Polymerase Chain Reaction Products Research Articles

Detection of Mycoplasma spp. from snakes from five different families

BackgroundMycoplasmas are an important cause of respiratory diseases in tortoises. In snakes, evidence of mycoplasma infections has been found almost exclusively in pythons. To better understand the occurrence of these bacteria in other snake species, samples submitted for routine testing for respiratory pathogens were also tested for mycoplasma by polymerase chain reaction (PCR). A total of 640 samples (mostly oral swabs) from snakes of 5 different families (Boidae n = 114, Colubridae n = 109, Elapidae n = 34, Pythonidae n = 301 and Viperidae n = 82) were included in the study. A genus-specific PCR (PCR1) developed for the detection of Mycoplasma [Mycoplasmopsis] agassizii and a pan-mycoplasma PCR (PCR2) were used. PCR products were sequenced for validation and phylogenetic analysis was performed. The sampled animals were from various owners and collections, all in human care at the time of sampling. Clinical background information was not provided.ResultsUsing PCR1, mycoplasmas were detected in 175 (175/640, 27%) samples (Boidae: 7/114, 6%; Colubridae: 3/109, 3%; Elapidae: 8/34, 24%; Pythonidae: 155/301, 51%; Viperidae: 2/82, 2%). A higher percentage of positive results were obtained using PCR2 (258/640, 40%; Boidae: 9/114, 8%; Colubridae: 25/109, 23%; Elapidae: 19/34, 56%; Pythonidae: 172/301, 57%; Viperidae: 33/82, 40%). The detected bacteria can be divided into at least 6 genetically diverse clusters representing different genera and species based on multiple sequence alignment and phylogenetic analysis.ConclusionsThese results show that diverse mycoplasmas are found in pythons and other snakes. Further investigations are necessary to evaluate the role of various mycoplasmas in respiratory diseases in snakes.

Epidemiology analysis of carbapenemase-producing Escherichia coli in a hospital in Henan Province from 2021 to 2023

Objective: To analyze the epidemiological characteristics of drug resistance genes of carbapenemase-producing Escherichia coli (CPECO) in Henan Province Hospital of Traditional Chinese Medicine from 2021 to 2023, providing data support and theoretical basis for controlling nosocomial infections of CPECO. Methods: Using a cross-sectional study, 30 carbapenem-resistant Escherichia coli (CRECO) strains confirmed by VITEK-2 Compact identification and drug sensitivity test in the Clinical Microbiology Laboratory of Henan Province Hospital of Traditional Chinese Medicine from 2021 to 2023 were tested, using carbapenemase inhibitor enhancement test to conduct preliminary screening of carbapenemases, and colloidal gold immunochromatography and polymerase chain reaction (PCR) were used to determine the phenotypes and genotypes of common carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA) respectively, and the genotypes (blaSHV, blaTEM, blaCTX) of common extended Spectrum beta-lactamases (ESBL) were confirmed using PCR. The PCR amplification products of carbapenemase and ESBL positive strains were Sanger-sequenced, and the sequencing products were compared on the Blast website to determine the exact carbapenemase and ESBL genotypes. Sequence typing (ST) was performed on CPECO using the Achtman multi-locus sequence typing scheme to determine the cloning relationship between different strains. Results: A total of 21 CPECO strains were screened. Drug sensitivity test results showed that CPECO strains showed widespread drug resistance, with the resistance rate to monocyclic (aztreonam) and trimethoprim/sulfamethoxazole being over 60%(16/21, 14/21), and the resistance rate to other antibacterial drugs being 100%. Only the sensitivity to aminoglycosides and fosfomycin remained relatively high, and no strains resistant to tigecycline and colistin were found. Colloidal gold immunochromatography detected 18 blaNDM types, 2 blaKPC types, and 1 blaIMP type. Sequencing of drug resistance gene PCR products classified 17 blaNDM-5 strains, 1 blaNDM-4 strain, 2 blaKPC-2 strain, and 1 blaIMP-4 strain, which were completely consistent with the results of screening test and colloidal gold immunochromatography. ESBL resistance gene testing showed that the detection rate of blaTEM was 42.9%(9/21), blaCTX-M was 33.3%(7/21), and blaSHV was 4.8%(1/21). The rate of blaNDM producing CPECO carrying both ESBL resistance genes was 27.8%(5/18). The MLST typing results revealed 11 sequence types (STs), including one ST155 clonal complex and nine singleton STs. Among these, there were seven strains of ST167, five strains of ST410, and one strain each of ST58, ST68, ST69, ST93, ST131, ST155, ST648, ST1114, and ST3268. Conclusion: The main resistance mechanism identified in this study for CPECO was the production of blaNDM-5 carbapenemase, with a high proportion of strains also carrying blaTEM-1D and/or blaCTX-M-15 ESBLs. MLST typing found that the epidemic strain of CPECO showed certain polymorphism, but there were clonal transmission of multiple clonal complexes between ST167 and ST410.

Genotype and Haplotype Analysis With In Silico Prediction of TMPRSS2 Gene in Jordanian Population.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global health concern. The entry of the virus into host cells is facilitated by the transmembrane protease serine 2 (TMPRSS2) receptor, and genetic variations in the TMPRSS2 gene may influence disease susceptibility. However, there is a lack of knowledge regarding TMPRSS2 genetic variants and haplotypes in the Jordanian population. This study aimed to characterize the genotype and haplotype variations in the TMPRSS2 binding domain with SARS-CoV-2 among Jordanian volunteers. The binding domain of TMPRSS2 with SARS-CoV-2 (Exons 9 and 10) was amplified using polymerase chain reaction (PCR) for a random sample of 120 healthy unrelated Jordanian volunteers, followed by Sanger DNA sequencing for the PCR products. The effect of the novel genetic variants on the TMPRSS2 protein structure was predicted using in silico methods. The results showed significant (p<0.05, chi-square) allele frequencies for known TMPRSS2 variants, with c.888C>T being the most prevalent among Jordanian volunteers. Novel genetic variants, including c.869A>G and c.923T>A, were also identified, with the latter being the most common novel variant. Haplotype analysis showed that the most prevalent TMPRSS2 haplotype is c.911G/1051A/1052T/1010+45C/1011-38T/1011-52C/1011-54A. In silico programs predicted that TMPRSS2 c.923T>A and c.1052T>A variants affect transmembrane proteins and catalytic sites. This research provides information about the gene structure of the TMPRSS2 binding domain in Jordanians. Some of the identified variants, especially c.923T>A, may influence protein function, warranting further in vitro and in vivo investigations. In addition, further clinical research studies are needed to link the identified TMPRSS2 variants with COVID-19 susceptibility and severity among Jordanians.

Inert splint-driven oligonucleotide assembly.

In this study, we introduce a new in vitro method for oligonucleotide fragment assembly. Unlike polymerase chain assembly and ligase chain assembly that rely on short, highly purified oligonucleotides, our method, named Splynthesis, uses a one-tube, splint-driven assembly reaction. Splynthesis connects standard-desalted "contig" oligos (∼150 nt in length) via shorter "splint" oligos harboring 5' and 3' blocking modifications to prevent off-target ligation and amplification events. We demonstrate the Splynthesis method to assemble a 741-bp gene fragment. We verify the assembled polymerase chain reaction product using standard molecular biology techniques, as well as long-read Oxford Nanopore sequencing, and confirm that the product is cloneable via molecular means, as well as Sanger sequencing. This approach is applicable for synthetic biology, directed evolution, functional protein assays, and potentially even splint-based ligase chain reaction assays.

Acinetobacter baumannii Co-Resistant to Extended Spectrum Beta-Lactamases and Carbapenemases in Six Peruvian Hospital Centers

Objectives: The aim of this study was to describe the phenotypic and molecular characteristics of Acinetobacter baumannii isolates carrying resistance genes to beta-lactams and carbapenems in six Peruvian public hospital centers. Materials and methods: The susceptibility of bacterial isolates was determined using the automated MicroScan system, with interpretation according to the M100 S30 CLSI 2020. Resistance genes were identified by conventional polymerase chain reaction (PCR), and PCR products were visualized by 1% agarose gel electrophoresis. Results: Nine strains (TRU1, PM1, PM2, CUS1, CUS2, CUS3, CAL1, CAL2 and CAL3) out of a total of 21 strains in the study were reactivated, showing resistance of 77.8% to imipenem, ciprofloxacin and cefepime, followed by 66.7% resistance to meropenem and ceftazidime, indicating marked multidrug resistance. In addition, the detection of the group A beta-lactamase genes blaCTX-M and blaTEM was confirmed, showing co-resistance in strains CUS1, CUS2 and CUS3, despite their unusual presence in this pathogen, also determined by the presence of the group D carbapenemase blaOXA in strain CUS3, the only strain to show co-resistance of the three groups. Conclusion: The prevalence of Acinetobacter baumannii resistant to extended-spectrum beta-lactamases and carbapenemases in Peruvian public centers represents a critical challenge for the treatment of infections. Rigorous surveillance, infection control strategies, and the development of alternative therapies are urgently needed to address this growing bacterial resistance.

Genomic Identification of Genital Ureaplasma urealyticum and Ureaplasma parvum Infection by Duplex Polymerase Chain Reaction in Symptomatic Women Attending a Tertiary Care Hospital: A Cross-sectional Study

Introduction: Ureaplasma urealyticum (U. urealyticum) and Ureaplasma parvum (U. parvum) are the two causative agents for Sexually Transmittable Diseases (STDs), they are often accompanied by Pelvic Inflammatory Disease (PID), vaginitis, endometriosis, etc. Aim: To detect the prevalent genotypes/biovars of Ureaplasma species by using Duplex Polymerase Chain Reaction (PCR) in symptomatic women attending the Obstetrics and Gynaecology clinic of a tertiary care hospital. Materials and Methods: This was a cross-sectional study, comprised of 200 symptomatic women aged from 18 to 45 years. This study was carried out in the Department of Microbiology, School of Allied Health Sciences, Department of Obstetrics and Gynaecology, Mahatma Gandhi Medical College and Research Institute, and Mahatma Gandhi Medical Advanced Research Institute, Sri Balaji Vidyapeeth, Puducherry, India for the period of January 2021 to January 2024. Duplex PCR was performed for the presence of U. urealyticum and U. parvum in endocervical samples. Amplified PCR products were analysed and the positive PCR products were sent for sequencing and the sequenced results were compared with GenBank and phylogenetic analysis was constructed. Data was analysed using Chi-square tests to test level of significance between the two groups (p-value≤0.05). Results: The overall results found that 48 (24%) of symptomatic women were positive for Ureaplasma species. In Duplex PCR, 24 (12.0%) were positive for both urease and upv genes; followed by 15 (7.5%) for urease and 9 (4.5%) for upv genes only. The phylogenetic tree concluded that the genetic sequences of Ureaplasma species are closely related to various isolates within a species. Conclusion: The study concludes that U. parvum is the most common pathogen in symptomatic women with predominant age of 26-35-year-old. The duplex PCR, increase the sensitivity and specificity by the application of two different genes in a single amplification reaction. Since the research will focus on different gene targets of Ureaplasma species by the application of multiplex in future study.

Analysis of composition of gut microbial community in a rat model of functional dyspepsia treated with Simo Tang.

To investigate composition of gut microbial community in a rat model of functional dyspepsia (FD) and to explore the interventional effects of Simo Tang (, SMT). A rat model of FD was established through the tail-clamping stimulation method. The rat model of FD was assessed by the state of rats, their weight, sucrose preference rate, and intestinal propulsion rate. The DNA was extracted from stool samples after treatment with SMT. Amplified polymerase chain reaction (PCR) products of the 16S rDNA were sequenced using NovaseQ6000 after construction of libraries. Composition of gut microbial community in the stool samples was determined and analyzed by cluster analysis, bioinformatic analysis, and analysis of α-diversity and β-diversity. The rat model of FD was successfully established using the tail-clamping stimulation method. The statistical results of cluster analysis of operational taxonomic units (OTUs) showed that the relative abundance of OTUs in the FD group was the lowest, while it was the highest in the normal (N) group. The composition of microbiome in the four groups was similar at phyla level. Compared with the FD group, the abundance of Firmicutes was downregulated, and the abundance of Proteobacteria and Bacteroidetes was upregulated in the Simo Tang (SMT) and high-dose Simo Tang (SMT.G) groups. The ratio of Bacteroidetes/ Firmicutes was also elevated. According to the analysis of α-diversity and β-diversity, the abundance of flora in FD rats was significantly reduced. The treatment using SMT appeared beneficial to improve the diversity of flora. SMT could improve the intestinal flora in FD rats. The results showed that FD rats had intestinal flora imbalance, and species diversity increased. The results suggested that SMT could regulate the disorders of intestinal flora caused by FD. SMT could restore gut homeostasis and maintain gut flora diversity by modulating the gut microbiota and its associated metabolites in rats, thereby treating gastrointestinal diseases.

Occurrence and molecular phylogeny of Fasciola species in camels of southwestern Iraq

Background and Aim: Fasciola spp. are important trematodes of public health concern in various animals, including camels. This study aimed to determine the occurrence of liver flukes in camels, to determine the molecular confirmation of Fasciola, and to perform phylogenetic analysis of study isolates to identify the species of Fasciola. Materials and Methods: In total, 107 slaughtered camels were inspected to collect liver flukes that were examined molecularly using polymerase chain reaction (PCR) to confirm Fasciola species. Then, the study isolates were sequenced, submitted to the National Center for Biotechnology Information (NCBI) database, and analyzed phylogenetically to identify the species of each study isolate. Results: Liver flukes were detected in 17.67% of the camels. Regarding the migratory stages of the collected worms, juvenile worms (73.91%) were significantly more prevalent than adult worms (26.09%). Regarding the risk factors, a significantly greater occurrence rate and risk of infection was detected in Al-Najaf compared with Al-Muthanna, as well as in younger camels (1–4 years) compared with older camels. Although the occurrence rate of liver flukes was insignificantly different between females (9.38%) and males (6.98%), females appeared to be at a significantly higher risk of infection than males. Molecularly, 33.33% of the worm samples were positive for species in the Fasciola genus. Phylogenetic analysis of all positive PCR products (total no = 19) confirmed that 63.16% of the local Fasciola spp. isolates were related to the NCBI-Basic Local Alignment Search Tool (NCBI-BLAST) Saudi Arabian Fasciola hepatica isolate at an identity range of 95.94%–99%; while 36.84% of the local Fasciola spp. isolates were related to the NCBI-BLAST Iranian F. gigantica isolate at an identity range of 97.73%–99%. Conclusion: This study found a 17.67% occurrence of Fasciola spp. in camels, with juvenile worms being more common than adult worms. Molecular analysis revealed that 63.16% of the isolates were related to F. hepatica from Saudi Arabia, while 36.84% matched F. gigantica from Iran. Younger camels and those from Al-Najaf were at higher risk, highlighting the need for targeted control measures. Keywords: camel trematode, conventional polymerase chain reaction, fascioliasis, one-humped camel, phylogenetic analysis.

Serologic and molecular identification of the variation on ABO*B.01 gene in ABO glycosyltransferases associated with Bw phenotype: a case report.

AB antigen is formed by glycosyltransferase enzyme, which catalyzes the corresponding substrates to be connected to the galactose of the precursor substance H antigen. To study the effect of the α-1,3-D galactosyltransferase (GTB) gene mutation on B antigen expression, we explored its molecular mechanism by combining molecular biological methods with bioinformatics. The ABO blood type of the patients was identified using conventional serologic methods, and the polymerase chain reaction (PCR) products of exons 1-7 of the ABO gene were directly sequenced using gene-specific primers and direct sequencing. Proteins in the secretory supernatant of transfected cells were collected in vitro, and GTB content was quantitatively analyzed using western blotting. Bioinformatics software was used to simulate the 3-dimensional structure of the mutant protein. In this case, the patient's serologic test results revealed subtype B. Gene sequencing results confirmed a mutation at base 278 of exon 6. The mutation (c.278C>T) changed the 93rd amino acid of the protein polypeptide chain from proline to leucine (p.P93L). The variant p.P93L did not affect the expression and secretion of GTB, but affected enzyme activity and stability, ultimately manifesting as weakened expression of the B antigen and reduced affinity.

Thelazia leesei Railliet & Henry, 1910 (Spirurida: Thelaziidae) of dromedary camel Camelus dromedarius: further morphological description, molecular characterization, and epidemiology in Iran

BackgroundIn camels, thelaziosis is mainly caused by Thelazia leesei Railliet & Henry, 1910, a little-known eyeworm species. Given the paucity of scientific data, this study aimed to provide new insights into the morphology, molecular characterization, and phylogenetic relationship of T. leesei and its occurrence in camels from Iran, where animals suffer from the high burden of eyeworms.MethodsFrom December 2020 to November 2022, slaughtered camels (n = 400) of different sex and age groups were examined in Sistan-va-Baluchestan province in Southeast Iran’s local abattoirs. Adult eyeworms were fixed and stored for morphological identification by light and scanning electron microscopy (SEM). Polymerase chain reaction (PCR) products corresponding to the partial sequences of the mitochondrial cytochrome c oxidase subunit I (cox1) of eyeworms were Sanger sequenced and analyzed phylogenetically.ResultsA total of 118 (29.5%) camels from all five counties examined were infected with eyeworms, with an abundance of 0.9 and a mean intensity of 3.2 (i.e., up to 18 worms from a single animal). The infection rate was higher in camels older than 4 years of age (P = 0.01901). Lachrymation was associated with infection in animals (P < 0.00001). The morphology of our specimens resembled that of T. leesei, with the exception of the position of the nerve ring and esophagus length. Genetic analysis showed that the cox1 partial sequences of our T. leesei specimens had genetic distances of 8.8% to 13.5% compared with other Thelazia species.ConclusionsOn the basis of the morphometrics and morphological characteristics, we identified our specimens as T. leesei. In the phylogenetic tree, T. leesei herein isolated formed a monophyletic group together with its congeners, and T. leesei formed a sister clade to T. lacrymalis. In addition, we demonstrated the epidemiology of the infestation of T. leesei in camels in the endemic areas of southeastern Iran. The data presented are crucial for better understanding the pathogenic role of T. leesei and developing effective treatment strategies. In particular, studies on the intermediate host(s) of T. leesei in these regions will support effective control strategies for this parasitosis.Graphical

Identification of mutation of MYOC (c.1099G>A), a pedigree pathogenic gene of juvenile open angle glaucoma (JOAG): A case report.

The MYOC gene is associated with juvenile open-angle glaucoma (JOAG). This study aims to provide genetic counseling for a Chinese JOAG family by detecting MYOC mutations to identify high-risk individuals for early JOAG intervention. It also supplements the clinical characteristics of glaucoma patients with MYOC gene mutations. A 43-year-old presented sought medical attention in a local hospital due to a 6-month decline in binocular vision. He was diagnosed as JOAG and underwent glaucoma surgery. The patient also had 11 family members with a history of JOAG. After sequencing the polymerase chain reaction products of the patient, MYOC c.1099 G > A (p.G367R) mutation was observed. That is consistent with a diagnosis of JOAG. Polymerase chain reaction analyses of 9 patients and 42 healthy family members were performed to explore potential mutations associated with familial JOAG. JOAG assisted in diagnosing the III-5 proband. Genetic detection indicated that III-5 was exposed to a novel heterozygous missense mutation of MYOC (c.1099 G > A [p.G367R]). The co-segregation of this gene with the trait observed in the pedigree was verified. All 10 participants exhibiting this mutation had JOAG phenotypes, whereas other participants did not show this mutation. In terms of MYOC mutation c.1099 G > A (p.G367R), this mutation occurred when the 1099th nucleotide in the encoding zone of MYOC changed from G to A. Moreover, the 367th amino acid coded by this base got mutated from glycine to arginine. DNAMAN sequence homology results showed that the G residues of MYOC: 367 were significantly conserved among different species. In addition, 3D protein conformation predicted that these mutations could decrease protein stability. MYOC c.1099 G > A was identified as a pathogenic gene of JOAG in this pedigree. The addition of the MYOC mutant spectrum to JOAG in the Chinese population facilitates a complete understanding of the molecular pathogenesis and clinical diagnosis of MYOC.

First Report of Root Rot Caused by Fusarium equiseti on Syrian rue (Peganum harmala) in China.

Syrian rue (Peganum harmala L) belongs to the family Zygophyllaceae and is mainly distributed in arid and semi-arid areas of Xinjiang, Gansu, Ningxia, Qinghai, and Inner Mongolia in China. It serves as a pioneer species in soil and water conservation, as well as in reclamation of wastelands, playing a crucial role in soil preservation and stabilization against sand encroachment. In 2023, a disease resembling root rot and wilt affected the quality and yield of Syrian rue in Xilingol (122.67°N, 42.78°E), Inner Mongolia, China. Symptoms ranged from yellowing to wilting, accompanied by darkening of the xylem in the roots. This disease was observed in approximately 50% of Syrian rue plants in desertification-prone grassland. A total number of 45 symptomatic root samples were collected from Syrian rue plants to isolate the pahogen. Small pieces of diseased tissue were surface sterilized with 75% alcohol for five seconds, rinsed twice with sterilized water, dried, and then placed on water agar and incubated for 72 h at 25℃ in darkness. A total of 45 isolates were obtained, showing identical morphology and sequences of the internal transcribed spacer region (ITS), translation elongation factor 1-alpha (EF1-α) and RNA polymerase II second largest subunit (RPB2) genes. Therefore, the isolate LTP-5 was used as the representative for further study. On potato dextrose agar (PDA), fungal colonies of LTP-5 exhibited yellow to light brown colore with white aerial mycelia and irregular growth after 7 days of incubation at 25℃. Conidia and chlamydospre development were observed in carboxymethylcellulose sodium medium. Chlamydospores were abundant, terminal or intergrown between hyphae, rough brown walls of 5.9 to 19.82 μm in diameter (n = 75) were observed in the culture. Macroconidia had 5 to 7 septa, thick in the middle and thin at both ends, measured 28.17 to 62.81 μm in length and 1.50 to 4.19 μm in width (n = 128). Microconidia were absent. The morphological characteristics were consistent with the descriptions of Fusarium equiseti (Nelson et al., 1983). The pathogen was further confirmed to be F. equiseti by sequence analysis of the ITS, EF1-α and RPB2 genes amplified using polymerase chain reaction (PCR) with the primers ITS4/ITS5 (White et al., 1990), EF-1/EF-2 (O'Donnell et al., 1998), and RPB2-5F2 /fRPB2-7cR (Zhen et al., 2017). The sequences showed 100% similarity to the F. equiseti strain in the NCBI GenBank with accession numbers MH054914.1, KJ396323.1, and KT213286.1 for ITS, EF1-α, and RPB2, respectively. The sequences of PCR products were submitted to GenBank with accession numbers PP814863.1 (ITS), PP831628.1 (EF1-α) and PP826937.1 (RPB2). Pathogenicity tests were conducted through a pot assay. Five Syrian rue seedlings were inoculated by immersing plant roots in a suspension of 1×107 spores/mL for 30 min and transplanting to pots containing autoclaved soil. Control plants were immersed in sterile water. The inoculated plants showed yellow and root rot but no symptoms were observed on control plants. The same fungus was successfully re-isolated and identified based on the morphological characterization and sequence analyses from the infected root tissue but not from non-inoculated plants. In addition, F. equiseti has been reported to cause disease on Sugar beet (Khan et al., 2021), Watermelon (Rahman et al., 2021), potato (Cui et al., 2021), and other plants. This is the first report confirming F. equiseti as causal agent of Syrian rue wilt and root rot in China.

Molecular Analysis of Gram-Negative Bacterial Strains in Patients from Koya City

This study addresses the critical issue of Gram-negative bacteria (GNB) in patients from Koya City, acknowledging their significant resistance to antibiotics and the consequent threat to public health. A total of 14 samples were collected from diverse patient demographics and sources, including urine, wounds, vaginal swabs, seminal fluid, and throat swabs. The samples were subjected to analysis through a multifaceted approach involving culturing and molecular identification. Following the isolation of bacterial samples, the 16SrRNA gene was targeted using polymerase chain reaction (PCR). Subsequently, the obtained PCR products underwent Sanger sequencing, providing a detailed analysis of the nucleotide sequences. The resulting sequences were then compared against existing prokaryotic DNA sequences in the BLAST database to determine the identity of the isolated bacteria. Out of the 14 samples, 13 yielded sequences of good quality, while one exhibited a noise sequence. The sequences were further aligned, and 11 samples demonstrated a 100% identity with previously identified bacteria such as Escherichia coli (100 %), Klebsiella pneumoniae (100 %), and Shigella flexneri (100 %) Notably, two samples displayed a &gt;99% identity with Escherichia coli (99.5%), highlighting the diversity within the bacterial strains. This study sheds light on the prevalence and identity of Gram-negative bacteria in Koya City patients. The findings underscore the necessity of molecular techniques, such as PCR and sequencing, in clinical diagnostics. The diverse bacterial strains identified highlight the complexity of bacterial infections, emphasizing the importance of targeted approaches in addressing antibiotic resistance.

APPLICATION OF PCR FOR THE DETECTION OF VIRAL INFECTIONS IN THE HONEYBEE Apis mellifera IN THE NORTHERN REGIONS OF UKRAINE

Aim. This study investigated viruses of the western honeybee (Apis mellifera) from the northern regions of Ukraine using polymerase chain reaction (PCR). Methods. To identify honeybee viruses, oligonucleotide primers, which are specific to a gene fragment encoding the capsid protein and a fragment of the RNA-dependent RNA polymerase gene, were used. The reaction mixture contained 12.5 μL of master mix solution (DNA polymerase, dNTPs, and buffer), 1 μL of each primer (20 pmol/μL), 2 μL of cDNA, and water to a total volume of 25 μL. PCR products were visualized in a 2% agarose gel. Results. In the Zhytomyr region in Apis mellifera was revealed for the first time the presence of four viruses representing different taxonomic groups, in particular Israeli Acute Paralysis Virus (IAPV), Sacbrood Virus (SBV), Deformed Wing Virus (DWV) and Chronic Bee Paralysis Virus (CBPV). Three of these viruses (SBV, DWV, and CBPV) were detected in the Kyiv region, while two (SBV and DWV) were identified in the Chernihiv region. Conclusions. The obtained data indicate the widespread distribution of Apis mellifera honeybee viruses in the northern regions of Ukraine. The use of PCR to detect bee viruses will contribute to the development of effective methods for improving the health of bee colonies.

A Polymerase Chain Reaction (PCR) Method to Detect Emerging Multidrug-Resistant Salmonella Infantis Harboring the pESI Plasmid in Seafood.

Salmonella Infantis is an emerging multidrug-resistant pathogen worldwide due to the acquisition of a megaplasmid, plasmid of emerging Salmonella Infantis (pESI). Reported initially in poultry, the distribution of pESI-harboring S. Infantis in other food types, including seafood, is unknown. This study aimed to develop and optimize a PCR assay for detecting the pESI in Salmonella and non-Salmonella Enterobacterales. A duplex PCR targeting the hilA gene and a pESI-associated gene of S. Infantis was designed, and the PCR conditions were optimized. The specificity and sensitivity of the assay were established using 119 Salmonella serovars and 51 non-Salmonella bacterial strains. All Salmonella isolates yielded hilA PCR product, while only pESI S. Infantis was positive for both hilA and pESI genes. No amplification product was obtained with the DNA of 51 non-Salmonella bacterial strains. The detection limit of the duplex PCR was 104 CFU/mL of pure culture of pESI S. Infantis. The sensitivity of detection in artificially spiked shrimp meat was 1 CFU/g after 6 h of enrichment in lactose broth, followed by 12 h of selective enrichment in the Rappaport-Vassiliadis medium. The duplex assay will help screen seafood for Salmonella in general and pESI S. Infantis in particular. Given its high sensitivity, the PCR will be a valuable tool for seafood quality assurance. This approach decreases the typical 3-6 day identification time of Salmonella to less than 24 h. S. Infantis carrying the highly transmissible megaplasmid (pESI) is a significant food safety concern. Given its rapid geographical spread and high antimicrobial-resistant traits, it is necessary to have a molecular tool that detects pESI-harboring Salmonella. This study successfully developed a duplex PCR assay that simultaneously detects Salmonella enterica and pESI S. Infantis. This molecular tool will help understand the distribution, sources, and spread of the multidrug-resistance (MDR) plasmid in the food environment.

Capillary flow velocity-based length identification of PCR and RPA products on paper microfluidic chips

This work demonstrates a novel, non-fluorescence approach to the length identification of polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) products, utilizing capillary flow velocities on paper microfluidic chips. It required only a blank paper chip, aminated microspheres, and a smartphone, with a rapid assay time and under ambient lighting. A smartphone evaluated the initial capillary flow velocities on the paper chips for the PCR and RPA products from various bacterial samples, where the pre-loaded aminated microspheres differentiated their flow velocities. Flow velocities were analyzed at different time frames and compared with the instantaneous flow velocities and interfacial tension (γLV) data. Subsequent error analysis justified the use of the initial time frames. A robust linear relationship could be established between the initial flow velocities against the square root of the product lengths, with R2 values of 0.981 for PCR and 0.993 for RPA. The assay seemed not to have a significant dependency on the cycle numbers and initial target concentrations. This novel method can be potentially used with various paper microfluidic methods of nucleic acid amplification tests towards rapid and handheld assays.

Effect of poly(dA) stretch in a template sequence on the rate and yield of polymerase chain reaction with electroactive modified 2′-deoxyuridine-5′-triphosphates

The effective incorporation of electroactive labeled nucleotides into DNA during its enzymatic amplification provides the ground for developing assays based on direct electrochemical detection of amplification products. Yet, whether the template sequence can affect the incorporation efficiency of modified nucleotides is still poorly understood. Here we examined the effects of a poly(dA) stretch in template sequence on the rate and yield of polymerase chain reaction (PCR) in the presence of modified 2′-deoxyuridine-5′-triphosphates (dUTP) in the reaction mixture. Four dUTP derivatives carrying 4-hydroxyphenyl (tyrosine; dUTP-Y1 and dUTP-Y2), 4-nitrophenyl (dUTP-N1), or indolyl (tryptophan; dUTP-W1) aromatic groups were tested as substrates for Taq and KTN (lacking 3′-5′ exonuclease activity) polymerases at 100 % substitution of 2′-deoxythymidine-5′-triphosphate (dTTP) in the PCR mixture. The 120 base pair long synthetic DNA templates contained poly(dA)·poly(dT) tracts of various length (dAn·dTn, where n = 2–10). In all cases, full-size amplicons have been obtained. Overall, in the presence of modified dUTP, the PCR yield was higher with KTN polymerase than that with Taq polymerase. The PCR yield slightly (for dUTP-Y1 and dUTP-Y2) or significantly (for dUTP-N1 and dUTP-W1) decreased with an increase of n in the dAn·dTn tract. In most cases, in the presence of modified dUTP, the PCR rate substantially decreased with n ≥ 4 in dAn·dTn. No changes in amplicon sequences due to potential incorrect nucleotide pairing during amplification with the complete substitution of dTTP with dUTP-Y2 were revealed. Electrochemical activity of tyrosine labeled PCR products dsDNA-Y2 differing in the length of stretches of modified nucleotides was similar. The findings demonstrate that the incorporation efficiency for base-modified nucleotides can in general depend on template sequence, in particular on the presence of long poly(dA) stretches when the modified dUTP are used. That has to be taken into account when developing electrochemical or other DNA assays with a specific modified nucleotide.

Analysis of Single Nucleotide Polymorphisms of Liver X Receptor Alpha (LXR-α) Gene in Diabetic Kidney Disease.

Introduction Genetic factors significantly contribute to the progression of diabetic kidney disease (DKD), which is one of the world's most common causes of chronic kidney disease (CKD). Inflammation, glucose homeostasis, and lipid metabolism are all significantly influenced by the liver X receptor alpha (LXR-α) gene. Single nucleotide polymorphisms in the liver X receptor alpha gene may result in the altered function of the gene, making the individual susceptible to DKD. The purpose of this article is to examine the frequency of expression of single nucleotide polymorphisms (SNPs) in exon 7 of the LXR-α gene in people with type 2 diabetes (T2DM) and DKD. Gaining an understanding of these genetic differences could improve risk assessmentand therapy approaches while providing fresh perspectives on the molecular mechanisms underlying DKD. Aims and objectives To identify specific SNPs in the LXR-αgene in T2DMand DKD patients. Methods The study was conducted between May 2022 and May 2023 and it involved 120 T2DM and 120 DKD patients. All the clinical history of the study participants were recorded. Blood samples were collected from the participants and genomic DNA was isolated and was followed by polymerase chain reaction (PCR) using exon 7 specified primers of LXR-α gene. To find mutations in the LXR-α gene, the PCR products were subsequently analyzed using the capillary-based Sanger sequencing technique. Results A total of seven SNPs were detected in T2DM patients, and 20 SNPs were detected in DKD patients. SNP rs7120118 was found with the highest frequency of 100 (83.33%) in T2DM patients and 80 (66.66%) in DKD patients. SNP rs1956299169 was found to have the second highest frequency of 60 (50%) among T2DM patients and SNP rs1252683195 was found to have a frequency of 20 (16.7%). Othercommon SNPs were rs1956299169, with 40 (33.33%) in DKD and 60 (50%) in T2DM, and rs12574081, with 20 (16.71%) in T2DM and 10 (8.33%) in T2DM. Conclusion The study detects the highest and the most common SNP rs7120118, which may be associated with T2DM and DKD. Thereby, ithelps to conduct furtherassociation studies with larger populations and to diagnose the disease.

Toxoplasma Gondii In Association with TLR4 Among Regular Hemodialysis Patients

Background: Hemodialysis patients can be considered immunocompromised as they have an increased risk of infections. The activity of Toll -like receptors in defense against infections was observed particularly for Toll like receptor4 molecule. Several single-nucleotide polymorphisms (SNPs) residing in genes encoding Toll -like receptors were reported as significant genetic modifications associated with infections. Aim: To determine the association between Toll- like receptors 4 polymorphisms and the susceptibility to infection with Toxoplasma gondii in hemodialysis patients. Materials and Methods: This case- control study was conducted on 150 patients with hemodialysis and 150 apparently healthy blood donors as control group, the samples were collected in the period from march 2021 to November 2021 from Al-Karama hospital and Al-Yarmouk Teaching Hospital. All patients were investigated for the presence of Toxoplasma gondii and Abs (IgG, IgM) by using ELISA technique and conformation by molecular technique (Real time-PCR). The selected SNPs in Toll- like receptors 4 were amplified by using conventional polymerase chain reaction PCR and then sequencing there polymorphism. Results: In the current study the comparison of median age between patients and controls was with no significant difference, while the laboratory tests revealed significant difference in median level of urea, creatinin , calcium , potassium ,albumin and hemoglobin, and insignificant difference for body mass index and sodium between studied groups. The ratio of females infected with Toxoplasma gondii was higher than infected males. The seropositive result in patients for Toxoplasma gondii - IgG and IgM was 15 and 6 respectively while 19 patients were positive by PCR. However in control group there were 8 patients seropositive for IgG and positive in PCR results while the results of IgM and PCR were negative for all control group. The Polymerase chain reaction (PCR) products of Toll- like receptors 4 genes were subjected for direct sequencing by using Bio edit software. And the resultant sequences were compared with reference sequences in NCBI. The analysis of TLR-4 rs4986790 genotypes were 24(80%) AA and 6(20%) AG in patients while 6 (60%) AA and 4(40%) AG in controls with insignificant difference, while the analysis of TLR-4 rs4986791 genotypes were 24(80%) CC and 6 (20%) CT in patients while 6 (60%) CC and 4(40%) CT in controls with insignificant difference. Conclusion: There was no significant association of Toxoplasma gondii with TLR4 genotypes of rs4986790 and rs4986791 SNPs in hemodialysis patients.

Repeat-mediated recombination results in Complex DNA structure of the mitochondrial genome of Trachelospermum jasminoides

BackgroundTrachelospermum jasminoides has medicinal and ornamental value and is widely distributed in China. Although the chloroplast genome has been documented, the mitochondrial genome has not yet been studied.ResultsThe mitochondrial genome of T. jasminoides was assembled and functionally annotated using Illumina and nanopore reads. The mitochondrial genome comprises a master circular molecular structure of 605,764 bp and encodes 65 genes: 39 protein-coding genes, 23 transfer RNA (tRNA) genes and 3 ribosomal RNA genes. In addition to the single circular conformation, we found many alternative conformations of the T. jasminoides mitochondrial genome mediated by 42 repetitive sequences. Six repetitive sequences (DRS01–DRS06) were supported by nanopore long reads, polymerase chain reaction (PCR) amplifications, and Sanger sequencing of the PCR products. Eleven homologous fragments were identified by comparing the mitochondrial and chloroplast genome sequences, including three complete tRNA genes. Moreover, 531 edited RNA sites were identified in the protein-coding sequences based on RNA sequencing data, with nad4 having the highest number of sites (54).ConclusionTo our knowledge, this is the first description of the mitochondrial genome of T. jasminoides. Our results demonstrate the existence of multiple conformations. These findings lay a foundation for understanding the genetics and evolutionary dynamics of Apocynaceae.