Genetic Analyzer Research Articles

Diagnosis of Olive Leaf Flay Daisenura oleae by PCR Technique in Nineveh Governorate

Through the PCR diagnostic technique, it found the Daisenura oleae, from the Cecidomyiidae family and the order Diptera. Olive leaf flay were characterized by their activity and continued presence during the activity season. The results of the electrophoresis of flys DNA, which was extracted from olive leaf adults. Obtained pure DNA, and the purity of the parasite DNA was confirmed by a successful operation. Electrophoresis using Agarose gel and showing the nucleic acid bundles clearly. The sequence of the nitrogenous bases of the samples (Cyanobacteria) that were under investigation was determined, as products were sent for PCR representation, and the sequence reading of the genes was completed by depending on the 3130 genetic analyzer that was equipped from the Japanese company Hitachi. BLAST software was used to compare the gene sequences that were sequenced with the gene sequences that were documented in the National Center for Biotechnology Information (NCBI). The findings of this comparison were then analysed. The products of the amplified packages resulting from the PCR reaction were sent outside Iraq to study the sequence and sequence of the nitrogenous bases and compare them with the original cytochrome c oxidase subunit I (cox1) gene stored in the Global Genome Bank with specific sites of the gene to be studied, and it was found that there are sites of variation or variation in the nucleotide sequence compared to with the original gene at the NCBI website, and the isolate match was 100%. The isolate was recorded in the Gen Bank with accession number LC757225.1, and the genetic tree was drawn to find out the relationship between the Iraqi insect isolate and the rest of the insect isolates, and the closest was the American isolate.

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.

Internal validation of the GA118-24B Genetic Analyzer, a stable capillary electrophoresis system for forensic DNA identification.

The GA118-24B Genetic Analyzer (hereafter, "GA118-24B") is an independently developed capillary electrophoresis instrument. In the present research, we designed a series of validation experiments to test its performance at detecting DNA fragments compared to the Applied Biosystems 3500 Genetic Analyzer (hereafter, "3500"). Three commercially available autosomal short tandem repeat multiplex kits were used in this validation. The results showed that GA118-24B had acceptable spectral calibration for three kits. The results of accuracy and concordance studies were also satisfactory. GA118-24B showed excellent precision, with a standard deviation of less than 0.1 bp. Sensitivity and mixture studies indicated that GA118-24B could detect low-template DNA and complex mixtures as well as the results generated by 3500 in parallel experiments. Based on the experimental results, we set specific analytical and stochastic thresholds. Besides, GA118-24B showed superiority than 3500 within certain size ranges in the resolution study. Instead of conventional commercial multiplex kits, GA118-24B performed stably on a self-developed eight-dye multiplex system, which were not performed on 3500 Genetic Analyzer. We compared our validation results with those of previous research and found our results to be convincing. Overall, we conclude that GA118-24B is a stable and reliable genetic analyzer for forensic DNA identification.

Concordance study on Y-STRs typing between SeqStudio™ genetic analyzer for HID and MiSeq™ FGx forensic genomics system

BackgroundMassively Parallel Sequencing (MPS) allowed an increased number of information to be retrieved from short tandem repeat (STR) analysis, expanding them not only to the size, as already performed in Capillary Electrophoresis (CE), but also to the sequence. MPS requires constant development and validation of the analytical parameters to ensure that the genotyping results of STRs correspond to those obtained by CE. Given the increased frequency of usage of Y-STRs as supplementary markers to the autosomal STRs analysis, it is urgent to validate the concordance of the typing results between CE and MPS analyses.Methods and resultsDNA extracted from 125 saliva samples of unrelated males was genotyped using Yfiler™ Plus PCR Amplification Kit and ForenSeq™ DNA Signature Prep Kit, which were analyzed by SeqStudio™ Genetic Analyzer for HID and MiSeq™ FGx Forensic Genomics System, respectively. For each shared Y-STR, allele designation, number of length- and sequence-based alleles per locus, stutter percentage, and the intra-locus balance of multicopy Y-STRs were screened.ConclusionsAlthough the number of forensic genetics laboratories that are applying the MPS technique in routine analysis is small and does not allow a global assessment of MPS limitations, this comparative study highlights the ability of MPS to produce reliable profiles despite the generation of large amounts of raw data.

A comparison of six adhesive tapes as tape lifts for efficient trace DNA recovery without the transfer of PCR inhibitors

Tape-lifting is a non-destructive method employed in the laboratory to recover and collect trace DNA evidence from crime scene exhibits with porous surfaces. The success of tape-lifting is a balance between capturing the biological material and compatibility with downstream DNA extraction processes to ensure efficient release of the tape-lifted material during DNA extraction. In this study, six commercially available low-, regular- and high-tack adhesive tapes were evaluated. The low-tack S183 tape and the highly adhesive S-Hold tape were compared for DNA recovery efficiency from different materials commonly encountered in casework. All tape-lifts were processed using PrepFiler Express™ BTA and AutoMate Express™ Forensic DNA extraction systems, DNA samples quantitated by Quantifiler TRIO, amplified using Powerplex® 21 and VeriFiler™ PLUS (VFP), and analysed on a 3500xl genetic analyser to evaluate the quality of the resultant STR profiles obtained.The more adhesive S-Hold tape recovered comparable or more DNA than the low-tack S183 tape from the majority of materials tested. However, STR profiles obtained from S183 tape-lifts were of markedly higher quality compared to S-Hold tape-lifts. This was most evident for towel, denim and printed chiffon, where S-Hold samples exhibited severe PCR inhibition, with VFP internal quality markers confirming the presence of inhibitors. The findings suggest that strong adhesion is not necessarily beneficial for tape-lifting, as the low tack S183 tape was able to efficiently recover cellular material from the surface of porous substrates commonly encountered in casework, while avoiding the co-transfer of PCR-inhibitory substances from the sampled material.

Development of Multiplex Polymerase Chain Reaction (PCR)-Based MSA Assay for Bladder Cancer Detection.

Several studies have shown that microsatellite changes can be profiled in the urine to detect bladder cancer. Microsatellite analysis (MSA) of bladder cancer detection requires a comprehensive analysis of up to 15-20 markers based on amplifying and interpreting many individual MSA markers, which can be technically challenging. To develop fast, efficient, standardized, and less costly MSA to detect bladder cancer, we developed three multiplex polymerase chain reaction (PCR) based MSA assays, all of which were analyzed by a genetic analyzer. First, we selected 16 MSA markers based on nine publications. We developed MSA assays based on triplet or three-tube-based multiplex PCR (Triplet MSA assay) using samples from Johns Hopkins University (JHU Sample, first set of samples). In the second set of samples (samples from six cancer patients and fourteen healthy individuals), our Triplet Assay with 15 MSA markers correctly predicted all 6/6 cancer samples to be cancerous and 14/14 healthy samples to be healthy. Although we could improve our report with more clinical information from patient samples and an increased number of cancer patients, our overall results suggest that our Triplet MSA Assay combined with a genetic analyzer is a potentially time- and cost-effective genetic assay for bladder cancer detection and has potential use as a dependable assay in patient care.

Genetic diversity and phylogenetic relationship of Angus herds in Hungary and analyses of their production traits.

This study aims to investigate the genetic structure and characteristics of the Angus cattle population in Hungary. The survey was performed with the assistance of the Hungarian Hereford, Angus, Galloway Association (HHAGA). Genetic parameters of 1,369 animals from 16 Angus herds were analyzed using the genotyping results of 12 microsatellite markers with the aid of PowerMarker, Genalex, GDA-NT2021, and STRUCTURE software. Genotyping of DNA was performed using an automated genetic analyzer. Based on pairwise identity by state values of animals, the Python networkx 2.3 library was used for network analysis of the breed and to identify the central animals. The observed numbers of alleles on the 12 loci under investigation ranged from 11 to 18. The average effective number of alleles was 3.201. The overall expected heterozygosity was 0.659 and the observed heterozygosity was 0.710. Four groups were detected among the 16 Angus herds. The breeders' information validated the grouping results and facilitated the comparison of birth weight, age at first calving, number of calves born and productive lifespan data between the four groups, revealing significant differences. We identified the central animals/herd of the Angus population in Hungary. The match of our group descriptions with the phenotypic data provided by the breeders further underscores the value of cooperation between breeders and researchers. The observation that significant differences in the measured traits occurred among the identified groups paves the way to further enhancement of breeding efficiency. Our findings have the potential to aid the development of new breeding strategies and help breeders keep the Angus populations in Hungary under genetic supervision. Based on our results the efficient use of an upcoming genomic selection can, in some cases, significantly improve birth weight, age at first calving, number of calves born and the productive lifespan of animals.

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.

Search for associations of bull sperm quality with <i>ESR1</i> gene polymorphism

Materials and methods. The semen of 53 bulls was collected at OJSC Nevskoe. A total of 110 bull semen samples were analyzed. Sperm quality was determined using Argus-CASA (ArgusSoft, Russia). Membrane integrity was determined by staining the samples with nigrosine-eosin dye (Diam, Russia) and a Motic BA 410 microscope. Spermatozoa respiration was determined using an Expert-001 instrument. The functional state of the energy system was assessed by the reaction of respiration to the addition of the uncoupler of respiration and phosphorylation, 2,4 dinitrophenol (2,4-DNF). DNA for genetic analysis was isolated from semen by the phenol-chloroform method. Sanger sequencing was performed on an Applied Biosystems 3500 Genetic Analyzer using commercial BigDye® Terminator v3.1 Sequencing Standard Kits (Applied Biosystems) according to the manufacturer's protocol.Results. Sperm quality were characterized by high individual variability. Thus, the volume of the ejaculate was from 2 to 15 ml, the concentration of spermatozoa was from 0.6 to 1.7 billion/ml, the total number of spermatozoa in the ejaculate was from 1.6 to 15 billion, and progressive motility was from 0 to 85%. Four SNPs were identified for the ESR1 gene. No significant associations of ESR1 gene polymorphism were found, except for a significant association of ESR1 665 G>C with spermatozoa concentration and the number of swollen acrosomes

Standardization of Euphorbia tithymaloides (L.) Poit. (Root) by Conventional and DNA Barcoding Methods.

Adulteration and substitution of medicinal plants have become a matter of great concern in recent years. Euphorbia tithymaloides is one such medicinal plant that has gained importance but is often confused with other plants of the same species. In order to address this issue, this study aimed to conduct a conventional and molecular pharmacognostic study for the identification of the root of E. tithymaloides. The root of the plant was studied for the macroscopic observations, and then, the root was ground into coarse powder for microscopic studies and to determine the physiochemical properties. The powder was subjected to extraction with solvents such as ethanol, ethanol/water (1:1), hexane, and ethyl acetate. The extracts were then used for qualitative and quantitative (phenol, alkaloids, and flavonoids) phytochemical analysis. The molecular study was performed with the DNA barcoding technique. The DNA was extracted from the root of the plant, and its purity was examined by gel electrophoresis (1% w/v). The DNA was then amplified using an Applied Biosystems 2720 thermal cycler for the rbcL, matK, and ITS primers. The amplified primers were sequenced with a 3130 Genetic Analyzer, and the generated sequences were searched for similarity in the GenBank Database using the nucleotide BLAST analysis. The micro- and macroscopic studies revealed the morphological and organoleptic characters as well as the presence of medullary rays, fiber, cork, sclereids, parenchymal cells, and scalariform vessels. The physiochemical properties were found within the limit. The phytochemical analysis revealed the presence of terpenoids, flavonoids, saponins, and alkaloids. In addition, the alkaloidal content was high in the ethanol extract (63.04 ± 3.08 mg At E/g), while the phenol content was high in the hexane extract (10.26667 ± 1.77 mg At E/g), and the flavonoid content was high in the ethyl acetate extract (41.458 ± 1.33 mg At E/g). After the BLAST analysis from the GenBank database, the rbcL, ITS, and matK primers showed a similarity percentage of 99.83, 99.84, and 100. The phylogenetic tree for the species closest to each primer was generated using the MEGA 6 software. The matK loci had the highest percentage similar to the rbcL and ITS loci, indicating that the matK loci can be used to identify the root of E. tithymaloides as a standalone. The results from this study can be used to establish a quality standard for E. tithymaloides that will ensure its quality and purity.

Evaluation of a novel real-time polymerase chain reaction assay for identifying H3 equine influenza virus in Kazakhstan.

Equine influenza (EI) is a highly contagious disease that causes fever and upper respiratory tract inflammation. It is caused by influenza virus A, belonging to the Orthomyxoviridae family, with subtypes H3N8 and H7N7. This study presents data on the development of a real-time polymerase chain reaction (RT-PCR) assay using TaqMan probes to detect the H3 subtype of EI virus (EIV). The evaluation of the developed RT-PCR assay involved five strains of EIV as positive controls and ten nasopharyngeal swab samples collected from horses. RNA was isolated using the GeneJet Viral DNA and RNA Purification Kit, and primers and probes were designed using the Integrated DNA Technology PrimerQuest Tool. The assay was optimized by investigating the annealing temperature, primer and probes concentrations, sensitivity, and specificity. Sequencing was performed using the Thermo Fisher 3130 Genetic Analyzer, and the evolutionary history was inferred using the Neighbor-Joining method. The designed primers and probes, targeting the H3 gene, were found to be specific to the EIV. The RT-PCR assay was capable of detecting as low as 50 femtogram (f) or 3 × 103 copies of genomic RNA. No cross-reactions were observed with other respiratory viral and bacterial pathogens, indicating the high specificity of the assay. To evaluate its effectiveness, ten nasopharyngeal swab samples collected from farms in North Kazakhstan regions during disease monitoring were analyzed. The accuracy of the analysis was confirmed by comparing the results with those obtained from a commercial RT-PCR assay for EI identification. The developed RT-PCR assay exhibited high sensitivity and specificity for detecting the EIV. The results demonstrate that the developed RT-PCR assay is suitable for diagnosing EI. This simple, highly sensitive, and specific assay for detecting H3 EIV can be a reliable tool for diagnosing and surveilling EI. Implementing this RT-PCR assay in veterinary practice will enhance and expedite the timely response to potential outbreaks of EI, thus positively impacting the overall epizootic well-being of EI in Kazakhstan.

Detection of Human Boca Virus in Gastric Adenocarcinoma.

Background: Gastric cancer is one of the most common cancers worldwide. Human bocavirus (HBoV), a recently isolated virus, has been investigated for its role in many respiratory and enteric diseases. Few studies have reported its presence in solid tumors, such as lung and colon cancers. The aim of this study was to detect the presence of the HBoV1 genome in gastric adenocarcinoma, which has not yet been evaluated. Methods: Formalin-fixed paraffin-embedded (FFPE) blocks of 189 gastric tumors and 50 blocks of non-tumor gastric tissue products from elective weight reduction operations were collected. DNA extraction and real-time polymerase chain reaction (PCR) were performed for HBoV1 detection. DNA sequencing was performed using ABI Genetic Analyzer series 3500. Results: The mean age of the patients was 60±13.33 years. Tumors were more common in males than females (2.5/1). HBoV1 PCR was positive in 34 (18%) cases of GC and 10 (20%) cases of chronic gastritis (P>0.05). There was no association between age, sex, stage, and histologic subtype of the tumor and HBoV1 positivity (P>0.05) in tumor samples. The rate of intestinal metaplasia and presence of lymphoid stroma were also not more frequent in HBoV1-positive tumors (P>0.05). Conclusion: The HBoV1 can be detected in a relatively high proportion of Iranian patients with gastric cancer (18%) with no predilection for specific subtypes and no association with the degree of lymphocytic infiltration. HBoV1 can also be observed in approximately 20% of chronic gastritis cases. Further comprehensive studies are needed to elucidate the role of HBoV1 in gastric cancer development.

Combination of a robotic solution pipetting device with a centrifugal molecular diagnostic platform for fully automatic respiratory infectious virus detection from nasopharyngeal swab samples

A novel molecular diagnostic platform is presented that enables complete automation for the genetic analysis of respiratory infectious virus including the solution loading, RNA purification, reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and real-time fluorescence detection. The overall structure consists of three parts: an integrated centrifugal microfluidic disc, a point-of-care (POC) genetic analyzer, and a robotic solution pipetting device. The integrated centrifugal microfluidic disc was designed to treat up to four samples simultaneously, with the capacity to detect seven targets in a single run. Additionally, a solution-loading cartridge is combined on the disc, facilitating the insertion of a nasopharyngeal swab sample as well as the storage and aliquoting of the washing solution, the elution solution, and the RT-LAMP master mix. The POC genetic analyzer is composed of a spinning motor for the automatic release of solutions, a couple of peltier heaters for gene amplification, a miniaturized fluorescence detector to generate an amplification curve, and an LED screen for displaying the experimental parameters and the diagnostic results. Importantly, we also integrate a robotic solution pipetting device with the POC genetic analyzer so that the designated solution can be loaded into the solution-loading cartridge without need of manual operation. Five targets (influenza A subtypes H1N1 and H3N2, influenza B, and SARS-CoV-2 (orf1ab and S gene)) of four samples were identified in 1.5 hr on a single device with a sample-in-answer-out manner.

Short Tandem Repeat (STR) Profiling of Earwax DNA Obtained from Healthy Volunteers.

The present study aimed to establish human earwax as a potential source of DNA evidence that could be effectively used in human identification. Sixty earwax samples were obtained from 15 healthy male and female Saudi volunteers living in Riyadh, Saudi Arabia. Four consecutive earwax swab samples were obtained from each volunteer and stored for 1, 15, 30 and 60 days. Earwax samples were stored at room temperature (20-22 °C). Reference oral swab was also taken from each volunteer. DNA was extracted by QIAamp DNA Mini kit and quantified by real-time polymerase chain reaction (RT-PCR) on 7500 Thermal Cycler. Autosomal STR loci were amplified using AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). Amplified fragments were size separated and analyzed on a 3500 Genetic Analyzer. Complete autosomal STR profiles were obtained from the earwax swabs of all the volunteers stored up to 30 days after the collection. Some STR profiles were partially obtained 60 days after the earwax collection. Allelic drop-out, allelic drop-in, and stutters were seen in earwax samples analyzed 60 days after the collection. The results have shown that human earwax can be a potential source of DNA evidence for human identification up to 30 days after the earwax collection. It is recommended to quickly analyze earwax samples or store them at room temperature or at -10 °C after their recovery from the crime scene.

Dissecting shared genetic architecture between obesity and multiple sclerosis.

Observational studies have associated obesity with an increased risk of multiple sclerosis (MS). However, the role of genetic factors in their comorbidity remains largely unknown. Our study aimed to investigate the shared genetic architecture underlying obesity and MS. By leveraging data from genome-wide association studies, we investigated the genetic correlation of body mass index (BMI) and MS by linkage disequilibrium score regression and genetic covariance analyser. The casualty was identified by bidirectional Mendelian randomisation. Linkage disequilibrium score regression in specifically expressed genes and multimarker analysis of GenoMic annotation was utilised to explore single-nucleotide polymorphism (SNP) enrichment at the tissue and cell-type levels. Shared risk SNPs were derived using cross-trait meta-analyses and Heritability Estimation from Summary Statistics. We explored the potential functional genes using summary-data-based Mendelian randomization (SMR). The expression profiles of the risk gene in tissues were further examined. We found a significantly positive genetic correlation between BMI and MS, and the causal association of BMI with MS was supported (β=0.22, P=8.03E-05). Cross-trait analysis yielded 39 shared risk SNPs, and the risk gene GGNBP2 was consistently identified in SMR. We observed tissue-specific level SNP heritability enrichment for BMI mainly in brain tissues for MS in immune-related tissues, and cell-type-specific level SNP heritability enrichment in 12 different immune cell types in brain, spleen, lung, and whole blood. The expressions of GGNBP2 were significantly altered in the tissues of patients with obesity or MS compared to those of control subjects. Our study indicates the genetic correlation and shared risk genes between obesity and MS. These findings provide insights into the potential mechanisms behind their comorbidity and the future development of therapeutics. This work was funded by the National Natural Science Foundation of China (82171698, 82170561, 81300279, and 81741067), the Program for High-level Foreign Expert Introduction of China (G2022030047L), the Natural Science Foundation for Distinguished Young Scholars of Guangdong Province (2021B1515020003), Natural Science Foundation of Guangdong Province (2022A1515012081), the Foreign Distinguished Teacher Program of Guangdong Science and Technology Department (KD0120220129), the Climbing Programme of Introduced Talents and High-level Hospital Construction Project of Guangdong Provincial People's Hospital (DFJH201803, KJ012019099, KJ012021143, and KY012021183), and in part by VA Clinical Merit and ASGE clinical research funds (FWL).

First report of a ring corneal ulcer caused by Cladorrhinum bulbillosum posing a diagnostic debacle: A case report

A young diabetic male patient presented with a ring infiltrate and hypopyon of 1-day onset in the left eye. Though we suspected acanthamoeba keratitis, the initial smear report showed fungal filaments. So, we started dual topical with systemic antifungals. On day 3, the ring was filled with a reddish-brown infiltrate, and the culture grew pale-gray fungal colonies. A clinical diagnosis of dematiaceous fungi was made, commonly observed at our clinic is Curvularia, which responds to the chosen treatment combination. Due to clinical worsening, we considered therapeutic penetrating keratoplasty that got delayed until diabetic control. With further worsening, we re-scraped with a day off medication and performed confocal microscopy and polymerase chain reaction (PCR). The organism was identified as Cladorrhinum bulbillosum from the DNA extract from colonies grown in blood agar when subjected to PCR-based sequencing carried out in the ABI 3130 genetic analyzer. The patient underwent therapeutic penetrating keratoplasty on day 11.

Paternity testing using 21 STR Loci in a biotechnology approach: case of Rwandan Population

INTRODUCTION: We focused on a sample size of 141 unrelated Rwandan persons to genotype21 STR loci that were relied up in establishing allele frequencies, heterozygosity and power ofexclusion. This study aims at exploring allele frequencies on a representative sample from Rwandanpopulation to determine probability of paternity for sampled families basing on polymorphic STRsloci, using 21 autosomal-STR loci by Genetic Analyzer 3500X.METHODS: This was an experimental study and global filer TM Express PCR Amplification kit wasused to amplify 21 autosomal STR loci.RESULTS: The total number of observed alleles was 270; the largest number of different alleleswas seen in SE33 and D18S51 loci. The locus with the highest heterozygosity was SE33, while locusTH01 had the lowest heterozygosity. The heterozygosity of the 21 STR loci ranged from 71.3%(TH01) to 91.6% (SE33) with an average of 81.1% a good indicator of high genetic variability. Forall microsatellites analyzed the power of exclusion ranged from 43.4% (TH01) to 78.1% (SE33)with an average of 58.2%. For seven of eight cases examined in paternity test alleged father wasnot excluded as biological father of child. The results found in examination of case 8 indicated thatthe alleged father was not the biological father of the child.CONCLUSION: Based on calculated statistical parameters, the population of Rwanda may usethese 21 STR loci as a vital tool for forensic identification and paternity testing.

Molecular detection of mononucleotide biomarkers of microsatellite instability in sporadic colorectal carcinoma patients with clinicopathological correlation

Objectives: To identify the frequency and types of microsatellite instability among a group of sporadic CRC patients and to correlate the findings with clinicopathological characteristics.
 Methods: During an 8-month period, all patients with sporadic CRC who attended to two teaching hospitals in Baghdad, Iraq were recruited to this cross-sectional study regardless of age, sex, ethnicity, or tumor characteristics. Demographic, clinical, and histopathological features were recorded. DNA was extracted from FFPE-blocks of the resected tumors and normal tissues. PCR amplification of five microsatellite mononucleotide repeat loci (BAT25, BAT26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide repeat control markers (Penta C and Penta D) was performed to determine the MSI status. Capillary electrophoresis and Genetic Analyzer 3500 (Applied Biosystem, Japan) were used to separate and examine the products. Data were analyzed by Genescan software (Promega, USA). Instability of two or more loci is considered MSI-H.
 Result: In this study, ages of the 45 recruited patients ranged between 20-80 years, with a mean±SD of 55±12.3 years; of them, 31(68.9%) were ≥50 years; 25 (55.6%) were males. Rectal bleeding was the most frequent presenting feature [22 (48.9%)] patients; 23 (51.1%) of CRCs were located at recto-sigmoid region, 29 (64.4%) were T3 tumors, 34(75.5%) were non-mucinous adenocarcinoma, 39(86.7%) were moderately differentiated, 17 (37.8%) patients had stage III tumors; and 25 (55.5%) had lymphovascular invasion. MSI-H was seen in 5/45 (11.1%) patients; 3(60%) of them were ≥50 years, 4(80%) were males, 3(60%) were smokers, 2 (40%) presented with intestinal obstruction and altered bowel habits each; 4(80%) had T3 tumors, 3(60%) had mucinous adenocarcinomas [p=0.004], 2(40%) had stage II tumor and stage III each.
 Conclusion: The frequency of MSI-H among the recruited patients with CRC was 5/45 (11.1%) and it was significantly associated with mucinous adenocarcinoma subtype. NR-24 and NR-21 were the most prevalent instable markers.

Analysis of 27 STR markers of the chromosome Y in the population of the jungle from Peru

Y-chromosome-specific short tandem repeat (Y-STR) markers reside on the non-recombinant portion of the Y chromosome, their inheritance is paternal, they are not detectable in female DNA. These properties make Y-STRs a useful tool in forensic investigations such as sexual assault, parenting, and other violent crimes; likewise they are also useful in genealogical and evolutionary studies. The objective of the research is to expand the scientific evidence of the distribution by region or country of the Y sex chromosome haplotypes. Similar studies in Peruvian populations are scarce due to the smaller number of Y-STR polymorphisms frequently used in Forensic and Population Genetics.Material and method: In the investigation, 141 DNA samples from the jungle of Peru were analyzed, of which comprised of 104 samples from Iquitos region (Loreto), 29 samples from Awajun (Amazonas) and 8 samples from Tambopata (Madre de Dios). The samples were processed using direct PCR with the Yfiler™ Plus PCR Amplification kit for 27 STRs, the amplified products were analyzed by capillary electrophoresis on the Applied Biosystem™ 3500XL Genetic Analyzer, and the data obtained was imported into the GeneMapper® ID-X v1.5 software to generate the genetic profiles. With the results obtained, the statistical analysis and the population structure were carried out.Results: Of the 141 samples, 106 unique haplotypes were observed. Gene diversities for each Y-STR marker ranged from .317 to .919. The haplotype diversity for the total sample was .9906. This study supports that the Y-STR haplotypes in this population are highly polymorphic in the analyzed population and, therefore, are very useful in forensic studies of human identification, as well as in population genetics when investigating groups or individuals from Latin America.

HLA-G susceptibility to hepatitis B infection and related hepatocellular carcinoma in the Japanese population

AimsHuman leukocyte antigen (HLA)-G plays a role in various physiological immunomodulatory functions. Aberrant HLA-G expression is observed in various disease states, including tumors, autoimmune disorders, and viral infections. The present study investigated the association between HLA-G functional gene polymorphisms (rs1736933 [-486 C > A], rs1049033 [+2018 C > T], 14 bp Insertion [Ins]/Deletion [Del] [+2961 Del > Ins], and rs1063320 [+3142 C > G]) and disease susceptibility, hepatocellular carcinoma (HCC) development, and hepatitis B surface antigen (HBsAg) clearance. MethodsAllele discrimination of the 3 SNPs (-486 C > A, +2018 C > T, +3142 C > G) was determined by a TaqMan 5′ exonuclease assay, while the 14 bp Ins/Del polymorphism was typed by fragment analysis using Genetic Analyzer and GeneMapper software. The above polymorphisms were analyzed for 325 Japanese hepatitis B virus (HBV) patients, 355 Japanese healthy subjects (Controls) as healthy controls, and 799 Japanese hepatitis C virus (HCV) patients as disease controls, respectively. ResultsThe 14 bp Insertion allele was significantly more frequent in HBV patients than Controls (27.1 % vs 20.6 %, odds ratio [OR] 1.43, P = 0.005) but did not differ between HCV patients and Controls. Similar results were found for the rs1063320 G allele (38.9 % vs 26.3 %, OR 1.78, P < 0.001) and the rs1736933 T allele (32.2 % vs 26.9 %, OR 1.29, P = 0.034) between HBV and Controls. The rs1049033 T allele showed a weak but significant association with HCC development in the dominant model (OR 1.95, P = 0.04). Regarding HBsAg clearance, the A allele at rs1736933 was significantly correlated in the recessive model (OR 3.23, P = 0.003). ConclusionsThis study revealed significant associations of HLA-G gene polymorphisms with disease susceptibility, HCC development, and HBsAg clearance in HBV patients.