Polymerase Chain Reaction Technique Research Articles

Seroprevalence and molecular detection of infectious laryngotracheitis virus (ILTV) in commercial chicken in Gazipur district of Bangladesh

Infectious laryngotracheitis (ILT) is one of the major diseases of chickens that cause great economic losses in commercial chicken farms. It is caused by the infectious laryngotracheitis virus (ILTV). This research work was designed to investigate the seroprevalence of ILTV in commercial chicken farms in Gazipur district. A total of 590 blood sera were collected randomly from commercial chicken farms from different locations in Gazipur district. An antigen-coated indirect ELISA was performed to determine the antibody titer against ILTV in these sera. Moreover, gross and histopathological changes in different organs were investigated. Polymerase chain reaction (PCR), a molecular detection method, was performed to detect the fragment of the glycoprotein ‘C’ (gC) gene of ILTV in the infected chicken. Overall, 195 out of 590 serum samples were positive for anti-ILTV IgG, representing 33.05% of positive cases. The highest mean titer of ILTV antibody was found in anti-ILTV-vaccinated layer chickens with a mean titer of 3554. In seasonal variation, the highest prevalence of ILTV was recorded during the winter, followed by the rainy and summer seasons. The prevalence of ILTV was the highest at the age of 10-30 weeks in layer chickens. Necropsy findings of dead chickens showed severe congestion and fibrosis in the lungs and trachea, with occlusion of the tracheal lumen by mucus, caseous exudates and blood. Microscopically, hemorrhage and huge infiltrations of inflammatory cells were found in trachea and lungs. Moreover, the nucleotide sequence of ‘gC’ gene of ILTV was amplified successfully and yielded 1.26 kbp amplicons. This study suggested that ILTV was endemic in Gazipur district and the PCR technique is a useful molecular tool for diagnosis of ILTV in the commercial chicken industry. Ann. Bangladesh Agric. (2023) 27 (1): 121-135

Dietary Restriction Improves Perioperative Neurocognitive Disorders by Inhibiting Neuroinflammation and Gut Microbial Dysbiosis

Anesthesia/surgery have been identified as potential factors contributing to perioperative neurocognitive disorders, with a notably heightened risk observed in aging populations. One of the primary drivers of this impairment is believed to be neuroinflammation, specifically inflammation of hippocampal microglia. Dietary restriction has demonstrated a favorable impact on cognitive impairment across various disorders, primarily by quelling neuroinflammation. However, the precise influence of dietary restriction on perioperative neurocognitive disorders remains to be definitively ascertained. This investigation aims to explore the effects of dietary restriction on perioperative neurocognitive disorders and propose innovative therapeutic strategies for their management. The model of perioperative neurocognitive disorder was induced through exploratory laparotomy under isoflurane anesthesia. Cognitive performance was evaluated using the open field test, Barnes maze test, and fear conditioning test. The enzyme-linked immunosorbent assay (ELISA) was employed to quantify concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in both serum and hippocampal samples. The Western blot technique was utilized to assess expression levels of hippocampal PSD 95, Synaptophysin, TLR4, MyD88, and NF-kB p65. Microglial polarization was gauged using a combination of reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence labeling techniques. We conducted 16S rRNA sequencing to investigate the impact of dietary restriction on the intestinal flora of aged mice following anesthesia/surgery. Our findings indicate that dietary restrictions have the potential to ameliorate anesthesia/surgery-induced cognitive dysfunction. This effect is achieved through the modulation of gut microbiota, suppression of inflammatory responses in hippocampal microglia, and facilitation of neuronal repair and regeneration.

Screening and evaluating microorganisms with broad-spectrum biogenic amine-degrading ability from naturally fermented dry sausage collected from Northeast China

This study aimed to screen autochthonous strains with broad-spectrum biogenic amine (BA) degradation ability from traditional dry sausages and to evaluate their BA-degrading ability in dry sausages. A total of 120 strains were isolated from dry sausages collected from various regions in Northeast China, and 35 of 120 isolates were identified as non-BA producing strains by the in vitro agar method. The random amplified polymorphic DNA polymerase chain reaction technique genotyped these 35 isolates into 18 biotypes. Moreover, high performance liquid chromatography (HPLC) quantification showed that six strains (Latilactobacillus sakei MDJ6; Lactiplantibacillus plantarum SH7; Weissella hellenica DQ9; Staphylococcus saprophyticus JX18 and SYS8; and Macrococcus caseolyticus SYS11) of the 18 biotypes exhibited broad-spectrum BA-degrading ability, all of which had various levels of amine oxidase activity with monoamine oxidase and diamine oxidase activities ranged of 6.60–619.04 and 26.32–352.81 U/mg protein, respectively. These six strains were subsequently inoculated into dry sausages and the results showed that they exhibited varying degrees of BA-degrading ability, of which strain Lat. sakei MDJ6 allowed to have less BA production on dry sausage with a final concentration of 61.33 mg/kg.

A importância da Biologia Molecular no diagnóstico do SARS-CoV-2

The pandemic caused by the etiologic agent SARS-CoV- 2, started at the beginning of 2020. And create a big challenge in the scientific community from all around the world. The symptoms can variate of levels manifestation to severs cases of pneumonia. Studies are seeking ways of prevention, diagnosis and treatment of the disease. In view of the pandemic scenario and the worsening number of cases and deaths due to the disease, the search for an accurate diagnosis is essential. Therefore, the aim of this study is demonstrating the importance of using molecular biology through the technique of Real Time Transcriptase Reverse Polymerase Chain Reaction (Real Time RT-PCR), for the detection of the genetic material of SARS-CoV-2 from data found in the literature. After a critical analysis of the literature, it was possible to confirm the importance and efficiency of using the Real Time RT-PCR technique in the diagnosis of infection, since this technique allows a good performance in the detection of viral genetic material, guaranteeing a high success rate when differentiating an infected patient from the non-infected. Concluding that until now this technique is considered as the main diagnostic method for the disease, remaining as a “gold standard”.

Pathogens in Pediatric Septic Arthritis: A Multi-Center Study in Turkiye (PEDSART Study).

Septic arthritis (SA) is a serious bacterial infection that must be treated efficiently and timely. The large number of culture-negative cases makes local epidemiological data important. Accordingly, this study aimed to evaluate the etiology, clinical characteristics, and therapeutic approach of SA in children in Turkiye, emphasizing the role of real-time polymerase chain reaction (PCR) techniques in the diagnosis. In this multi-center, prospective study, children hospitalized due to SA between February 2018 and July 2020 in 23 hospitals in 14 cities in Turkiye were included. Clinical, demographic, laboratory, and radiological findings were assessed, and real-time PCR was performed using synovial fluid samples. Seventy-five children aged between 3 and 204 months diagnosed with acute SA were enrolled. Joint pain was the main complaint at admission, and the most commonly involved joints were the knees in 58 patients (77.4%). The combination of synovial fluid culture and real-time PCR detected causative bacteria in 33 patients (44%). In 14 (18.7%) patients, the etiological agent was demonstrated using only PCR. The most commonly isolated etiologic agent was Staphylococcus aureus, which was detected in 22 (29.3%) patients, while Streptococcus pyogenes was found in 4 (5.3%) patients and Kingella kingae in 3 (4%) patients. Streptococcus pyogenes and Kingella kingae were detected using only PCR. Most patients (81.3%) received combination therapy with multiple agents, and the most commonly used combination was glycopeptides plus third-generation cephalosporin. Staphylococcus aureus is the main pathogen in pediatric SA, and with the use of advanced diagnostic approaches, such as real-time PCR, the chance of diagnosis increases, especially in cases due to Kingella kingae and Streptococcus pyogenes.

Molecular Detection and Identification of oprl and lasb Genes Isolated from Pseudomonas aeruginosa

: Species Pseudomonas aeruginosa is a distinguished opportunistic nosocomial bacterium that is commonly dispersed in nature. A pathogen that causes a large range of infections i.e. chronic as well as acute, with considerable levels of mortality and morbidity. Infections of P. aeruginosa are extremely hard to eliminate because of the presence of several virulence determinants as well as the intrinsic resistance against antibiotics. The present study investigated the detection of the virulence genes by polymerase chain reaction (PCR) namely oprL and lasB in the P. aeruginosa that were isolated detection from both human and animal specimens. 120 specimens of P. aeruginosa including samples were then collected from hospitals and veterinary laboratories, from which 55 isolates of P. aeruginosa were identified and selected. Pseudomonas aeruginosa strains were present in 40% of animal sample isolates and 86% of human sample isolates. The PCR results showed that oprL genes were detected in 54.16% of isolates obtained from animal samples and 80.76% of isolates obtained from human samples. While, the lasB gene was present at 58.33% and 92.30% in the isolates obtained from animal and human samples, respectively. Our results also showed the coexistence of both virulence genes in 32.34% of animal sample isolates and 25.10% of human sample isolations. The detection of gene oprL and lasB by the PCR technique can be effectively used for the molecular-level identification of P. aeruginosa. The identification of such virulence genes in P. aeruginosa isolates suggests that these may be linked with varying degrees of the pathogenesis and inherent virulence in this pathogen.

Microbiología clásica y molecular en el diagnóstico de la endocarditis infecciosa

Introduction and objectivesIn recent years, molecular techniques for the aetiological diagnosis of infective endocarditis (IE) have attained relevance; however, they are not implemented in all laboratories. Our objective is to evaluate the usefulness of broad-spectrum polymerase chain reaction (PCR) technique followed by sequencing (universal PCR) on valve tissue as part of the IE diagnostic practice and to compare the results with classical methods. ResultsUniversal PCR showed a higher sensitivity than valve culture (80.8 vs 27.1%, P<.001) and allowed the identification of the causative agent or the confirmation of the aetiological diagnosis in case of discordance in 7.7% of patients. Regarding conventional diagnosis, blood culture stands out as the reference technique and Gram staining due to its high positive predictive value. The highest sensitivity and specificity was obtained considering classical and molecular microbiology together. The anatomopathological study presented a sensitivity of 83.9% and made it possible to assess the results of the microbiological diagnosis when there were discrepancies. ConclusionsUniversal PCR proved to be a valuable technique that completes the classical diagnosis. In an entity as complex as IE, it is necessary to use all the diagnostic tools at our disposal. All areas of laboratory diagnosis are complementary and not exclusive and should be assessed together.

Investigating Mutations of 288 Genes in a Family with Charcot-Marie-Tooth Disease by the Whole-Exome Sequencing Method in Khuzestan Province

Background: Genetic factors play an important role in the occurrence of many diseases, including skeletal diseases. Skeletal diseases are sometimes caused by a mutation in a gene that passes on to children according to the Mendelian inheritance pattern, and sometimes several genes are involved in this path and cause heterogeneous disease. The whole-exome sequencing (WES) technique can be a powerful tool in diagnosing this disease. Methods: Peripheral blood samples (5 mL) were collected from the individuals in EDTA tubes. After sequencing the patient's genome using the WES technique, the candidate genes were checked using the polymerase chain reaction (PCR) technique and Chromas software to confirm the variant in the family. Results: After studying the genome using the WES method and analyzing the results, mutation NM_001005373.4:c.1985C&gt;T was identified by creating a nonsense change in the LRSAM1 gene. Conclusions: The data obtained by the WES technique can help diagnose the disease faster. In addition, these data can be used as background information in the development of a special panel to diagnose this disorder.

Impact of Multiplex PCR in the Therapeutic Management of Severe Bacterial Pneumonia.

Pneumonia is a common and severe illness that requires prompt and effective management. Advanced, rapid, and accurate tools are needed to diagnose patients with severe bacterial pneumonia, and to rapidly select appropriate antimicrobial therapy, which must be initiated within the first few hours of care. Two multiplex molecular tests, Unyvero HPN and FilmArray Pneumonia+ Panel, have been developed using the multiplex polymerase chain reaction (mPCR) technique to rapidly identify pathogens and their main antibiotic resistance mechanisms from patient respiratory specimens. Performance evaluation of these tests showed strong correlations with reference techniques. However, good knowledge of their indications, targets, and limitations is essential. Collaboration with microbiologists is, therefore, crucial for their appropriate use. Under these conditions, and with standardized management, these rapid tests can improve the therapeutic management of severe pneumonia faster, more precisely, and with narrow-spectrum antibiotic therapy. Further randomized controlled trials are needed to address the many unanswered questions about multiplex rapid molecular testing during the diagnosis and the management of severe pneumonia. This narrative review will address the current knowledge, advantages, and disadvantages of these tests, and propose solutions for their routine use.

TMEM64 aggravates the malignant phenotype of glioma by activating the Wnt/β-catenin signaling pathway

Transmembrane protein 64 (TMEM64), a member of the family of transmembrane protein, is an α-helical membrane protein. Its precise role in various types of tumors, including glioma, is unclear. This study used immunohistochemical (IHC) staining, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) techniques to show that TMEM64 expression was significantly higher in glioma cells and tissues compared to normal cells and tissues, respectively. Additionally, a correlation between high TMEM64 expression and higher grade as well as a worse prognosis was found. TMEM64 enhanced cell proliferation and tumorigenicity while inhibiting glioma cell apoptosis in vitro and in vivo, according to loss- and gain-of-function studies. Mechanistically, it was discovered that TMEM64 increased the malignant phenotype of gliomas by accelerating the translocation of β-catenin from the cytoplasm to the nucleus, thereby activating the Wnt/β-catenin signaling pathway. Stimulation with the Wnt/β-catenin signaling pathway activator CHIR-99021 successfully reversed the malignant phenotype of glioma; however, these effects were inhibited upon TMEM64 silencing. Stimulation with the Wnt/β-catenin signaling pathway inhibitor XAV-939 successfully rescued the malignant phenotype of glioma, which was promoted upon TMEM64 overexpression. Our results provide that TMEM64 as a novel prognostic biomarker and a potential treatment target for glioma.

The Influences of Chrysin on Stress-Induced Changes of Melanin-Concentrating Hormone and Orexin Gene Expression in Rats

Introduction: Chrysin is a bioactive component of herbal medicines such as Passiflora incarnate, Passiflora caerulea, and Oroxylum indicum. Although evidence has demonstrated the neuroprotective, anti-inflammatory, and pain-relieving effects of chrysin, the intra-hypothalamic molecular mechanisms underlying the anxiolytic effects of chrysin are still unclear. This study aimed to explore the effects of chrysin on hypothalamic orexin and melanin-concentrating hormone (MCH) gene expression in a rat model of stress. Methods: Twenty male Wistar rats (200±10 g) were segregated into four groups (n=5). For the induction of stress, the animals were placed in the restraint cage for 2 hours. The intact and stressed groups received saline. Thirty minutes before the induction of stress, chrysin was injected into the other two groups of the stress model at a dosage of 20 or 40 µg via the third cerebral ventricle. Hypothalamic samples were removed and frozen, and the relative gene expression of orexin and MCH was measured using the real-time polymerase chain reaction technique. Results: The induction of stress significantly increased mRNA levels of orexin and MCH compared to the control rats. The mRNA levels of MCH and orexin significantly declined in rats receiving chrysin compared to the stress group. Conclusion: The inhibition of the hypothalamic MCH and orexin neuronal circuits may be involved in the preventive effects of chrysin against stressful situations. Chrysin may be a potential target to manage anxiogenic behaviors due to the down-regulation of MCH and orexin gene expression upstream the hypothalamic corticotropin-releasing hormone.

Circular PCR as an efficient and precise umbrella of methods for the generation of circular dsDNA with staggered nicks: Mechanism and types.

Here, we introduce the highly versatile circular polymerase chain reaction (CiPCR) technique, propose a mechanism of action, and describe a number of examples demonstrating the versatility of this technique. CiPCR takes place between two fragments of dsDNA with two homologous regions, as long as one of the fragments carries said regions at its 3'- and 5'-ends. Upon hybridization, elongation by a polymerase occurs from all 3'-ends continuously until a 5'-end is reached, leading to stable circular dsDNA with staggered nicks. When both dsDNA fragments carry the homology at their 3'- and 5'-ends (Type I CiPCR), all four 3'-ends effectively prime amplification of the intervening region and CiPCR products can function as template during the reaction. In contrast, when only one of the two dsDNA fragments carries the homologous regions at its 3'- and 5'-ends and the other carries such regions internally (Type II CiPCR), only two 3'-ends can be amplified and CiPCR products possess no template activity. We demonstrate the applicability of both CiPCR types via well-illustrated experimental examples. CiPCR is well adapted to the quick resolution of most of the molecular cloning challenges faced by the biology/biomedicine laboratory, including the generation of insertions, deletions, and mutations.

Development of a high-throughput DNA isolation method for livestock and poultry meat based on mesoporous metal-organic framework-coated solid-phase microextraction device

A rapid, portable, simple, low-cost, and high-throughput DNA isolation method was developed for the quantitative identification of meat adulteration based on mesoporous UiO-66 coated solid phase microextraction (SPME) devices. The high-throughput DNA extraction system consists of 96 UiO-66 SPME devices and a polyformaldehyde plate, which can extract DNA from 96 livestock and poultry meat samples simultaneously. At the same time, several parameters of the high throughput DNA isolation method were optimized, including lysis buffer volume, elution buffer volume, lysis time and elution time, etc. Compared with the single DNA isolation device, the 96-SPME high-throughput extraction system maintains the original good universality, reproducibility, chemical stability, and reusability. On this basis, it obtained higher DNA concentration (80 ± 34 ng μL−1), satisfactory DNA purity (A260/A280=2.4) and shorter extraction time (less than 7.2 s per sample), which are much more convenient and efficient than the conventional kit method and the single SPME DNA isolation method. In addition, the conventional multiplex polymerase chain reaction and real-time polymerase chain reaction techniques were used for the qualitative and quantitative detection of meat adulteration. The DNA obtained from this extraction method is highly reproducible and sensitive for the identification of duck ingredients in beef, with a detection range of 1–100 % and a minimum detection limit of 1 % (w/w).

Baseline susceptibility of Anopheles gambiae to clothianidin in northern Ghana

BackgroundClothianidin, an insecticide with a novel mode of action, has been deployed in the annual indoor residual spraying programme in northern Ghana since March 2021. To inform pragmatic management strategies and guide future studies, baseline data on local Anopheles gambiae sensu lato (s.l.) susceptibility to the clothianidin insecticide were collected in Kpalsogu, a village in the Northern region, Ghana.MethodsPhenotypic susceptibility of An. gambiae mosquitoes to clothianidin was assessed using the World Health Organization (WHO) insecticide resistance monitoring bioassay. The WHO cone bioassays were conducted on mud and cement walls sprayed with Sumishield 50 wettable granules (WG) (with clothianidin active ingredient). Daily mortalities were recorded for up to 7 days to observe for delayed mortalities. Polymerase chain reaction (PCR) technique was used to differentiate the sibling species of the An. gambiae complex and also for the detection of knock down resistance genes (kdr) and the insensitive acetylcholinesterase mutation (ace-1).ResultsThe WHO susceptibility bioassay revealed a delayed killing effect of clothianidin. Mosquitoes exposed to the cone bioassays for 5 min died 120 h after exposure. Slightly higher mortalities were observed in mosquitoes exposed to clothianidin-treated cement wall surfaces than mosquitoes exposed to mud wall surfaces. The kdr target-site mutation L1014F occurred at very high frequencies (0.89–0.94) across all vector species identified whereas the ace-1 mutation occurred at moderate levels (0.32–0.44). Anopheles gambiae sensu stricto was the most abundant species observed at 63%, whereas Anopheles arabiensis was the least observed at 9%.ConclusionsAnopheles gambiae s.l. mosquitoes in northern Ghana were susceptible to clothianidin. They harboured kdr mutations at high frequencies. The ace-1 mutation occurred in moderation. The results of this study confirm that clothianidin is an effective active ingredient and should be utilized in malaria vector control interventions.

Factors associated with human papillomavirus persistence after loop electrosurgical excision procedure in patients with cervical squamous intraepithelial lesion.

To seek the high-risk factors of human papillomavirus (HPV) persistence and residual lesion or recurrence after loop electrosurgical excision procedure (LEEP) focus on the predictive value of intraoperative human papilloma virus (IOP-HPV) testing. Intraoperative endocervical sample was obtained with a cytobrush from the remained cervix of 292 patients immediately after LEEP. HPV Genotyping was performed using a polymerase chain reaction technique. All patients followed by HPV genotyping and cytology every 3-6 months. The IOP-HPV testing results and possible risk factors such as age, cytology grade, menopause status, margin involvement, preoperative HPV status, and cervical lesion grade were assessed in predicting persistence of HPV and residual lesion or recurrence after surgery. There were 61 (20.9%) patients presented persistent HPV infection. Multivariate analyses showed that IOP-HPV positive, post-menopause and preoperative HPV multiplex infection was strongly associated with HPV persistence after LEEP, IOP-HPV positive and post-menopause was also associated with residua or recurrent disease after LEEP. IOP-HPV positive, post-menopause, and preoperative HPV multiplex infection are independent predictors of HPV persistence in patients with cervical squamous intraepithelial lesion treated by LEEP. IOP-HPV test is a new approach that may potentially allow for early identification of patients at high risk of HPV persistence and residua or recurrent disease after LEEP, thereby possibly facilitate an attenuated follow-up schedule for negative patients those at low risk of persistent HPV infection.

CircERCC6 Positively Regulates the Induced Activation of SHF Stem Cells in Cashmere Goats via the miR-412-3p/BNC2 Axis in an m6A-Dependent Manner.

The cashmere, a kind of nature protein fiber, is one of the main use of cashmere goats. The induced activation of secondary hair follicle (SHF) stem cells by the dermal papilla cell-derived signals is a key biological process for the morphogenesis and growth of cashmere fiber in cashmere goats. Previously, the circRNA-ERCC6 (circERCC6) was identified from cashmere goat SHFs; however, its biological significance is unclear in the SHF physiology process of cashmere goats. In this study, we found that circERCC6 exhibited significantly higher expression at anagen SHF bulge compared with the counterpart of telogen and harbored three m6A modified sites (named m6A-685, m6A-862, and m6A-995) through methylation immunoprecipitation using a real-time quantitative polymerase chain reaction (Me-RIP-qPCR) technique. The knockdown experiments of circERCC6 in SHF stem cells showed that circERCC6 positively regulates the induced activation of SHF stem cells in cashmere goats. Through a dual-luciferase reporter assay, we demonstrated that m6A-modified circERCC6 (m6A-circERCC6) sponged miR-412-3p to upregulate the expression of BNC2 mRNA in SHFstem cells. Through m6A-deficient mutant assay in circERCC6 knockdown SHF stem cells, we further showed that m6A modification within circERCC6 is required to mediate the miR-412-3p/BNC2 axis to finally promote the proper induced activation of SHF stem cells in cashmere goats.

The Influence of Genetic Polymorphisms on the Expression of Interleukin-1beta, Prostaglandin E2 and Tumor Necrosis Factor Alpha in Peri-Implant Crevicular Fluid: A Cross-Sectional Study.

The aim of this study was to evaluate the possible relationships between polymorphisms in the interleukin-1 (IL-1) A, IL-1B, and IL-1RN genes and concentrations of the inflammatory mediators IL-1β, tumor necrosis factor-alpha (TNF-α), and prostaglandin E2 (PGE2) in peri-implant crevicular fluid (PICF). A cross-sectional analytical study was conducted on 51 patients with dental implants. Samples from the buccal mucosa were obtained, and genetic analysis was performed using the real-time polymerase chain reaction (PCR) technique for IL-1A and IL-1B and PCR and restriction fragment length polymorphism analysis for IL-1RN. For the biochemical analysis, the concentrations of IL-1β and TNF-α were analyzed using multiplexed fluorescent sphere immunoassays, and PGE2 by enzyme-linked immunosorbent assay. In patients with detected IL-1RN polymorphism, there was an increase in the concentration of the three mediators with statistically significant differences in the mean values of TNF-α and PGE2, regardless of peri-implant health status (p = 0.002 and p = 0.049, respectively). The concentrations of all three mediators were positively and significantly correlated (IL-1β vs. TNF-α Rho = 0.480, p < 0.001; IL-1β vs. PGE2 Rho = 0.382, p = 0.006; and TNF-α vs. PGE2 Rho = 0.528, p < 0.001). We can conclude that the IL-1RN polymorphism exerts an influence on the PICF immune response, which may explain the influence of this genetic polymorphism on the occurrence of peri-implantitis.

Different OXA-Carbapenemases in Genetically Unrelated Klebsiella pneumoniae Strains Isolated in a North Italian Hospital During Multidrug Resistance Screening.

Klebsiella pneumoniae is one of the main opportunistic pathogens that cause a broad spectrum of diseases with increasingly frequent acquisition of resistance to antibiotics, namely carbapenems. This study focused on the characterization of 23 OXA-48-like carbapenemase-producing K. pneumoniae isolates using phenotypic and molecular tests. Phenotypic determination of the presence of β-lactamases was performed using the extended-spectrum beta-lactamase (ESBL) NP test, and phenotypic determination of the presence of carbapenemase was based on the Carba NP test. Antimicrobial susceptibility tests were performed to assess the resistance against carbapenems. Molecular characterization of ESBL genes and carbapenemase genes (blaOXA-48, blaKPC, blaVIM, and blaNDM) was performed using polymerase chain reaction (PCR) techniques. In addition, K. pneumoniae strains were analyzed for their relatedness using multilocus sequence typing PCR analysis based on the Institut Pasteur protocol, which produces allelic profiles that contain their evolutionary and geographic pattern. Following further Sanger sequencing of the blaOXA-48 genes, no genetic mutations were found. Some OXA-48-producing K. pneumoniae isolates coharbored blaKPC, blaNDM, and blaVIM genes, which encode other carbapenemases that can hydrolyze carbapenem antibiotics. The final part of the study focused on the characterization of the plasmid profiles of all isolates to better understand the spreading of the IncL/M blaOXA-48 plasmid gene. The plasmid profile also revealed other incompatibility groups, suggesting that other plasmid genes are spreading in K. pneumoniae isolates, which can coharbor and spread different carbapenemases simultaneously.

Molecular study of hpmA and ureR genes among Proteus mirabilis isolates inpatient with urinary tract infection

The most frequently occurring human bacterial infections are urinary tract infections (UTIs). P. mirabilis is one of the common causes of UTIs in humans with complicated UTIs, the formation of stones in the kidney, and long-term indwelling catheters. This study aims to isolate, and identify Proteusmirabilis from patients with UTIs using traditional methods and 16s rRNA gene sequencing techniques, as well as detect some virulence genes (hpm A and ureR) in Proteus mirabilis by using polymerase chain reaction (PCR). A total of five isolates of Proteus mirabilis were isolated from patients with urinary tract infections in June 2022. Identification of isolates depends on microscopic and cultural characteristics and traditional biochemical tests, finally, isolates are identified by target gene 16s rRNA using PCR technique, in addition, this study investigates the genes responsible for virulence factors such as hpmA and hpmR.In this study laboratory diagnosis and molecular analysis (16s rRNA gene) revealed that the five isolates are Proteus mirabilis also this study shows the presence of the hpmAgenes by PCR techniques in the isolates 100%. Our study has shown that 16s rRNA was a strong eclectic power and an excellent method for the identification of bacteria also these study frequency ofureR and hpmA in P.mirabilis isolated from UTIs and these genes have a manifest role in the infection.

Detection of new single nucleotide polymorphisms in plasma cells carcinoma (myeloma) by polymerase chain reaction–single strand conformation polymorphism and sequencing technical methods

Abstract Background: MicroRNA (miRNA) is a short, single-stranded, non-coding sequence of RNA that does not translate into proteins. Because of their ability to control gene expression, recent studies have shown that these short, non-coding sequences play an important role in cancer. So that, detection the polymorphisms or variants in miRNA genes may help to identify their correlation to cancer susceptibility. Objectives: Study the polymorphisms in miRNA genes or variants miRNA genes and their correlation with myeloma disease. Materials and Methods: Twenty samples were collected from people who visited Marjan Hospital in Province of Babylon, Iraq, from February to July 2022. In addition, 20 samples from healthy people were collected as control. Genotyping of miRNA was accomplished using the polymorphism techniques of polymerase chain reaction and single strand conformation polymorphism (SSCP). DNA sequencing was also applied to categorize all the ranges of SSCP models detected using only gel imaging. Results: The genotypes obtained in this experiment verified that several single nucleotide polymorphisms were obtained between 5-band and 6-band miRNA National center for biotechnology information Primer3 plus reference. Conclusion: The results explain that the miRNA gene polymorphisms may have correlation with the susceptibility to myeloma disease.