. This is particularly the case for the diagnosis of synchronous endometrioid adenocarcinoma of the ovaries and uterine corpus, which histogenetically belong to the same germ layer and have similar histological structure. Until recently, clinicians relied only on morphological examination in these cases, but with the development of molecular genetic technologies, new diagnostic possibilities have emerged.Aim. Is the detection of the status of microsatellite instability in tumors of patients with endometrioid adenocarcinoma of the ovaries and/or uterine corpus.Materials and Methods. A pilot retrospective molecular genetic study (n = 48) was conducted to determine the status of microsatellite instability (MSI) in the tumors of the ovaries and/or uterine corpus: it involved 33 patients with solitary endometrioid ovarian cancer and 15 patients with synchronous endometrioid adenocarcinoma of the ovaries and uterine corpus. Microsatellite instability status was detected using PCR method with subsequent fragment analysis performed on ABI PRISM 3500 genetic analyzer (8 capillaries, Applied Biosystems). DNA was isolated from paraffin blocks of surgical specimens using DNAsorb B extraction kit (AmpliSens, Russia), according to the manufacturer’s manual. DNA concentration was estimated fluorometrically using Qubit 2.0 (Life Technologies, USA). The obtained data were analyzed using GeneMapper program (Thermo Fisher, USA). In case of polymorphism of two and more markers high-level microsatellite instability (MSI-H) was observed.Results. The incidence of MSI-H in solitary endometrioid ovarian cancer (n = 33) was 12,1 % (4 cases), while in synchronous ovarian and uterine corpus tumors (n = 15) MSI-H incidence made up 20 % (n = 3). Herewith, there have been only cases of a combination of endometrioid histotypes of ovarian and endometrial cancer with identical status of microsatellite instability. Thus, the incidence of MSI-H in synchronous endometrioid adenocarcinoma of the ovaries and uterine corpus (20 %) is comparable to that in solitary endometrial cancer.Conclusion. Our pilot study became a significant complement to the previously published materials, as it allowed to confirm the clonal origin of tumors in patients with endometrioid adenocarcinoma of the ovaries and uterine corpus, that can affect the stratification of treatment strategy for this category of patients.

Development of a Sensitive, Easy and High-Throughput Compliant Protocol for Maize and Soybean DNA Extraction and Quantitation Using a Plant-Specific Universal Taqman Minor Groove Binder Probe.

We report the optimization of a high-throughput, compliant DNA extraction method that uses standard format 96-well plates and a commercial automated DNA purification system (ABI PRISM® 6100 Nucleic Acid PrepStation). The procedure was set up for maize and soybean, the most common GMO crops and the main ingredients of several foodstuffs, and compared with an EU-validated CTAB-based method. Optimization of the DNA extraction was achieved by applying self-prepared buffers (for DNA extraction, binding, and washing) on the PrepStation loaded with proprietary glass-fiber-coated purification plates. Quantification of extracted DNA was performed by real-time PCR using previously reported endogenous soybean lectin and maize starch synthase genes and a novel plant-specific universal TaqMan MGB probe that targets the 18S rRNA multiple copy gene. Using serial dilutions of both maize and soybean genomic DNAs, we show low PCR sensitivity and efficiency for the official TransPrep DNA extraction protocol compared to the CTAB-based one. On the other hand, using serial dilutions of a standard reference plasmid containing a 137 bp sequence cloned from the 18S rRNA plant-specific ribosomal gene, we demonstrate the high PCR sensitivity and efficiency of the optimized DNA extraction protocol setup with self-prepared buffers. The limits of detection and quantification of the 18S rDNA reiteration were consistent with the calculated values, supporting the suitability of the DNA extraction procedure for high-throughput analyses of large populations and small amounts of tissue.

A study of genetic profiles of grape plants preserved under the name of Dagestan variety ‘Khatmi’

Background. Traditionally, the description of grape varieties is a task of ampelographic studies. However, several different grape cultivars have similar phenotypic traits. Molecular genetic characterization is the most accurate tool for cultivar identification. The development of DNA fingerprinting of varieties is the first step in this direction. An extensive database of DNA profiles of grape genotypes makes it possible to determine the varietal affiliation of unknown forms, confirm or refute the varietal correspondence of planting material. ‘Khatmi’ is an autochthonous grape variety from Dagestan. The profile of ‘Khatmi’ is presented in the VIVC international database of DNA fingerprints for grape varieties. However, an application of DNA analysis methods in grape variety studies has determined that several ancient varieties were cultivated under one name, while for others a certain variability of genotypes was observed. The objectives of the work were to study samples of ‘Khatmi’ plants from different places of growth in Dagestan by standard microsatellite loci used for grape genotyping, to assess the level of genetic similarity of the samples, and to refine the DNA profile of ‘Khatmi’. Materials and methods. Molecular genetic study was carried out on 10 samples from different ‘Khatmi’ populations. The material was picked from the collection sites of Dagestan breeding experimental station of viticulture and vegetable growing and the Dagestan Experiment Station of VIR, as well as from production plantations. DNA was extracted from herbarium specimens of young grape shoot tips by the CTAB method. The samples were genotyped by polymerase chain reaction using a standard set of primers for 9 microsatellite markers VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62 and VrZAG79 recommended by the International Organization of Grapes and Wine (OIV) for grapevine DNA fingerprinting. Amplification products were separated, and their sizes were assessed using capillary electrophoresis on an ABI Prism 3130 genetic analyzer. Results. Genotyping was done for 10 samples of grapes growing in Dagestan under the name ‘Khatmi’, including samples from different collections and places of industrial cultivation, as well as clonal variations of this variety and putative clonal variations. The two base pair differences within one of the loci distinguished the DNA profiles of the analyzed samples from that of ‘Khatmi’ presented in the international grape varieties database VIVC. It was determined that the sample under the name ‘Khatmi krupnoyagodnyi’ is closely related to ‘Khatmi’ variety by its genotype, but probably represents a clonal variation of ‘Koz uzyum’, another local variety of Dagestan. Conclusion. The DNA profile of the local Dagestan grape variety ‘Khatmi’ has been refined.

Pasteurella multocida Pneumonia with Hemoptysis in an Immunocompetent Case

Pasteurella species are gram-negative bacilli found in healthy pets' oropharynx and gastrointestinal tract flora. In humans, skin and soft tissue infections develop most frequently with the bite or scratching of animals such as cats or dogs. At the same time, they cause infections in the respiratory tract, mainly in patients with chronic lung disease or immunosuppressive patients. In this case report, a rare case of pneumonia caused by P.multocida bacteria in a patient with bronchiectasis was presented. A young male patient was admitted to the emergency department of our hospital with complaints of hemoptysis, cough with phlegm, and weight loss. The patient's blood pressure was 140/82 mmHg and SO2= 94%. Rales and rhonchi were detected in the lower left lung during the examination. Standard thorax tomography revealed prominent cystic structures and pneumonic infiltrates in the left lower lobe. Laboratory findings were normal. The Coronavirus disease-2019 (COVID-19) quantitative real-time polymerase chain reaction (qRt-PCR) test was found to be negative in the nasopharyngeal swab sample taken from the patient. Fiberoptic bronchoscopy was performed on the patient to investigate the presence of endobronchial lesion or foreign body aspiration. Culture and cytological evaluation was requested from the bronchial lavage taken. Gram-negative coccobacilli were seen among dense polymorphonuclear leukocytes in the Gram stain of the sample. Acid-fast bacilli were not detected with Ehrlich Ziehl Neelsen stain. In the lavage culture evaluated after 24 hours, colonies growing in blood and chocolate media were stained and gramnegative coccobacilli were observed. The isolate was identified as 96.0% P.canis with the automated Vitek 2 (Biomerieux, France) system. It was determined that the isolate was susceptible to levofloxacin, trimethoprim-sulfamethoxazole, amoxicillin-clavulanic acid, penicillin, ciprofloxacin and cefotaxime in the antibiogram performed by disc diffusion test according to EUCAST v13.0 guideline criteria. Sequence analysis of the isolate obtained from the culture was performed on the ABI Prism 310 Genetic Analyzer (Applied Biosystems, USA). Sequence analysis of the isolate revealed 99.85% homology with P.multocida (GenBank accession no: NG_115137.1). Although Pasteurella multocida pneumonia is not commonly observed, the presence of underlying bronchiectasis in this patient facilitated the establishment of the bacteria. In order not to miss the diagnosis of pneumonia due to P.multocida, microbiological evaluation and molecular typing should be performed in the samples taken from the respiratory tract in patients with chronic respiratory diseases such as bronchiectasis.

Increasing trend of transmitted integrase inhibitor resistance in a cohort of antiretroviral therapy-naive people living with HIV.

Drug regimens containing integrase strand transfer inhibitors (INSTIs) are widely recommended by international guidelines for ART-naive people living with HIV (PLWH).1,2 However, INSTI resistance mutations are uncommon and therefore their surveillance is not suggested before starting ART. The aim of the study was to evaluate the prevalence trend of transmitted INSTI drug resistance (DR) and to assess factors associated with transmitted INSTI resistance among ART-naive PLWH over the past decade. We conducted a time-trend, single-centre study over the period 2009–19 on adult naive PLWH with a genotypic resistance test (GRT) performed before the ART initiation. GRT was determined by Sanger sequencing using an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA); we included in the analysis the immediate pre-ART GRT available in people newly diagnosed with HIV infection. The degree of resistance to each INSTI drug was calculated using the Genotypic Resistance Interpretation Algorithm of the Stanford HIV Drug Resistance Database Program (version 9.1.1) (https://hivdb.stanford.edu/hivdb/by-patterns/). The obtained scores were used to classify the presence of low-level, intermediate or high-level resistance to each INSTI drug and to the INSTI drug class. DR within the INSTI class was defined by the presence of at least low-level resistance to ≥1 drug of the INSTI class.

Isolation of Cystic Echinococcosis Causative Agents of Echinococcus equinus and Echinococcus ortleppi Species from Humans in the Central Anatolia Region of Türkiye

Cystic Echinococcosis (CE) caused by Echinococcus granulosus sensu strico is neglected in Türkiye despite being one of the common diseases due to agriculture being at the forefront, low socioeconomic status and unhygienic animal slaughter. Considering the morbidity, mortality, and difficulties in treatment, more studies and precautions are needed regarding this disease. In this study, it was aimed to genotype Echinococcus isolated from CE patients in the Central Anatolia region. DNA isolation from tissue samples taken from 60 CE patients was performed using the QIAamp DNA FFPE tissue kit. Cytochrome c oxidase subunit gene region of Echinococcus was targeted and JB3/JB4.5 primers were used for genotyping. Polymerase chain reaction (PCR) products were purified according to the instructions for use of the QIAquick PCR purification kit. PCR products were prepared using the ABI Prism BigDye Terminator V3.1 Cycle sequencing kit and the nucleotide sequences in the samples were evaluated with the ABI 3100 sequencing device. The nucleotide sequences obtained in the study were analyzed using MetaPIGA2, MRBAYES v.3.1.2, phyogenetic analysis using parsimony, Unigen programs, maximum likelihood, Bayesian and parsimony methods. It has been found that 88.4% (53/60) of Echinococcus isolates were E.granulosus s.s. in this study. It has been genotyped as 41.7% (25/60) G1, 30.0% (18/60) G3 and 16.7% (10/60) G2 genotype. It has been determined that 6.6% (4/60) of the other Echinococcus isolates were E.equinus and 5.5% (3/60) were E.ortleppi. It was observed that E.equinus and E.ortleppi were isolated from atypically located cysts and from those living in rural areas. The E.equinus and E.ortleppi species were not found in CE patients living in urban areas. CE cases are common in the Central Anatolia region due to dog and cattle breeding, and the disease agent Echinococcus species vary. Genotyping of Echinococcus species is effective in the development of CE treatment and control strategies. Study results can play an active role in the fight against CE, which has formed the basis of the "one health" approach in the world and in Türkiye in recent years.

DNA Polymorphism of 13 Autosomal Short Tandem Repeats (STR) Loci in Mentawai Populations.

&lt;b&gt;Background and Objective:&lt;/b&gt; Indonesia consists of various ethnic groups with diverse cultures. However, research on allele frequencies and genetic variations of each ethnic group in Indonesia has not been well established. Therefore, a study was conducted on 13 STR loci panels to analyze the mentawai population as an enhancement of the Indonesian population's genetic data library and genetic variation. Mentawai is one of the Indonesian tribes who live on Mentawai Island, West Sumatera. &lt;b&gt;Materials and Methods:&lt;/b&gt; The PrepFiler kit to extract DNA from blood and the GlobalFiler kit for amplifying the 13 loci were used. The genetic analyzer ABI PRISM 3500 was used to detect PCR products. Data were processed and analyzed by EasyDNA and FORSTAT software. &lt;b&gt;Results:&lt;/b&gt; The allele frequency data for 13 autosomal loci in Mentawai populations were obtained. Expected heterozygosity was found with a minimum of 0.607 for TPOX and a maximum of 0.866 for FGA. Power of discrimination (PD) values ranges from TPOX (0.792) to FGA (0.968). Polymorphic information content (PIC) values for all loci were high, ranging from 0.642 for TPOX to 0.921 for vWA. Three off-ladder alleles in these findings were detected. Allele 6.3 at the TH01 loci and allele 14.2 at the D18S51 loci are exquisite. The mean expected heterozygosity and mean power of discrimination value across the 13 loci were 75.9 and 90.1%, indicating striking gene diversity. &lt;b&gt;Conclusion:&lt;/b&gt; These 13 STR loci can be used for population genetic studies and forensic identification.

SNP-Profiles of &lt;i&gt;Yersinia pestis&lt;/i&gt; Strains of the Medieval Biovar from the Caspian Sea Region Plague Foci

The SNP-typing method based on the detection of stable genetic markers in the genome, i.e., single nucleotide polymorphisms, is successfully used for genotyping of pathogenic microorganisms and can be applied for SNP-profiling of Yersinia pestis strains and molecular-genetic certification of focal areas. The aim of the study was to determine the SNP profiles of Y. pestis strains of the medieval biovar isolated in the Caspian Sea region plague foci in 1912–2015 and to develop a method for identifying unique SNPs using the Sanger sequencing for molecular-genetic certification of these territories. Materials and methods. A comprehensive study of the phenotypic and genotypic properties of 190 Y. pestis strains from plague foci in the Caspian Sea region was carried out. Phylogenetic reconstruction by the Maximum Likelihood method (GTR model) in the SeaView 5.0.4 software was performed on the basis of 1621 SNPs identified among 50 Y. pestis strains according to WG-SNP analysis in the snippy 4.6 program. Primers for PCR amplification of the SNP loci selected as target were calculated using the Vector NTI program. Sanger sequencing of SNPs loci was conducted on an ABI PRISM 3500XL genetic analyzer (Applied Biosystems, USA). Results and discussion. According to phenotypic characteristics, all studied strains from the Caspian foci belonged to a highly virulent and epidemically significant medieval biovar of the main subspecies of Y. pestis. According to the results of the WG-SNP analysis, 9 SNP genotypes were identified based on the polymorphism of single nucleotides of 24 genes characteristic of the main phylopopulations, which include strains isolated during various periods of epidemic and epizootic activity in the Caspian plague foci. Determining of SNP genotypes of Y. pestis strains of the medieval biovar, obtained over a hundred years in the Caspian foci, creates the prerequisites for defining the canonical SNP profile (canSNP) and for developing an algorithm for molecular epidemiological monitoring of the foci in which this highly virulent biovar circulates.

First report of youcai mosaic virus in firecracker flower (Crossandra infundibuliformis) in Taiwan.

Firecracker flower or crossandra (Crossandra infundibuliformis), an ornamental native to southern Asia, is commonly grown as bedding plants in the garden. In January 2021, crossandra plants showing mosaic, chlorotic ringspot, and leaf deformation (Suppl. Fig. 1) were observed at a recreational farm in Zhuolan Township (Miauli County, Taiwan) (E120°82'62'', N24°33'30'') . Transmission electron microscope (JEM-1400, JOEL, Japan) examination by negative staining of 10 affected plants indicated the presence of particles resembling a tobamovirus in all examined affected samples. However, no tobamovirus-like particles were observed in the crude sap prepared from healthy crossandra leaf tissues. Total RNA was extracted from affected leaves and used for RT-PCR amplification using the tobamovirus group-specific primer pair Tob Uni1 (5'-ATTTAAGTGGAGGGAAAACCACT-3') and Tob Uni2 (5'-GTYGTTGATGAGTTCGTGGA-3') (Letschert et al., 2002). A cDNA fragment of about 650-bp was amplified and Sanger sequenced (ABI PRISM 3730 DNA Sequencer, Biotechnology Center at National Chung Hsing University, Taichung, Taiwan ), revealing 98% sequence identity to that of a Brassica isolate of youcai mosaic virus (YoMV, AY318866). The virus was isolated through mechanical inoculation onto Chenopodium quinoa to yield two pure isolates, designated FC-1 and FC-2. Mechanical inoculation of FC-1 and FC-2 back to virus-free Crossandra infundibuliformis plants (5 for each isolate) resulted in systemic mosaic, chlorotic ringspot, and leaf deformation. All mock and healthy controls were symptomless and failed to obtain any RT-PCR products with YoMV-specific primers CPF1 (5'- ATGGTTTACAACATCACGAG-3') and CPR1 (5'-CTATGTAGCTGGCGCAGTAG-3'). Systemic symptoms of mild mosaic, ringspot, leafroll, and necrosis appeared on some of the tobaccos (Nicotiana benthamiana, N. tabacum, N. rustica), and cruciferous vegetables (Brassica rapa subsp. chinensis cv. Known-You No.2), B. rapa subsp. pekinensis cv. Autumn Sun), B. oleracea var. italica cv. Ching Hua), B. oleracea var. capitata cv. Green Peak), and Raphanus sativus cv. Snow Lady). However, inoculation of FC-1 and FC-2 on pepper (Capsicum annuum cv. Blue Star) resulted in severe necrosis on leaves and necrotic sunken spots on petioles and stems causing acute wilting and quick death. In stark contrast, FC-1 and FC-2 only induced local lesions on inoculated leaves of Chenopodium quinoa, Gomphrena globose, and Carica papaya. The infectivity of FC isolates to all plants used in host range tests were further confirmed by RT-PCR as mentioned. Oligonucleotide primers (Suppl. Table 1) specifically complementary to YoMV sequence were designed and used to amplify full-length genomic sequences of FC-1 and FC-2 isolates by RT-PCR and Sanger sequencing. Sequence analysis revealed that the genome of both FC-1 and FC-2 isolates consists of 6302 nucleotides. The viral genome has four open reading frames encoding a small replicase subunit, a RNA-dependent RNA polymerase , a movement protein, and a coat protein (CP), respectively. Both sequences, which shared 99.6% identity with each other, have been deposited in the NCBI database (Genbank Accession Numbers LC701592 and LC701593). FC-1 and FC-2 and the deduced amino acid sequences of CP shared 91.2% - 98.9% and 93.4 - 99.4% similarities, respectively, to those of published YoMV strains, confirming the identity of FC-1 and FC-2. RT-PCR analyses detected YoMV in all (100%) crossandra samples collected from the field (Suppl. Fig. 2). YoMV, formerly named Chinese rape mosaic virus (CRMV) or oilseed rape mosaic virus (ORMV) (Zhu et al., 2001), has been reported to infect cruciferous, solanaceous, and ornamental crops in Asia and Europe (Ju et al., 2019). The firecracker flower is a common and popular ornamental in Taiwan, even though its economic values are not so important. However, finding YoMV in firecracker flower may have epidemiological impacts as YoMV can infect economically important cruciferous and solanaceous crops. To our knowledge, this is the first report of YoMV infecting firecracker flowers in Taiwan.

Molecular analysis of human adenoviral keratoconjunctivitis cases: Results of a 2-year survey

Objective: This study aimed to determine the adenovirus genotypes and their epidemiological features between January 2018 and&#x0D; November 2019, in Istanbul, Turkey.&#x0D; Material and Methods: Conjunctival swab samples were obtained from patients who were clinically diagnosed with keratoconjunctivitis.&#x0D; Samples were screened with an Adeno Detector kit (Rapid Pathogen Screening, RPS Inc., South Williamsport, PA). Nucleic acid&#x0D; extraction and amplification were performed with the ADENOVIRUS ELITe MGB® kit in the ELITe In Genius instrument (Elitech&#x0D; Group, Torino, Italy). For subtyping of the strains, sequencing primers targeted the ‘Hypervariable Region 7’ (HVR-7) of the hexon&#x0D; gene were used. DNA sequence analysis (n:72) was performed with ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, USA),&#x0D; and subtyping was done by BLAST analysis.&#x0D; Results: The median viral load in the samples (n: 77) was 7 log10 copies/mL (IQR: 4.5-7.4 log10 copies/mL). The clinical finding score&#x0D; was found to be significantly higher in the high viral load group (Adenovirus DNA≥6 Log 10 copies/mL) than in the low viral load&#x0D; group (Adenovirus DNA

The Mutations on embA, embB and embC Gene Region in Ethambutol-Resistant and Susceptible Clinical Mycobacterium tuberculosis Complex Isolates

Ethambutol (EMB) is one of the first-line drugs used in the standard combination therapy for tuberculosis (TB) caused by Mycobacterium tuberculosis complex (MTC), and resistance to drugs that play a key role in treatment is increasing worldwide. Mutations in the embCAB operon that have been confirmed to be associated with resistance are responsible for EMB resistance. In this study, it was aimed to determine the frequency and patterns of mutations in embA, embB and embC gene regions in clinical MTC isolates found to be phenotypically resistant and susceptible to EMB. A total of 64 MTC isolates, 44 of resistant to EMB and 20 of susceptible to EMB, isoniazid, rifampicin, and streptomycin by conventional phenotypic drug susceptibility test, were included in the study. Following the DNA isolation, embA, embB and embC gene regions associated with EMB resistance were amplified with specific primer sequences. The PCR products were cycle sequenced using the Bigdye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, USA) and electrophoretically separated on the ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA). Mutated gene regions were identified by aligning sequence analysis data in multiple sequence analysis programs. In the study, genomic mutations in the embCAB operon were detected in 68.2% (30/44) of the EMB resistant isolates. Mutations in the embB gene region were detected in 66% (29/44) of the resistant isolates, 76% (22/29) of these mutations were at codon 306 and the most common mutation patterns in this codon were determined as ATG→GTG (M306V; 58.6%; 17/29), ATG→ATA, ATC or ATT (M306I; 17.2%; 5/29). Other mutations in the embB gene region were determined as Y334H (3.4%; 1/29), D354A (6.9%; 2/29), E378A (3.4%; 1/29), G406C (3.4%; 1/29), M423I (3.4%; 1/29) and E521A (3.4%; 1/29). Of the 44 EMB-resistant isolates, mutations were detected in one (2.3%) of the isolate in the embA gene region (L330L) and in two (4.5%) of the isolates in the embC gene region (T270I in one isolate and T270I and E305E in the other isolate). Of the phenotypically EMB susceptible isolates, mutation was detected in only one (5%) of the isolates in the embA gene region (E180G). In our study, it was determined that mutations frequently occur in codon 306 of the embB gene in EMB-resistant MTC isolates and this mutation has a potential role in the development of EMB resistance. However, it was concluded that the absence of mutations does not exclude phenotypic EMB resistance. Our results will shed light on the molecular epidemiology of embCAB operon mutations that cause EMB resistance in our country.

ALLELIC AND GENOTYPIC FREQUENCIES OF THE CSN-2 GENE IN A POPULATION OF DAIRY CATTLE IN THE NORTHERN REGION OF THE STATE OF TOCANTINS

The present study aimed to verify allelic and genotypic frequencies of the ß-casein genes in a population of dairy cattle in the north of the state of Tocantins in order to identify animals capable of producing ß-casein A2 in preference to its counterpart. To this end, five herds belonging to the cities of Araguaína, Arapoema, Araguatins, Colinas do Tocantins, and Palmas were selected, totaling 1,109 dairy cattle of mixed and/or Gir dairy breed. Samples for two markers of polymorphic regions were characterized and confirmed by real-time PCR, using an ABI Prism® 7500 sequence detection system (Applied Biosystems). Allelic and genotypic frequencies were determined using the TaqMan ™ detection system, in which the primer and probe emit different fluorescence signals for each allele of the polymorphism. The observed frequencies indicated a prevalence of the A2 allele (72.59%) compared to the A1 allele (27.41%). Considering the genotypic constitution of the animals, there was a greater correspondence for A2A2 (54.55%) compared to A1A1 (9.38%) and A1A2 (36.06%). This denotes a promising future for the development of economic activities focused on the production of type-A2 milk in the northern region of the state of Tocantins. Keywords: Beta casein A1. Beta casein A2. Bovine milk. Hypersensitivity.

Molecular and Genetic Study for Detection of Helicobacter pylori Virulence Genes among Patients with Diffuse Large B-Cell Lymphoma

Background: The bacteria Helicobacter pylori (Hp) has supposed to confirm an oncogenic consequence with the evolution of malignancy events in stomach, effectiveness of Hp consider a top risk factor of gastric-diffuse large B-cell lymphoma (DLBCL). Aims: the aim of this study was to observe the correlation between virulence factors, especially cytotoxin association gene A (CagA) and the progress of gastric DLBCL. Materials and Methods: Biopsy samples (50) involved in this study (28 were men, and 22 women, with a mean age up to 57.64 years), among gastric-DLBCL patients, with (25) control (were 14 men and 11 women) without DLBCL, tested at Medical City Teaching Hospital and Teaching Laboratories, study conducted from January 1st to September 30, 2022. Diagnosis was by molecular methods included DNA extraction for Hp by STR-Format kit (BIORON Diagnostics GmbHm In den Rauhweiden 20,67354 Germany), and “real-time polymerase chain reaction” methods using the (ABI PRISM 7700) Sequence Detection System, also applied the primers and probes, for Hp-CagA. Results: The mean of age was 55.7 ± 2 in DLBCL, while in control were 61.82 + 6, CagA +ve 66.66 + 2. In gastric DLBCL, the red blood cell count, white blood cell count, platelets count, and hemoglobin were all decline in DLBCL, not in the control group, lactate dehydrogenase levels in gastric DLBCL patients were rise (322 U/L), while less in control, while albumin (322g/dl) was less in gastric DLBCL patients. Hp 16S rRNA was detected relating to CagA variety, 66% (33/50) were CagA positive with highly significant in 23 patients with DLBCL (P = 0.004), and 10 in control patients. CagA gene expression was highly expression in gastric DLBCL patients with range up to (+), 8 (++), and 12 (+++), while in the control group was 5 (+), 2 (++), and 3 (+++), respectively. Conclusions: This present study fixed high frequency of CagA gene in gastric DLBCL patients. Moreover, confirmed this gene is significantly associated with gastric DLBCL in patients with Hp. Moreover, genotype EPIYA-ABC motif is related with the high risk of gastric DLBCL.