Polymerase Chain Reaction Products Research Articles

Novel Gammaherpesvirus Infections in Narrow-Ridged Finless Porpoise (Neophocaena asiaeorientalis) and False Killer Whales (Pseudorca crassidens) in the Republic of Korea

Abstract: A female narrow-ridged finless porpoise (Neophocaena asiaeorientalis) stranded on a beach on Jeju Island showed epithelial proliferative skin lesions on its body. Two false killer whales (Pseudorca crassidens), caught using nets near Gangneung and Samcheok, respectively, had multiple plaques on their penile epidermis. Histological examination of the epidermis revealed that all the lesions had common features, including accentuated rete pegs, ballooning changes, and eosinophilic intranuclear inclusion (INI) bodies. Based on the histopathological results, herpesvirus infection was suspected, and thus further analysis was conducted using herpesvirus-specific primers. Based on nested polymerase chain reaction (PCR) tests using the herpesvirus-detectable primers, the PCR products demonstrated two fragments: a 222-base-pair (bp) sequence of the DNA polymerase gene, SNUABM_CeHV01, showing 96.4% identity with a bottlenose dolphin herpesvirus from the Jeju narrow-ridged finless porpoise; and a 222 bp sequence of the DNA polymerase gene, SNUABM_CeHV02, showing 95.95% identity with the same bottlenose dolphin herpesvirus from the Gangneung and Samcheok false killer whales. The significance of this study lies in its ability to demonstrate the existence of novel cetacean herpesviruses in South Korean seawater, representing an important step forward in studying potentially harmful pathogens that affect endangered whale and dolphin populations.

Clinical Characteristics and Molecular Insights of Carbapenem-Resistant Klebsiella pneumoniae Isolates from Patients in Intensive Care Units.

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP), a significant worldwide public health threat, is common in patients in intensive care units. Methods: A retrospective study was conducted over a period of 22 months to assess the risk factors associated with infection caused by CRKP isolates. Strain identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and antimicrobial sensitivity was assessed using the micro broth dilution method and Kirby-Bauer test. The genes blaKPC, blaOXA-48, blaNDM, blaVIM, and blaGES were amplified using polymerase chain reaction (PCR), followed by sequencing of the PCR products. The polymerase hypermucoviscosity phenotype was determined using the string test. Capsular serotypes (K1, K2) and presence of the virulence gene (rmpA) in positive isolates were investigated using phenotypic tests followed by PCR. Results: Length of hospitalization and use of carbapenems were associated with CRKP infection. CRKP isolates exhibited extensive drug resistance, but retained sensitivity to colistin and ceftazidime-avibactam (CZA). The main gene detected in 35 CRKP isolates was blaKPC-2. In addition, 11 strains were positive in the string test, and two of these strains carried rmpA. Conclusions: Prolonged hospitalization and carbapenem exposure increased the risk of CRKP infection in intensive care unit (ICU) patients. The prevalence of CRKP carrying the blaKPC-2 gene was high, and suspected hypervirulent carbapenem-resistant K. pneumoniae isolates were scattered.

L-DNA-Based Melt Analysis Enables Within-Sample Validation of PCR Products.

The melt analysis feature in most real-time polymerase chain reaction (PCR) instruments is a simple method for determining if expected or unexpected products are present. High-resolution melt (HRM) analysis seeks to improve the precision of melt temperature measurements for better PCR product sequence characterization. In the area of tuberculosis (TB) drug susceptibility screening, sequencing has shown that a single base change can be sufficient to make a first-line TB drug ineffective. In this study, a reagent-based calibration strategy based on synthetic left-handed (L)-DNA, designated LHRM, was developed to confirm validation of a PCR product with single base resolution. To test this approach, a constant amount of a double-stranded L-DNA melt comparator was added to each sample and used as a within-sample melt standard. The performance of LHRM and standard HRM was used to classify PCR products as drug-susceptible or not drug-susceptible with a test bed of nine synthetic katG variants, each containing single or multiple base mutations that are known to confer resistance to the first-line TB drug isoniazid (INH). LHRM achieved comparable classification to standard HRM relying only on within-sample melt differences between L-DNA and the unknown PCR product. Using a state-of-the-art calibrated instrument and multiple sample classification analysis, standard HRM was performed at 66.7% sensitivity and 98.8% specificity. Single sample analysis incorporating L-DNA for reagent-based calibration into every sample maintained high performance at 77.8% sensitivity and 98.7% specificity. LHRM shows promise as a high-resolution single sample method for validating PCR products in applications where the expected sequence is known.

UNVEILING THE GENETIC BASIS OF BACTERIOCIN PRODUCTION FROM Enterococcus faecium ATCC 19434

The prevalence of resistance to traditional antimicrobials is increasing rapidly, leading to a significant rise in annual deaths from antibiotic-resistant illnesses. In 2020, the global death toll from such diseases was approximately 500,000. If current trends continue, this figure could exceed 10 million by 2050, with associated economic damages surpassing 100 trillion USD. These alarming statistics highlight the urgent need for alternative antimicrobials. Bacteriocins are considered among the most promising options. Enterococcus faecium ATCC 19434 is known to produce a potent bacteriocin effective against Listeria monocytogenes and Staphylococcus aureus, though its specific bacteriocin and genetic properties have not been fully explored. This study aimed to identify the gene responsible for bacteriocin production in Enterococcus faecium ATCC 19434. DNA was extracted from the bacterium, and polymerase chain reaction (PCR) was conducted. The PCR products were analyzed through agarose gel electrophoresis, sequenced, and subjected to homology searches using the BLASTN and BLASTX tools from the National Center for Biotechnology Information (NCBI). Results revealed that Enterococcus faecium ATCC 19434 contains a gene encoding enterocin B. Additionally, it was found to harbor the gene for enterocin A immunity protein production. This discovery paves the way for future genetic modification of Enterococcus faecium ATCC 19434 to enhance enterocin B synthesis, facilitating its commercial application.

First Report of Botrytis cinerea Causing Stem Rot on Hydrangea macrophylla in China.

Hydrangea (Hydrangea macrophylla), commonly referred to as big leaf hydrangea, is a species within the Hydrangeaceae family notable for its ornamental value. Characterized by its vividly colored sepals and lush, striking inflorescences, this species is globally esteemed as both a potted and landscape plant. Notably, in 2022, an alarming incidence of stem rot was observed in approximately 40% of H. macrophylla plants aged between six and twelve months within 16 greenhouses situated in Nanjing City (N 31°14', E 118°22'), Jiangsu Province, China. Initial symptoms of the disease manifested as wet gray-black spots at the base of the seedlings and stems, progressing to a necrotic gray-white discoloration in the stems and accompanied by the growth of gray mold on the affected parts. This infection ultimately led to the wilting of the leaves and the death of the seedlings. For pathogen identification, stem tissues at the interface of diseased and healthy sections were excised, surface-sterilized with 75 % ethanol for 30 s, followed by a 2 - 3 min treatment with 3% sodium hypochlorite, and subsequently rinsed three times with sterile water before air drying. Sections measuring 2 - 3 mm were then cultured on potato dextrose agar (PDA) medium, supplemented with 50 mg/mL rifampicin (RFP), and incubated at 25 ℃ for 3 - 5 d (Zhou et al. 2022). Upon 2 - 3 days of incubation, notable growth of fungal colonies was observed. Mycelial clusters from the periphery of these colonies were subsequently transferred to fresh PDA plates and incubated at 25 ℃ for an additional 5 - 7 d. A particular colony, designated JSNJ2022-2 and now preserved at the Jiangsu Academy of Agricultural Sciences, was selected for detailed examination. This colony exhibited a flocculent texture, with a coloration ranging from grey-white to light brown. It was characterized by the presence of irregularly formed, hard sclerotia within the hyphae. The conidiophores were observed to be slender and erect, featuring dendritic branches at their extremities. The conidia were clustered on the conidiophore like grapes. These conidia were generally colorless or grey, oval in shape, smooth and transparent, and measured between 6.4 - 12.2 × 7.3 - 18.2 μm (n = 50). For genetic analysis, genomic DNA (gDNA) was extracted using the DNA secure Plant Kit (Tiangen Biotech, Beijing, China). Polymerase chain reaction (PCR) amplification was performed using a set of universal primers of ITS1/ITS4 (White et al. 1990), primers corresponding to the specific sequences of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) (Yang et al. 2020). The resultant PCR products were sequenced, and the resulting sequences were submitted to the GenBank database, under the accession numbers OP131597, OP142320, OP142321, and OP142322, respectively. BLAST analysis of the sequences obtained from the isolate JSNJ2022-2 revealed a high degree of genetic similarity, ranging from 99 to 100%, with known sequences of Botrytis cinerea (accessions MK051124.1, MH796662.1, MH479931.1, and KU760986.1). To elucidate the phylogenetic position of the isolate, a phylogenetic tree was constructed using the maximum likelihood method, supported by 1,000 bootstrap replications, in the Mega7 software (Kumar et al. 2016). The results of this analysis confirmed that the strains under study clustered within the same branch as B. cinerea. To establish the pathogenicity of the isolate, Koch's postulates (Falkow 1988) were employed. Healthy potted H. macrophylla seedlings, approximately three months old, were wound inoculated at the base of the seedlings with a 6 mm diameter mycelium plug of JSNJ2002-2 cultivated on PDA for 3 days, which was subsequently covered with moistened degreasing cotton. Control plants were treated with moistened degreasing cloths minus the pathogen. Post-inoculation, these plants were placed in a growth chamber maintained at 25 ℃ with a relative humidity range of 60 - 80%. After a 3-d incubation period, the inoculated plants displayed symptoms identical to those initially observed in the greenhouse. The pathogen was successfully re-isolated from these inoculated plants and was morphologically re-confirmed as B. cinerea, thus satisfying the criteria of Koch's postulates. To our knowledge, this report represents the first documented incidence of B. cinerea causing stem rot in H. macrophylla in China.

Genotype-phenotype analyses of Iranian patients with hemophilia B (Leyden -) and hemophilia B (Leyden +): A single-center study

BackgroundThere is a high prevalence of inherited bleeding disorders in Iran, such as hemophilia A (HA) and hemophilia B (HB). This study aimed to analyze the molecular and clinical profiles of patients with HB. MethodsA single-center study was conducted among patients with severe HB between March 20, 2000, and June 31, 2023. The polymerase chain reaction (PCR) amplification was used for all of the major regions, such as the promoter, the exons, the adjacent intronic regions, and the untranslated regions of the F9 gene. Finally, Sanger sequencing was performed on the PCR products. ResultsA total of 111 HB patients (17 with HB [Leyden +] and 94 with HB [Leyden -]) were enrolled in this study. Among 94 patients with HB (Leyden -), 59 (62.8%) had missense, 21 (22.3%) had nonsense, and 8 (8.5%) had frameshift mutations. Moreover, the most frequent pathogenic variant in HB (Leyden +) was c.-17 A>G in this study. ConclusionThe results of this study confirm that HB is caused by a wide range of molecular defects in Iran. Thus, by knowing the genotypes and phenotypes, we would be able to stratify the patients which is important in terms of their management and outcome.

Molecular identification of Entamoeba spp. in humans and cattle in Baghdad, Iraq.

A total of 10% of the global population succumbs to amoebiasis yearly, equating to 50,000-100,000 recorded fatalities. It is closely associated with contaminated food and water supplies due to human feces. The disease's pathophysiology remains a subject of ongoing debate among experts. Some experts attribute the role of the host's conditions, parasite species and strain, and infection intensity in eliciting clinical symptoms. The aim of this study was to perform molecular identification of Entamoeba species isolated from humans and cattle. Stool samples from three hundred patients and one hundred cattle were collected from different regions, age groups, and sexes in Baghdad for microscopic examination. One hundred randomly chosen patient and cattle stool samples underwent microscopic examination and conventional polymerase chain reaction (PCR) targeting the 18S rRNA gene. Phylogenetic tree analyses were performed for Entamoeba species identification. The infection rate in humans differed significantly (p < 0.05) between age groups and genders, totaling 38%. The infection rate in cattle, determined by conventional PCR, differed significantly (p < 0.05) between age groups and genders, amounting to 58%. Ten PCR products with positive results were sequenced and deposited in GenBank database. Sequence analysis detected that Eight sequences belong to E. histolytica (OM268853.1, OM268854.1, OM268855.1, OM268857.1, OM268858.1, OM268860.1, OM268861.1, and OM268862.1) and two sequences belong to E. dispar (OM268856.1 and OM268859.1) in humans, while 10 sequences (ON724165.1 to ON724174.1) belongs to E. histolytica in cattle. The increased susceptibility of cattle to E. histolytica suggests a considerable role in human infection and substantial public health risks. Further research should be conducted on the many virulence factors such as HM1:IMSS strain, cysteinprotease (Cp1), Gal/lectin, etc. of E. histolytica and E. dispar.

Long-range and real-time PCR identification of a large SERPINC1 deletion in a patient with antithrombin deficiency.

Congenital antithrombin (AT) or serpin C1 deficiency, caused by a SERPINC1 abnormality, is a high-risk factor for venous thrombosis. SERPINC1 is prone to genetic rearrangement, because it contains numerous Alu elements. In this study, a Japanese patient who developed deep vein thrombosis during pregnancy and exhibited low AT activity underwent SERPINC1 gene analysis using routine methods: long-range polymerase chain reaction (PCR) and real-time PCR. Sequencing using long-range PCR products revealed no pathological variants in SERPINC1 exons or exon-intron junctions, and all the identified variants were homozygous, suggesting a deletion in one SERPINC1 allele. Copy number quantification for each SERPINC1 exon using real-time PCR revealed half the number of exon 1 and 2 copies compared with controls. Moreover, a deletion region was deduced by quantifying the 5'-upstream region copy number of SERPINC1 for each constant region. Direct long-range PCR sequencing with primers for the 5'-end of each presumed deletion region revealed a large Alu-mediated deletion (∼13kb) involving SERPINC1 exons 1 and 2. Thus, a large deletion was identified in SERPINC1 using conventional PCR methods.

Molecular Characterization of Methicillin-resistant Staphylococcus aureus (MRSA) Isolates from Ruminants

Background: Methicillin-resistant Staphylococcus aureus (MRSA) infection is one of the leading causes of infections in animals as well as human beings and is associated with significant morbidity, mortality, length of stay and cost burden. Methods: In this study, 725 samples of nasal swabs and milk were collected randomly from cattle, buffalo, sheep and goats and these samples were inoculated on Mannitol salt agar mixed with Oxacillin Resistance Selective Supplement for the molecular characterization of MRSA isolates through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and polymerase chain reaction (PCR) test. Result: Out of 725 samples, 171 were found positive for Oxacillin resistant S. aureus. The SDS-PAGE showed different bands of molecular weight 13, 17, 20, 24, 26, 28, 33, 36, 39, 43, 47, 59, 64, 72, 86, 97 and 121 kDa. An amplified mec A DNA fragment (137) of 533 base pairs (bp) and PCR product of fem A (133 isolates) of 510 bp were detected in isolates.

Thermally resistant nuclease in Serratia marcescens hinders PCR reactions and degrades PCR products.

Polymerase chain reaction (PCR) is an important tool for exogenous gene acquisition and recombinants identification. There exist two problems when using Serratia marcescens as a template for PCR amplification: amplified PCR products are rapidly degraded, and the results of PCR amplification are unstable. The aim of the present work was to elucidate the reasons for this. By mixing PCR products amplified from Escherichia coli DH5α with S. marcescens supernatant or pellet, we found that the DNA-degrading substance in S. marcescens is thermally resistant and present both intracellularly and extracellularly. We then determined that it is protein, and most likely S. marcescens nuclease, that degrades PCR products since the addition of SDS and EDTA can effectively inhibit or block the degradation of PCR products. By knocking out the S. marcescens nuclease encoding gene, nucA, we confirmed that the nuclease is responsible for the degradation of PCR products and the instability of PCR amplification. This work is the first to show that the S. marcescens nuclease is temporarily and partially inhibited by high temperatures during PCR and recovers rapidly at room temperature after PCR.

First Report of Neofusicoccum parvum Causing Seedpod Blight on Lotus (Nelumbo nucifera) in China.

Lotus (Nelumbo nucifera Gaertn.) is a widely cultivated plant in China, and the fruit lotus variety has a high economic value attributed to the exquisite flavor of its fresh seeds. During the summer of 2023, an unidentified blight was observed affecting lotus seedpods in Jiande City, Zhejiang province, with approximately 65% of seedpods impacted in a 130-hectare area. The initial symptoms included dark purple spots on the lotus seedpod surface, which gradually expanded over time. After 5 to 7 days, the entire seedpod turned black, withering, and rendering the lotus seeds inedible. To identify the causal agent, tissues from symptomatic seedpods were excised and disinfected in 75% ethanol for 60 s, and washed twice in sterile distilled water. The disinfected symptomatic tissues (5 × 5 mm) were plated on potato dextrose agar (PDA), incubated at 25 ℃, transferred hyphal tips to obtain pure isolates after 3 days. Fungal colonies exhibiting Botryosphaeriaceae morphology were isolated from 33% of the samples (n = 15). Pure cultures were grown on PDA for both morphological and molecular identification. The colonies displayed a white aerial mycelium, turning olivaceous grey after 7 days. Pycnidia were produced within 3 weeks on PDA with added sterilized healthy lotus seedpod pieces on the surface. Conidia were hyaline, unicellular, ellipsoidal, 12.65 to 20.72 × 3.92 to 9.38 μm in size (mean 16.67 × 6.24 μm, n = 100). To determine the fungal species, genomic DNA was extracted from one representative isolate (ZJUP1112-1), to amplify four gene loci through polymerase chain reactions (PCR): rDNA internal transcribed spacer (ITS) with primers ITS1/ITS4, rDNA large subunit (LSU) with LR0R/LR5, the translation elongation factor 1-alpha gene (tef1) with EF1-728F/EF1-986R, and β-tubulin gene (tub2) with Bt2a/Bt2b. The PCR products were Sanger sequenced in Zhejiang Shangya biotechnology co., LTD, and the resulting sequences were assembled and deposited in GenBank (ITS: OR740546; LSU: OR740547; tef1: OR776996; tub2: OR776997). BLAST searches indicated the highest nucleotide sequence identity with the reference strains of Neofusicoccum parvum CMW 9081 (ITS: 98.8%, AY236943; LSU: 100%, AY928045; tef1: 99.6%, AY236888; tub2: 99.3%, AY236917). Multi-locus phylogenetic analyses revealed that isolate ZJUP1112-1 formed a highly supported clade with N. parvum. Pathogenicity tests were performed on healthy lotus seedpods using mycelial plugs (5 mm diameter) from actively growing colonies of ZJUP1112-1 that were placed onto the front and side of the seedpods (6 each). Controls received PDA plugs. Treated seedpods were wrapped with parafilm and incubated at 25 ℃ and the experiment was repeated three times. After 5 days, dark purple lesions were observed on the inoculated seedpods, whereas controls remained symptomless. The same isolate was recovered from the margin of resulting lesions and confirmed by morphology, thus fulfilling Koch's postulates. N. parvum is a polyphagous pathogen causing blights and fruit rot on multiple economically important fruit crops, such as cacao (Puig et al. 2019), walnut (Chen et al. 2019), pistachio (Lopez-Moral et al. 2020), chestnut (Seddaiu et al. 2021), blueberry (Spetik et al. 2023) and mango (Polizzi et al. 2022), among others. To the best of our knowledge, this is the first report of N. parvum causing seedpod blight on lotus seedpods in China, which contributes to a better understanding of the pathogens affecting this plant species in China.

Spatial Distribution and Seasonal Variation of Antibiotic-Resistant Bacteria in an Urban River in Northeast China

As the largest freshwater river flowing through Harbin, the Songhua River is a standby water source. It is very important to know the species and distribution of antibiotic-resistant bacteria (ARB) in the river. In this study, five antibiotics were selected to screen and identify ARB in spring and autumn. The results showed that the concentration of cefotaxime-resistant bacteria was the highest, and the maximum concentration at S6 in spring was up to 1.40 × 104 CFU/mL. In spring and autumn, bacteria resistant to three antibiotics were screened at S1 of the Songhua River, and bacteria resistant to five antibiotics were screened at S6. No multiple antibiotic-resistant bacteria (MARB) were screened in the other four sites in autumn, while MARB were screened in the other three samples except S2 in spring. In all sample areas in spring and autumn, the probability of screening MARB at S1 and S6 was the highest, reaching 100%. The identification results of 16S rDNA polymerase chain reaction (PCR) products of ARB showed that a total of 51 ARB strains from 15 bacterial genera were screened in the Songhua River, of which 20 ARB strains were from Pseudomonas. Among the 15 bacterial genera, bacteria from 8 bacterial genera have pathogenicity. The results of this study revealed the concentration, spatial distribution, and seasonal variation of culturable ARB in the Songhua River, providing data support for the remediation of antibiotic resistance gene (ARG) pollution in the river.

Short-homology-mediated PCR-based method for gene introduction in the fission yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe is a commonly utilized model organism for studying various aspects of eukaryotic cell physiology. One reason for its widespread use as an experimental system is the ease of genetic manipulations, leveraging the natural homology-targeted repair mechanism to accurately modify the genome. We conducted a study to assess the feasibility and efficiency of directly introducing exogenous genes into the fission yeast S. pombe using Polymerase Chain Reaction (PCR) with short-homology flanking sequences. Specifically, we amplified the NatMX6 gene (which provides resistance to nourseothricin) using PCR with oligonucleotides that had short flanking regions of 20bp, 40bp, 60bp and 80bp to the target gene. By using this purified PCR product, we successfully introduced the NatMX6 gene at position 171 385 on chromosome III in S. pombe. We have made a simple modification to the transformation procedure, resulting in a significant increase in transformation efficiency by at least 5-fold. The success rate of gene integration at the target position varied between 20% and 50% depending on the length of the flanking regions. Additionally, we discovered that the addition of dimethyl sulfoxide and boiled carrier DNA increased the number of transformants by ~60- and 3-fold, respectively. Furthermore, we found that the removal of the pku70+ gene improved the transformation efficiency to ~5% and reduced the formation of small background colonies. Overall, our results demonstrate that with this modified method, even very short stretches of homologous regions (as short as 20bp) can be used to effectively target genes at a high frequency in S. pombe. This finding greatly facilitates the introduction of exogenous genes in this organism.

KRAS and BRAF genetic alterations in lung cancer: A case - control study.

Lung cancer (LC) is one of the most critical neoplastic abnormalities, having globally a high mortality rate. Knowledge about its genetic mutations and their association with clinically pathological features of LC is very important. Here, we describe the epidemiological molecular study of genetic mutations in KRAS and BRAF genes and their relationship with the demographic and clinical characteristics of Pakistani patients with lung adenocarcinoma. To analyze the mutations of KRAS and BRAF in LC patients among Pakistani population. The study has been carried out at universities and health institutes of Islamabad, Pakistan. Deoxyribonucleic acid (DNA) was extracted from the patient samples by using the standard protocol and amplified by using the specific primers. Later on, the Polymerase Chain Reaction (PCR) products were examined with the help of single stranded conformational polymorphism (SSCP). Relationship between KRAS, BRAF mutations, and LC risk was accessed by conditional logistic regression using SPSS version 24.0. Results were illustrated by odds ratio (OR), 95% confidence interval (CI), and P value. LC is more common in male population and smoking is one of the leading risk factors for (p < 0.0001) LC. KRAS and BRAF mutations were found to be contributing factors toward LC development and showed statistically significant results along with conformation through computational analysis. It can be concluded that smoking is lethal and cancer causing. The concomitant mutations found in KRAS and BRAF were infrequent, and they probably have a very unusual effect on the clinical management of Pakistani patients with lung adenocarcinoma.

Molecular Detection, Antibiotic Resistance, and Biofilm Formation of Clinical Pseudomonas aeruginosa Isolates from Patients in Al-Hillah, Babylon Province, Iraq

Abstract Background: Pseudomonas aeruginosa is known as one of the main causes of nosocomial infections and is repeatedly related to opportunistic infections among hospitalized patients worldwide. Antibiotic resistance (AR) and biofilm-forming ability (BFA) are two major virulence factors of P. aeruginosa isolates that are involved in the continuation of infections. Objectives: This study aimed to isolate and identify P. aeruginosa isolates from patients and to elucidate the correlation between AR and BFA among isolates. Materials and Methods: A total of 130 clinical P. aeruginosa isolates were obtained from Al-Hillah Al-Sadiq Hospital in Babylon province, Iraq during the period from March to July 2022. AR profile and BFA of P. aeruginosa isolates were proceeded by Kirby-Bauer and Congo red agar (CRA) methods, respectively. Polymerase chain reaction (PCR) screening, targeting the ecfX gene, was conducted to identify species-specific ecfX genes in P. aeruginosa. Results: Multidrug-resistant (MDR) P. aeruginosa accounted for 96.1% (125/130) and 85.4% (111/130) of isolates showed BFA. Statistical analyses showed a nonsignificant correlation between AR and BFA in P. aeruginosa isolates. The ecfX PCR product analyses affirmed their allocation to P. aeruginosa isolates. Conclusion: The high incidence of MDR and BFA among P. aeruginosa isolates might require a plan for regular evaluation and controlling policy programs.

Milk Protein Genes Polymorphism in Indigenous and Crossbred Cattle from a private Dairy Farm in Ethiopia

Kappa casein and beta lactoglobulin genes are major milk proteins which have a direct effect on protein content in dairy cattle. Molecular-based selection through the identification of genetic polymorphism of major protein genes can be used to gain genetic improvement of milk protein yield. The objective of this study was to identify kappa casein (CSN3) and beta lactoglobulin (LGB) genes polymorphisms in indigenous and crossbreed cattle. A total of 90 whole blood samples were collected from individual animal in a private dairy farm. DNA extraction and quality assessment were done salting out procedure and gel electrophoresis, respectively. Polymerase chain reaction (PCR) was performed with gene specific primers. For genotyping, PCR products of CSN3 gene was digested with HinfI and HindIII while LGB gene was digested with HaeIII restriction enzymes. Two haplotypes A and B; three genotypes, AA, AB and BB were observed at CSN3 of HinfI site and LGB HaeIII site, but only AA and AB were observed at CSN3 of HindIII site in crossbred and indigenous cattle populations. However, at CSN3 locus, A allele was found to be more common (0.65) in indigenous cattle than the B allele (0.35), while B (0.51) allele and AB genotype (0.81) were more frequent in crossbred cattle. But, LGB B allele was higher in indigenous cattle (0.67) compared to Allele A (0.33). LGB BB genotype (0.57) is higher followed by AA genotype in indigenous cattle while both allele and genotypic frequencies are equal in crossbred cattle. Both CSN3 and LGB loci were polymorphic in studied populations. Expected heterozygosity was higher in crossbred (0.49, 0.50) than in indigenous (0.38, 0.33) cattle at CSN3 and LGB locus, respectively which might be due to breed variation. The current findings showed that both CSN3 and LGB genes could be promising diagnostic markers in selecting dairy cattle breed. However, further investigations with large sample size and association study with milk composition is required to substantiate the current result.

Detection of Bartonella schoenbuchensis (sub)species DNA in different louse fly species in Saxony, Germany: The proof of multiple PCR analysis necessity in case of ruminant-associated bartonellae determination.

Hippoboscid flies are bloodsucking arthropods that can transmit pathogenic microorganisms and are therefore potential vectors for pathogens such as Bartonella spp. These Gram-negative bacteria can cause mild-to-severe clinical signs in humans and animals; therefore, monitoring Bartonella spp. prevalence in louse fly populations appears to be a useful prerequisite for zoonotic risk assessment. Using convenience sampling, we collected 103 adult louse flies from four ked species (Lipoptena cervi, n=22; Lipoptena fortisetosa, n=61; Melophagus ovinus, n=12; Hippobosca equina, n=8) and the pupae of M. ovinus (n= 10) in the federal state of Saxony, Germany. All the samples were screened by polymerase chain reaction (PCR) for Bartonella spp. DNA, targeting the citrate synthase gene (gltA). Subsequently, PCRs targeting five more genes (16S, ftsZ, nuoG, ribC and rpoB) were performed for representatives of revealed gltA genotypes, and all the PCR products were sequenced to identify the Bartonella (sub)species accurately. The overall detection rates for Bartonella spp. were 100.0%, 59.1%, 24.6% and 75.0% in M. ovinus, L. cervi, L. fortisetosa and H. equina, respectively. All the identified bartonellae belong to the Bartonella schoenbuchensis complex. Our data support the proposed reclassification of the (sub)species status of this group, and thus we conclude that several genotypes of B. schoenbuchensis were detected, including Bartonella schoenbuchensis subsp. melophagi and Bartonella schoenbuchensis subsp. schoenbuchensis, both of which have previously validated zoonotic potential. The extensive PCR analysis revealed the necessity of multiple PCR approach for proper identification of the ruminant-associated bartonellae.

Simultaneous detection of SARS-CoV-2 and influenza A/B viruses on an electromagnetically-driven, integrated microfluidic system

Accurately distinguishing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from other respiratory diseases with similar clinical manifestations, including influenza A (Inf A) and influenza B (Inf B), is 1) challenging due to their similar early-stage respiratory symptoms and 2) important because different treatments are required for each. Herein an electromagnetically-driven, compact (35 × 25 × 25 cm), integrated microfluidic system (IMS) was developed that incorporated fluorescence detection and quantification of real-time, reverse transcription polymerase chain reaction (RT-PCR) products for the detection of three viruses simultaneously. The integrated microfluidic system could automate and perform the entire diagnostic process, including virus capture, viral lysis, RT-PCR, and optical quantification from fluorescence signal, thus enabling the identification and differentiation of Inf A/B from SARS-CoV-2 within only 50 μL of sample for 2 hrs. The limits of detection for E and RdRp genes of SARS-CoV-2, NP gene of Inf A, and M gene of Inf B were 200, 200, 700, and 160 copies/mL, respectively. The sensitivity and accuracy were comparable to conventional approaches with developed integrated microfluidic systems. The integrated microfluidic system is considered to utility for rapid diagnosis of all three types of infectious viruses

Confirmatory Assay for Laboratory Diagnosis of Malaria Using Molecular Approach.

Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination.

The frequency of NUDT15 rs116855232 and its impact on mercaptopurine-induced toxicity in Syrian children with acute lymphoblastic leukemia.

Polymorphisms in NUDT15 may result in differences in mercaptopurine-induced toxicity. This study aimed to identify the frequency of the NUDT15 (c.415C>T; rs116855232) polymorphism and investigate the effect of this polymorphism on mercaptopurine-induced toxicity in a population of Syrian patients with childhood acute lymphoblastic leukemia (ALL). This is a retrospective study that included children with ALL reaching at least 6 months of maintenance therapy. The NUDT15 genotyping was determined using standard targeted sequencing of polymerase chain reaction products. The odds ratio (OR) for the association between toxicity and genotype was evaluated. A total of 92 patients were enrolled. The majority of the patients in the study population were low-risk (63.04%), followed by intermediate-risk (25%), and high-risk (11.96%). There were 5 patients (5.4%) with NUDT15 (c.415C>T; rs116855232) CT genotype, and 1 patient (1.08%) with NUDT15 TT genotype, with allele frequencies of C=0.962 and T=0.038. The mercaptopurine median dose intensity was 100%, 54.69%, and 5% for the genotypes CC, CT, and TT, respectively (P=0.009). Early onset leukopenia was significantly associated with the NUDT15 polymorphism (OR: 6.16, 95% CI: 1.11-34.18, P=0.037). There was no association between the NUDT15 genotype and hepatotoxicity. Approximately 6.5% of the study population exhibited reduced NUDT15 activity. The mercaptopurine dose intensity was considerably low in NUDT15 rs116855232 TT genotype compared with CT and CC. The dosage of mercaptopurine should be adjusted according to the NUDT15 genotype in pediatric patients with ALL.