Polymerase Chain Reaction Products Research Articles

Puccinia suaveolens Causing Leaf Rust on Cirsium setosum in China.

Thistle, Cirsium setosum (Willd.) M. Bieb., is widely distributed in China as a common weed in fields. It is also used as a traditional Chinese medicine for cooling blood, stopping bleeding, dispelling stasis, detoxifying, and resolving carbuncle. In 2023, we found a rust disease on plants of Cirsium setosum in the experimental field of Hebei Agricultural University, Baoding, Hebei Province, China, with incidence of 15% - 25% (Fig. S1 A, B). The diseased leaves turned yellow, and the leaf edges were slightly rolled. The yellow, oil-like pycnia and pycniospores covered the baxial surface of leaves, and brown pustules were produced after 2-3 weeks. On the adaxial surface of the leaves, the brown rust pustules were mainly along the leaf veins. Stems were also be infected later, and dark pustules were scattered. The diseased plants were relatively short and small, and produced relatively small or no flowers compared to healthy plants. A total of 100 plants with typical leaf rust symptoms and signs were collected. To confirm the pathogenicity, healthy plants of thistle were sprayed with 5 ml of urediospores suspension (2.6×105/ml), and plants sprayed with sterile distilled water were treated as control. The sprayed plants were incubated under high moist conditions at 18°C for 24 h, and the inoculated plants were grown at 20°C in a greenhouse. Ten days after inoculation, the plants inoculated with urediniospores showed rust symptoms with uredinia and urediniospores on the leaves (Fig. S1 C), while the control plants were healthy. For morphological characterization, urediospores were picked from the naturally infected plants and placed in a drop of sterile water on a glass slide using a sterile needle, and observed and measured under a microscope. Urediospores were nearly spherical, brown-yellow, and measured 15 - 25 μm in diameter (n=100) (Fig. S1 D). Telia were scattered on the baxial surface of the naturally infected leaves, and teliospores were oval, yellow-brown, double-celled, with very short hyaline pedicels, and measured 15-20 × 15-30 μm (n=100) (Fig. S1 E). For molecular characterization, about 200 μg of urediniospores was collected and placed in a 1.5 ml sterile centrifuge tube, and genomic DNA was extracted using the cetyl-trimethylammonium bromide method (Gawel et al. 1991). The internal transcribed spacer (ITS) region of the rDNA and the D1/D2 domain were amplified using primer pairs ITS1/ITS4 (White et al. 1990) and NL1/NL4 (Borhani et al. 2013) in polymerase chain reaction (PCR), respectively. The PCR products were sequenced, and their sequences were aligned and compared with those deposited in GenBank. The obtained sequences were deposited in GenBank (OR600240 for ITS and OR598614 for D1/D2), which were 100% identical with 100% coverage to the ITS sequence (ON063373.1) and the D1/D2 sequence (ON063379.1) of Puccinia suaveolens (Menzies 1953). Based on the morphological characteristics and DNA sequences, the isolates were identified as P. suaveolens (Fig. S1 and Fig. S2). Thistle rust caused by Puccinia obtegens has been reported in some other parts of China (Zhang 2012). To the best of our knowledge, this is the first report of P. suaveolens causing leaf rust on C. setosum in China. This discovery is helpful for control of leaf rust on thistle grown for Chines medicine and other purposes, and the rust species could be used for biological control of thistle as a weed in crop fields.

Comparison of pre-labelled primers and nucleotides as DNA labelling method for lateral flow detection of Legionella pneumophila amplicons

Labelling of nucleic acid amplicons during polymerase chain reaction (PCR) or isothermal techniques is possible by using both labelled primers and labelled nucleotides. While the former is the widely used method, the latter can offer significant advantages in terms of signal enhancement and improving the detection limit of an assay. Advantages and disadvantages of both methods depend on different factors, including amplification method, detection method and amplicon length. In this study, both methods for labelling PCR products for lateral flow assay (LFA) analysis (LFA-PCR) were analysed and compared. It was shown that labelling by means of nucleotides results in an increase in label incorporation rates. Nonetheless, this advantage is negated by the need for post-processing and competitive interactions. In the end, it was possible to achieve a detection limit of 3 cell equivalents for the detection of the Legionella-DNA used here via primer labelling. Labelling via nucleotides required genomic DNA of at least 3000 cell equivalents as starting material as well as an increased personnel and experimental effort.

Ultrasensitive electrochemiluminescence biosensor based on polymethylene blue nanoparticles and DNA network for Staphylococcus aureus detection

A novel bipolar electrode (BPE)-electrochemiluminescence (ECL) device was constructed for the ultra-sensitive detection of Staphylococcus aureus (S. aureus) by combining polymerase chain reaction (PCR) amplification and DNA network-loaded polymethylene blue nanoparticles (pMB NPs). The presence of target triggered the dissociation of double-stranded DNA on Fe3O4 NPs and the release of T strand, which initiated the PCR. The PCR product contains two protruding single-stranded DNA fragments that serve as bridges to connect Au NPs labeled probes. The PCR-Au products were captured by the probes on cathode of BPE to form three-dimensional DNA networks, which offer multiple adsorption sites for pMB NPs, leading to the remarkable enhancement of ECL intensity. Under optimal circumstances, a wide linear range from 10 to 108 CFU/mL and a low detection limit of 0.78 CFU/mL were achieved. This research opens new horizons for the application of PCR-based biosensors for the accurate and sensitive measurement of pathogenic bacteria.

Use of the its Region in the Identification of Endophytic Mycota in Fruits of Syagrus Coronata (MART.) BECC., Caatinga Bioma

Objective: The study aimed to identify the endophytic fungi present in fruits of S. coronata (licuri) through sequencing of the ITS (Internal transcribed spacer) region. Theoretical framework: The Syagrus coronata palm tree, known as licuri, plays a significant role in the Caatinga, both in socioeconomic and cultural terms. The fruits of this plant host several types of fungi, including saprophytes, phytopathogens, and endophytes. The complexity of fungi taxonomic identification, due to the diversity and morphological similarity between distinct taxa, often requires the use of molecular biology as an essential tool for accurate characterization. Method: The study used 14 isolates of endophytic fungi from fruits of S. coronota (licuri) preserved in the Mycoteca of the Mycology Laboratory of the Universidade do Estado da Bahia/DEDC - Campus VIII, Paulo Afonso. The DNA of these isolates was extracted to perform PCR (Polymerase Chain Reaction) using the primers ITS1F and ITS4. The PCR products were sequenced and analyzed with the Blastn tool, revealing 98 to 100% similarity in the accessions. Results and conclusion: From the 14 isolates, four presented themselves as Chlamydospores and one as Mycelia sterilia, making morphological identification unfeasible, and four were from taxa without taxonomic identification. The taxa identified using ITS region sequencing belong to the genera Curvularia, Diplodia, Fusarium, Lasiodiplodia, Neodeightonia, and Penicillium. Research implications: This study highlights the importance of using molecular techniques to complement the taxonomy based on morphological markers. Originality/value: The primary purpose of this study was to use the ITS region as a tool for identifying endophytic fungi in licuri fruits, since in the literature the mycota of this botanical substrate, which is a biocultural heritage of Bahia, is practically unknown.

Sex Identification of the Sun Conure (Aratinga solstitialis) Using Calamus Based on Polymerase Chain Reaction

Sex identification of Sun Conures (Aratinga solstitialis) is crucial for breeding and preservation, as well as increasing sun conure populations. These birds are sexually monomorphic. Therefore, Determination between male and female carried out by their morphology examination. The Polymerase Chain Reaction (PCR) method, utilizing molecular-based technology, was employed to determine the sex of Aratinga solstitialis in this study. The P2 and P8 primers were utilized in this method, which has been deemed suitable and accurate for sex identification through calamus samples. The research focused on two 28-month-old Aratinga solstitialis birds. Calamus samples were collected and subjected to PCR amplification using the extracted calamus. The resulting PCR products were then visualized using electrophoresis with a 1% agarose gel. In the electrophoresis photo, the presence of two bands indicated a female specimen, whereas a single band indicated a male specimen. The result of the gel electrophoresis research showed that both of the Aratinga solstitialis were male with one band of each bird on ranged from 300-400 base pairs. The result show that the Polymerase Chain Reaction method in terms for sex identification on monomorphic birds, especially Aratinga solstitialis birds is very effective to differentiate the sex of young birds and the adults.

Towards pharmacogenomics-guided tuberculosis (TB) therapy: N-acetyltransferase-2 genotypes among TB-infected Kenyans of mixed ethnicity

BackgroundThough persons of African descent have one of the widest genetic variability, genetic polymorphisms of drug-metabolising enzymes such as N-Acetyltransferase-2 (NAT2) are understudied. This study aimed to identify prevalent NAT2 single nucleotide polymorphisms (SNPs) and infer their potential effects on enzyme function among Kenyan volunteers with tuberculosis (TB) infection. Genotypic distribution at each SNP and non-random association of alleles were evaluated by testing for Hardy-Weinberg Equilibrium (HWE) and Linkage Disequilibrium (LD).MethodsWe isolated genomic DNA from cryopreserved Peripheral Blood Mononuclear Cells of 79 volunteers. We amplified the protein-coding region of the NAT2 gene by polymerase chain reaction (PCR) and sequenced PCR products using the Sanger sequencing method. Sequencing reads were mapped and aligned to the NAT2 reference using the Geneious software (Auckland, New Zealand). Statistical analyses were performed using RStudio version 4.3.2 (2023.09.1 + 494).ResultsThe most frequent haplotype was the wild type NAT2*4 (37%). Five genetic variants: 282C > T (NAT2*13), 341 T > C (NAT2*5), 803A > G (NAT2*12), 590G > A (NAT2*6) and 481C > T (NAT2*11) were observed with allele frequencies of 29%, 18%, 6%, 6%, and 4% respectively. According to the bimodal distribution of acetylation activity, the predicted phenotype was 76% rapid (mainly consisting of the wildtype NAT2*4 and the NAT2*13A variant). A higher proportion of rapid acetylators were female, 72% vs 28% male (p = 0.022, odds ratio [OR] 3.48, 95% confidence interval [CI] 1.21 to 10.48). All variants were in HWE. NAT2 341 T > C was in strong complete LD with the 590G > A variant (D′ = 1.0, r2 = − 0.39) but not complete LD with the 282C > T variant (D′ = 0.94, r2 = − 0.54).ConclusionThe rapid acetylation haplotypes predominated. Despite the LD observed, none of the SNPs could be termed tag SNP. This study adds to the genetic characterisation data of African populations at NAT2, which may be useful for developing relevant pharmacogenomic tools for TB therapy. To support optimised, pharmacogenomics-guided TB therapy, we recommend genotype-phenotype studies, including studies designed to explore gender-associated differences.

Incidence of Yq Microdeletion among Chattishgarh Population and Cast based distribution

Abstract Background: Millions of individuals in their reproductive years are affected by infertility on a global scale, potentially exerting a significant influence on their lives and family dynamics. The coexistence of abnormal seminogram and Yq microdeletion synergistically affects infertility. Therefore, the study was designed to determine the frequency of distribution of Yq microdeletion in abnormal semen parametric infertility cases. Methodology: Seventy-five cases of infertility and 78 controls with known fertility were enrolled for the cross-sectional study. In the collected blood sample, DNA was isolated and a polymerase chain reaction (PCR) mix for various markers was prepared. After running in a thermocycler, PCR products were analyzed by gel electrophoresis. Results: The distribution of deletion among different subtypes: azoospermic, severe oligozoospermic, oligozoospermic, and normozoospermic cases was 35%, 33%, 35%, and 33%, respectively. The most common deletion type in the Chhattisgarh population was azoospermia factor c. Caste-based distribution among the study group was quite uniform. Conclusion: Y chromosome microdeletion would be an essential test after seminogram in cases of male infertility, especially to prevent the transmission or inheritance of infertility to offspring. Due to the high frequency of microdeletions, it is a very useful test to identify male infertility in Chhattisgarh.

Molecular appraisal of Giardia intestinalis from Western India: A prospective observational study.

Giardia intestinalis is an intestinal protozoan which commonly causes parasitic gastroenteritis globally. It is a species complex consisting of at least eight assemblages (genotypes). In India, Giardia is mostly underreported and missed in asymptomatic cases. The aim of this study was to genotype the G. intestinalis isolates from stool samples of patients at a tertiary care center in Rajasthan, India, and to clinically correlate it. This prospective pilot cross-sectional study was conducted from 2019 to 2021 in a tertiary care center in western India. Patients who were microscopically positive for giardiasis were enrolled. DNA was extracted from their stool samples and amplified by polymerase chain reaction (PCR) using 4E1-HP as the target sequence. Anthropometric measurements and analysis were done for children by using Anthrocal application. A total of 50 patients were enrolled. Diarrhea was present in 18 patients (36%). Among these, 6 were immunocompromised and had different comorbidities. Among the children <12 years of age, 55.17% (n = 16/29) were stunted (<-2 S.D.), and among <5 years, 44.4% (n = 4/9) showed wasting (<-2 S.D.). A PCR product corresponding to assemblage B of G. intestinalis was amplified in 47 stool specimens. Only three stool samples were negative for both assemblages A and B and posed an interesting enigma. In this study, a predominance of assemblage B of G. intestinalis was detected in 94% of the isolates. Furthermore, the possibility of zoonotic transmission could not be ruled out.

Development of antibodies against recombinant staphylococcal enterotoxin B from food poisoning cases.

Staphylococcal enterotoxin B (SEB) is the most common serotype involved in food poisoning. The aim of this study was to develop immunoassay detection methods using a recombinant enterotoxin B antigen protein to produce recombinant polyclonal antibodies in vivo. Staphylococcus aureus isolated from a food poisoning case (strain JH5800) was analyzed by polymerase chain reaction (PCR) and confirmed to contain a seb gene of 477 bp. A SEB segment was amplified, cloned, sequenced, and aligned. The PCR product corresponding to the predicted mature SEB peptide was inserted into Escherichia coli BL21 (DE-3) expression vector and expressed as a hexahistidine-SEB fusion protein. Antiserum against recombinant SEB protein was produced by immunization of Balb/c mice. In the indirect enzyme-linked immunosorbent assay (ELISA), the polyclonal antibodies produced had a titer of 1:3200. The seb gene of Staphylococcus aureus isolated from a poisoning case (JH5800) had a molecular size of about 477 bp and a band of recombinant SEB toxin was observed at approximately 30 kDa on SDS-PAGE gel. The polyclonal anti-SEB antibody titer, as revealed by indirect ELISA, was 1:3200 at 59 days. SEB recombinant protein could be used to produce polyclonal antibodies. ELISA and Western blotting were used to analyze the specificity and sensitivity of the recombinant polyclonal antibodies. Polyclonal antibodies produced could be used to detect SEB on a large-scale.

Prevalence of Wolbachia endosymbiont in Culex molestus mosquitoes from South Korea

AbstractWolbachia, an endosymbiotic bacterium, has a significant impact on the reproductive biology and pathogen transmission capabilities of various arthropods, including mosquitoes. This study aimed to assess the prevalence of Wolbachia in Culex molestus, a vector mosquito species, in South Korea. Mosquito samples were collected from different locations and taxonomically identified. Individual mosquito DNA was extracted, and polymerase chain reaction (PCR) was conducted targeting the Wolbachia surface protein gene (wsp) and three multi‐locus sequence typing (MLST) (gatB, hcpA, and ftsZ) genes. Gel electrophoresis was performed to determine the prevalence of Wolbachia by analyzing PCR products. Our findings revealed that Wolbachia was present in 100% of the tested Culex mosquitoes from South Korea, with one major Wolbachia strain (supergroup B) detected using the wsp gene and subgrouping determined with the gatB as well as hcpA, and ftsZ. These results underscore the widespread occurrence of Wolbachia in Cx. molestus mosquito within the studied region, thus suggesting the potential for leveraging this endosymbiont to devise novel strategies for mosquito‐borne disease control. Future research should investigate the influence of Wolbachia on mosquito biology and vector competence in South Korea.

Identification of Eimeria acervulina (Apicomplexa: Eimeriidae) infecting the broiler chicken Gallus gallus domesticus through morphology and molecular analyses.

Coccidiosis is an intestinal protozoan disease that affects the poultry industry worldwide. The severity of this disease varies depending on the identity of the infectious agents. Therefore, this study was carried out to identify the Eimeria species that affect broiler chickens, Gallus gallus domesticus, through morphological and molecular phylogenetic analyses. Twenty-five faecal samples were collected from the broiler chickens in a commercial poultry farm in Riyadh (Saudi Arabia). Using the floatation technique, faeces were examined microscopically for the Eimeria occurrence. Identification of Eimeria species was performed based on morphological criteria and molecular tools (DNA amplification for the partial small subunit ribosomal RNA (18S rRNA), internal transcribed spacer (ITS)-1, and mitochondrial cytochrome c oxidase I (COI) genes. In this study, 32% (8 out of 25) of collected samples were found to be positive for coccidiosis. After sporulation in potassium dichromate (K2Cr2O7), the sporulated oocysts were observed as ovoid and measured 18.37-23.19 µm (19.87) long and 15.07-18.67 µm (16.46) wide, with the anterior location of a polar granule and absence of micropyle. These Eimeria oocysts were assumed to size and shape characteristics of Eimeria acervulina. Molecular analysis was conducted on the sequences of the polymerase chain reaction products from the three genes studied (18S rRNA, ITS-1, and COI). At the three genes, results showed that the resultant sequences clustered with E. acervulina from different regions confirming morphological description. This study highlighted the importance of molecular techniques to detect avian Eimeria species more than the traditional morphology-based tool to optimise the appropriate anticoccidial strategies for long-term control in the studied area.

Phylogenetic monitoring of Escherichia coli in medical wastewater/Baghdad City

Clean drinking water access is a main factor that supports general health worldwide, where important investment is made to maintain water quality. The gene sequence of 16S rRNA was applied to study bacterial phylogenesis and classification. This studyaimed to isolate new bacterial strains from wastewater environments. Thisstudy collected water samples from differentwastewater sites for Baghdad Medical City’s Hospital. The results of Vitek 2Compact were with a probability of99% forEscherichia coli isolates .The bacterial isolates were identified using Polymerase chain reaction (PCR) based on the universaldiagnostic gene 16SrRNA, and the PCR product was obtained with a molecular weight of 1250bp. The PCR productsequencingshowed 166 isolates of E. coli different from the isolates registered in the NCBI database of E. coli after alignment between them.The present study reported 16 Iraqi isolates in NCBI with an accession number: OM032663.1; OM 032664.1; OM294659.2;ON724178.1; ON724264.1; ON724331.1; ON725076.1; ON725091.1; ON725139.1; ON725141.1; ON739200.1; ON739201.1;ON739207.1; ON739208.1; ON739209.1; ON739211.1. The identity was then observed(96-100%) with strains in Gene Bank.There were many different sequence position substitutions (transition, deletion, transversionfor the registered local isolates whenconducting multiple alignments and comparing themwith the isolates in the Gene Bank. According to the phylogenetic treebuilding by joining-the neighbor method, the local Iraqi isolates clustered into sub-descents. Moreover, local isolates of E.coliappeared more convergent to Saudi Arabia isolates (KY780347.1). This may be due to the geographical proximity of the two countries to the same Arab Gulf region .The present study identified 16 isolates of E. coli from the Iraqi aquatic environment (medical wastewater). The novelty of this study was represented to monitoring the evolution of E. coli in medical wastewater.

New Records in Iraq and Arab Nations for some Fungi Isolated from Al-Barakia wastewater treatment plant in Al-Najaf Province

This study was conducted in 2020 in the wastewater treatment plant in Al-Barakia Najaf, where samples were taken in November from the Bioshft unit and the compact unit from the plant to know the efficiency of the plant in treatment. The process of isolation and purification was Microbiology Laboratory_ Ecology and Pollution Department - College of Science - University of Kufa. Its diagnosis was carried out at the Plant Virus Laboratory - College of Agriculture - the University of Karbala, and (19) fungal isolates isolated from wastewater treatment plants were diagnosed. These isolates were analyzed using the PCR technique and determination of the nucleotide sequences of the polymerase chain reaction products that were amplified from those isolates using ITS4 and ITS1 primers. It belonged to the fungus A. caespitosus. and isolated A flavus (7,8), T. asperellum (9-11) A.tubingensis (12), A.terrus(13), A.niger (14,15), A. alternata (16), C.sphaerospermum (17), A.oryzae (18), Acremonium sp (19), similarity rate of 100% with isolates registered with the NCBI. The results show that all the isolated fungi diagnosed in this study are recorded globally. However, they are not recorded in Iraq, and (5) isolates are not recorded in the Arab nation and Iraq, including A. tubingensis, C.sphaerospermum, A. alternate, and A.oryzae, while the isolate Acremonium sp. According to the National Center for Biotechnology Information NCBI, it is registered only in Germany under the number (AJ557731), and the similarity rate is 100% with the isolate diagnosed in this study. Keywords: wastewater; Molecular Identification; fungi; Polymerase chain reaction (PCR); DNA sequence analysis.

Streamlining Genetic Diagnosis With Long-Range Polymerase Chain Reaction (PCR)-Based Next-Generation Sequencing for Type I and Type II Collagenopathies.

In the practice of clinical genetics, gene testing is usually guided by clinical diagnosis. When dealing with rare diseases, it is often necessary to create new test systems. The handling of a gene with a substantial number of exons poses a challenge both in sequential Sanger sequencing for each exon, and in the setup of capture probes to each exon for next-generation sequencing (NGS). We present very long amplicon sequencing (vLAS), an optimized long-range polymerase chain reaction (PCR)-based NGS method that overcomes this challenge. By utilizing approximately 20 Kb long PCR products and short-read NGS, vLAS is emerging as a highly adaptable and effective solution, especially for genes with numerous exons concentrated in a limited genomic region. Here, we demonstrate vLAS in the analysis of five patients with type I and two with type II collagenopathies. The integration of user-friendly NGS methods into genetic diagnosis enhances the practicality of clinical genetics.

Expression of Melanoma Antigen Genes A11 and A12 in Non-Small Cell Lung Cancer

Highlights:1. In this study, new primers designed using the semi-nested polymerase chain reaction (PCR) method were utilized to identify MAGE-A11 and MAGE-A12 expressions in specimens collected from core biopsy, forcep biopsy, and bronchoalveolar lavage.2. The histopathological analysis revealed positive expressions of MAGE-A11 and MAGE-A12 in specimens diagnosed with non-small cell lung cancer (NSCLC) as well as in specimens with no malignant cells. AbstractThe melanoma antigen gene (MAGE) belongs to the group of cancer testis antigens that are exclusively expressed in germ cells but may be re-expressed in cancer cells. The highly expressed MAGE-A subfamily in lung cancer may potentially be a diagnostic and prognostic biomarker. This study aimed to identify MAGE-A11 and MAGE-A12 expressions in lung tumors obtained from core biopsy, forceps biopsy, and bronchoalveolar lavage specimens. A cross-sectional observational study was conducted on 90 patients clinically diagnosed with lung tumors. These patients received core biopsy, forceps biopsy, and bronchoalveolar lavage interventions after ethical approval was obtained. The complementary deoxyribonucleic acid (cDNA) quality was assessed by the polymerase chain reaction (PCR) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The assessment was performed to ascertain if all specimens exhibited positive PCR amplification of the GAPDH gene. MAGE-A11 and MAGE-A12 were identified through a semi-nested reverse transcription PCR. The positive results were detected by measuring the PCR products, with MAGE-A11 and MAGE-A12 measuring at base pairs (bp) of 858 and 496 in the first and second rounds, respectively. The expressions of MAGE-A11 and MAGE-A12 were observed in 3 (3.33%) and 40 (44.44%) out of 90 specimens, respectively. The prevalence rate of non-small cell lung cancer (NSCLC) was 31.11% (28/90). Among these cases, 3.57% (1/28) showed the expression of MAGE-A11, while 32.14% (9/28) exhibited the expression of MAGE-A12. Sixty-two (68.89%) out of 90 patients were diagnosed with no tumor cell malignancy. Out of 62 cases, 2 (3.23%) exhibited the expression of MAGE-A11, while 31 (50%) demonstrated the expression of MAGE-A12. MAGE-A11 and MAGE-A12 were detected in NSCLC and in certain specimens with a pathological diagnosis that indicated the absence of malignant cells. In conclusion, MAGE A11 and MAGE A12 have potential markers that can improve the pathological diagnosis of lung cancer. Further investigation is necessary to explore the expression of MAGE-A in correlation with lung cancer progression.

A 3D-printed oscillatory polymerase chain reaction system using a single heater

Nucleic acid amplification is a process that arises in all organisms. Polymerase chain reaction (PCR) is a standard laboratory nucleic acids amplification technology in vitro widely used in medical detection and biometric-related research. This device represents the first demonstration of 3D-printed oscillatory thermocycling within a portable PCR device. This study uses a single Peltier element to create the three temperature regions required for PCR. The overall control system uses an Arduino microcontroller with the advantages of easy development, abundant resources, and easy access to device software design. The sample-driving system integrated with the 3D printed object and the laser-cut acrylic component to achieve the linear reciprocating motion of the sample is conducive to the realisation of the portable design of the device. After system optimisation, the reaction tube moves from the denaturation zone to the aluminum block for cooling, and it moves to the groove of the denaturation zone from the annealing zone to accelerate the sample temperature rise. The time duration reduces from 180 s to 85 s to complete one thermal cycle. Combining the 5 mm instead of 3 mm thickness of the acrylic material and the configuration of the acrylic end exposed in the air creates a more significant temperature difference from 25 °C to 40 °C. Using this oscillatory PCR device, we successfully amplified a 219 bp PCR product of turmeric DNA after 30 thermal cycles within 50 min. The developed PCR device will cost less than $300 USD in our work. Compared with previous works, the cost of this research device is relatively low, and the total volume of the device is quite small. This work should be interesting to persons involved in diagnosing the infectious diseases of viruses and bacteria and identifying the fake healthy food in the low-cost bioreaction chambers in fields and remote areas.

Molecular Analysis of Bacterial Meningitis in Suspected Cases

One hundred and seventy-six cases of suspected meningitis (SMN) were included in a cross-sectional study. Their ages ranged from less than 1 year to 80 years, of whom 44.3% were male. The aim was to assess bacterial meningitis (BMN) in terms of incidence and types of causative bacteria. Cerebrospinal fluid (CSF) specimens were collected and polymerase chain reaction (PCR) analysis was conducted with universal primers designed to amplify a DNA fragment (996 bp) of the 16S rRNA gene of eubacteria. Resolving PCR products in agarose-gel electrophoresis revealed that 37.5% of CSF specimens were PCR positive, while 62.5% of CSF specimens showed no band and were considered PCR-negative. Eighty percent of the latter specimens were noted to have pleocytosis, in addition to having protein and/or glucose concentrations lower or higher than those in the normal range (abnormal CSF). The remaining 20.0% of CSF specimens were considered normal regarding pleocytosis, protein, and glucose (normal CSF). When the amplified DNA of PCR-positive specimens was subjected to sequencing and alignment with reference sequences, both Gram-negative (GN) and Gram-positive (GP) bacteria were identified (23 and 9 types, respectively). Acinetobacter spp. and Pseudomonas spp. were the most common GN bacteria (each with 17.8%), while Staphylococcus spp. was the most common GP bacteria (43.75%). The study concluded that BMN presents an important public health challenge, and PCR analysis of the CSF was an effective method for diagnosing pathogenic bacteria in SMN. In addition, leukocytes, glucose, and protein are valuable CSF parameters that may aid in the diagnosis of BMN.

Molecular Characterization and Zoonotic Significance of Cryptosporidium spp. and Giardia duodenalis in Asymptomatic Adult Stray Cats and Dogs in Turkey

Background: Cryptosporidium species and Giardia duodenalis are important protozoan parasites due to their wide host range and potential as zoonotic diseases. These parasites, responsible for Cryptosporidiosis and Giardiasis, are significant health concerns and have garnered attention from both the public and veterinary fields. Pets, such as dogs and cats, are susceptible to these infections and play a crucial role in transmitting these protozoans to humans. This study was conducted to determine the prevalence, species, genotypes and zoonotic significance of Cryptosporidium and Giardia in asymptomatic adult stray cats and dogs. Methods: Fecal samples from 75 adult stray animal, 42 dogs and 33 cats, were collected in Diyarbakýr city, southeastern Anatolia, Turkey. Direct immunofluorescence test was used to confirm the presence of G. duoenalis cysts and cryptosporidium spp. oocysts in collected fecal samples. Microscopic analysis was used to count the number of cysts/oocysts per gram as specified by the manufacturer. Molecular confirmation was done with Polymerase chain reaction (PCR) using Giardia SSU-rDNA and Cryptosporidium spp. SSU rRNA gene. The secondary PCR products of all positive samples were sequenced in one directions on an automated sequencer. Nucleotide sequence analysis was performed by BLAST alignment using the National Center for Biotechnology Information database. Result: The prevalence of Cryptosporidium was 7.14% in 42 cats and 9.09% in 33 dogs. The prevalence of G. duodenalis was 23.80% in cats and 54.54% in dogs. The average number of Cryptosporidium spp. oocysts per gram of cat and dog feces was 1866.3 and 1813.3, respectively. The average number of G. duodenalis cysts per gram of cat and dog feces was 1011.6 and 4786, respectively. All Cryptosporidium isolates were determined as C. canis in dogs and C. felis in cats. C. canis and C. felis isolates identified in the study, MT329018.1 and MN696800.1, AF159113.1 and KM977642.1 showed similarity with Genbank number, respectively. Assemblages A, E, C, D were found in dogs and assemblages A and E in cats in the result of the sequence of 130 bp gene fragments obtained from G. duodenalis isolates. This study sequence analysis of G. duodenalis cat isolates identified assemblages A (50%/10) and E (50%/10). In this study, assemblage E was the most common genotype isolated in dogs (38.8%), followed by Assemblage C (27.7%), Assemblage D (16.6%) and Assemblage A (15.5%). Assemblage A, which has zoonotic importance, was detected in cats and dogs, while assemblage E in cats and dogs was reported for the first time in Turkey.

Species Identification of Sea Bamboo (Isis hippuris) using COI-based DNA Barcoding

Conservation and effective management of marine ecosystems and biodiversity requires accurate species identification. This study classifies sea bamboo (Isis hippuris) specimens using DNA barcoding, a technique widely recognized for its speed, accuracy, and objectivity. This study examines the cytochrome c oxidase subunit I (COI) gene analysis for species identification. Isis hippuris was collected from two stations (coral and seagrass areas) of Tanjung Tiram Waters, South Konawe, Southeast Sulawesi. Genomic DNA was extracted from the base, main, and lateral stem of I. hippuris. Polymerase chain reaction (PCR) was used to amplify the mtDNA of I. hippuris with HCO2198 and LCO1490 primers. The highest quality PCR product based on the COI gene was chosen for sequencing analysis. The study revealed that COI gene analysis could only be performed on the base and main stem of the I. hippuris. Samples from coral and seagrass-coral areas on lateral stems were not further analyzed due to low concentration and purity values, which could potentially fail DNA sequencing. Each part of I. hippuris may have unique genetic differences. This study highlights the advantages of DNA sequencing in providing a unique genetic fingerprint for each species, enabling accurate species identification. This research provides insight into using DNA barcoding for sea bamboo species identification.

A New Duplication in 668 to 672 Sites of UL54 Gene in Cytomegalovirus Ganciclovir Resistant Liver Transplanted Patients

Background: In liver transplant recipients, human Cytomegalovirus (CMV) infection is a significant concern. Ganciclovir is the preferred medication for treating widespread CMV infections. In some cases, patients might require high doses of treatment for CMV infections that are resistant to ganciclovir, although liver transplant patients who have received extended ganciclovir and valganciclovir prophylaxis have reported infrequent cases of ganciclovir-resistant CMV infections. Mutations in the UL54 gene, responsible for encoding deoxyribonucleic acid (DNA) polymerase, can result in resistance. Objectives: In this study, the focus was on UL54 mutations and their association with ganciclovir resistance. Methods: In this study, 23 liver transplant recipients who were admitted to the transplant departments of Namazi and Abu Ali Sina hospitals within 2015 and 2017 were examined. Cytomegalovirus infection was then confirmed in them using the quantitative real-time polymerase chain reaction (PCR) method. The UL54 mutations were found after electrophoresis using the nested PCR method, and the PCR products were subsequently sequenced using the Sanger method. Sequence analysis and locating of UL54 mutations were performed using Finch software (version 1.4.0). Results: After sequencing 52 samples from 23 patients, 25 mutations in the UL54 gene were identified in 9 patients who were CMV-infected, occurring at a median of 32 days after transplant. These mutations, including S655L (10/9, 40%), N685S (8/9, 32%), F669L (4/9, 16%), A688V (2/9, 8%), and the novel AK124703.1: p.V668-G672dup (1/9, 4%), were detected in 9 liver transplant recipients over a median period of 2 years in the UL54 gene. Furthermore, a phylogenetic analysis was conducted to investigate the origins of these mutations in CMV isolated from the Iranian population. Conclusions: Considering that treatment with the drug ganciclovir has led to resistance mutations, particularly the new AK124703.1:p.V668-G672dup mutation, and inefficiency in treatment, it is necessary to determine drug-resistant CMV strains and closely monitor these patients. This includes determining viral load, assessing response to treatment, and identifying non-response at regular intervals until the viral load is completely eradicated in order to ensure the effectiveness of the treatment.