Polymerase Chain Reaction Products Research Articles

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.

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.

Molecular Determination of Resistance Gene in Methicillin Resistant Staphylococcus aureus (MRSA) Isolated from Skin and Wound

The wide spread use of antibiotic resulted in the development of resistance to antibiotics through acquisition of the mobile cassette chromosome carrying the Methicillin-resistant gene mecA. The study was aimed to characterize the resistance gene in Methicillin resistant Staphylococcus aureus (MRSA) isolated from skin and wound samples in Kano Metropolis, Northwestern, Nigeria. A total of 235 S. aureus isolates were identified and subjected to MRSA screening. MRSA were phenotypically identified by antibiotic susceptibility testing using agar disc diffusion method. The suspected MRSA were subjected to polymerase chain reaction (PCR). The PCR products were subjected to gel electrophoresis and a DNA ladder were loaded into the gel wells. The gel was examined for the presence of specific amplicons of the expected size for mecA, which is 192bp. Out of a total of 235 isolates of Staphylococcus aureus, only 11 (4.7%) strains were found to be Methicillin resistant Staphylococcus aureus (MRSA). Five out of the 11 isolates show the presence of DNA bands of the expected size for MecA gene which indicated the presence of the resistance genes in the bacterial isolates. It is concluded that MecA gene is one of the gene responsible for methicillin resistance in MRSA.

Genetic diversity of vector-borne zoonotic pathogens in companion dogs and cats, Tianjin, China.

Dogs and cats are the hosts of many vector-borne human pathogens that can be transmitted to humans. Given their direct and intimate contact with humans, companion dogs and cats are considered direct sentinels of vector-borne human pathogens. However, limited information is currently available regarding canine and feline zoonotic pathogens in China. This study detected canine and feline vector-borne human pathogens to better understand the potential risk to humans. Blood samples were collected from 275 domestic companion animals (117 dogs and 158 cats) living in Tianjin city, China, and the presence of DNA from Anaplasma, Babesia, Bartonella, and Rickettsia was detected by semi-nested polymerase chain reaction (PCR). The PCR products of the expected size were sequenced, and these newly generated sequences were subjected to BLASTN, nucleotide identity, and phylogenetic analyses. A total of 24 blood samples tested positive for vector-borne pathogens in companion dogs and cats in Tianjin city, China, with a relatively low positive rate of 8.7%. Specifically, seven human pathogens, including Rickettsia raoultii, Candidatus Rickettsia jingxinensis, Rickettsia sibirica, Rickettsia felis, Babesia venatorum, Bartonella tribocorum, and Bartonella Henselae, were identified. In addition, Anaplasma ovis with zoonotic potential and Candidatus A. cinensis were detected. Our results indicate substantial genetic diversity in the vector-borne human pathogens circulating in companion dogs and cats. Interventions based on "One Health" should be taken to reduce the potential risks of contracting infection from companion dogs and cats in Tianjin, China.

Bacteriophage-assisted lysis and eluted genomic DNA-based detection of pathogenic bacterial contamination in food

Foodborne diseases represent a significant global health concern, with a substantial number of illnesses and fatalities reported annually owing to the consumption of contaminated food. Current methods for identifying foodborne pathogens cannot effectively distinguish live from dead bacteria or efficiently detect multiple targets. Therefore, we developed a selective detection method for three pathogenic bacteria in food samples that leverages bacteriophage-assisted live-cell lysis and exposed genomic DNA amplification. The antimicrobial activity and host specificity of these three bacteriophages were employed to confirm the target bacteria selectivity. Bacterial DNA purification using bacteriophage-assisted lysis demonstrated the superior activity of the selected phages in extracting DNA from Escherichia coli O157:H7 and Salmonella Typhimurium. The results from reactions involving the three strains, including Bacillus cereus, highlighted that a 1 h incubation period yielded the most efficient DNA extraction and purification. The polymerase chain reaction (PCR) products obtained were comparable in terms of concentration and purity with those generated using bacterial DNA prepared using a commercial kit. In the practical food sample application experiment, bacterial strains were successfully recovered and concentrated using a polyethersulfone filter, resulting in a concentration factor >3 for all samples. Furthermore, the concentration of purified bacterial DNA from contaminated kimchi cabbage exceeded 10 ng/μL. Notably, we could detect E. coli O157:H7, S. Typhimurium, and B. cereus spiked at 103 colony-forming units/mL using multiplex PCR. This innovative bacteriophage-based approach holds substantial promise for the selective and practical detection of live foodborne pathogens, thereby significantly advancing food safety standards.

A rapid and low-cost platform for detection of bacterial based on microchamber PCR microfluidic chip.

Polymerase chain reaction (PCR) has been considered as the gold standard for detecting nucleic acids. The simple PCR system is of great significance for medical applications in remote areas, especially for the developing countries. Herein, we proposed a low-cost self-assembled platform for microchamber PCR. The working principle is rotating the chamber PCR microfluidic chip between two heaters with fixed temperature to solve the problem of low temperature variation rate. The system consists of two temperature controllers, a screw slide rail, a chamber array microfluidic chip and a self-built software. Such a system can be constructed at a cost of about US$60. The micro chamber PCR can be finished by rotating the microfluidic chip between two heaters with fixed temperature. Results demonstrated that the sensitivity of the temperature controller is 0.1℃. The relative error of the duration for the microfluidic chip was 0.02s. Finally, we successfully finished amplification of the target gene of Porphyromonas gingivalis in the chamber PCR microfluidic chip within 35min and on-site detection of its PCR products by fluorescence. The chip consisted of 3200 cylindrical chambers. The volume of reagent in each volume is as low as 0.628 nL. This work provides an effective method to reduce the amplification time required for micro chamber PCR.

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.