Amplification Of Target Genes Research Articles

Role of rs5743708 SNP in the risk of Bacterial Respiratory Infections in Children of Ramadi city/Iraq

Background and Aim: Respiratory Infections are a major global health and financial burden, so among cause the five most common and deadly for respiratory illnesses is acute lower respiratory tract infections (RTIs), these infections claim over 4 million lives annually. Toll-like receptors (TLRs) are key components of the innate immune system that recognize pathogen which associated a molecular patterns) PAMPs) and initiate an immune response. Understanding the role of TLR polymorphisms in susceptibility to respiratory infections in children is important for developing strategies to prevent and treat these infections. So, the current study aims to "Investigate the potential association between TLR2 gene polymorphisms and susceptibility to respiratory infections in children". Methodology: The study included collect of 300 samples with respiratory infection from both sex with range ages(1-12) years, and other 50 healthy control samples. The samples were collected from general and private hospitals in Ramadi city, Iraq. PCR was carried out using primer for gene target and amplification of target gene. Sanger Sequencing conducted with both direction for all samples. Results and Conclusion: The result of sputum culture indicate that 100 of suspected samples were positive culturing while other demonstrate a negative growth. The results analysis of sequencing according to Query and subjected samples indicated that 4 of patient have SNP of rs5743708 G>A, while there is not found in control samples, and the results of genotype frequencies analysis it was found that TLR2 polymorphism was associated with increased risk for bacterial respiratory infections in children according to under study samples, but no statistical significant differences correlation of rs5743708 G>A and infection, with adjusted odds ratio [OD:5.3; 95%Cl: 0.3-101; P: 0.2].

NEW PRIMER DESIGN of MULTIPLEX POLYMERASE CHAIN REACTION (PCR) for DETECTION of Escherichia Coli and Salmonella Enterica in FOOD SAMPLE

Background: Multiplex PCR techniques are used to detect multiple pathogens at the same time simultaneously. In order to achieve the specificity and sensitivity of detection, it is important to optimize the multiplex PCR method properly. Developing suitable primers and optimizing PCR temperature to boost particular genes from pathogens are key factors for this optimization. Methods: For the simultaneous detection of Escherichia coli and Salmonella enterica in food samples, this research aims to develop a set of primers for multiplex polymerase chain reaction (PCR). Using Primer-BLAST software, the study utilizes specific primer designs for the phoA gene (Eschericia coli) and invA gene (Salmonella enterica). DNA isolation has confirmed successful extraction from the two bacterial samples. PCR was performed under different conditions, including Singleplex and Multiplex PCR, using two annealing temperatures of 53oC and 50oC. Results: The results showed that this method can effectively amplify target genes and indicate their specificity and reliability at both temperature levels. Given these results, it has successfully conducted multiplex PCR using the built primer pairs. Both annealing temperatures of 50oC and 53oC can be used to perform multiplex PCR to detect E. coli and Salmonella enterica in one PCR reaction. Conclusion: Through this research, we have created a new set of Multiplex PCR Primers and an optimized multiplex PCR technique for the simultaneous detection of E coli and Salmonella enterica in food samples. The rapid and simultaneous screening of E. coli and S. enterica, which contributes to improved food safety measures and pathogen detection in the food industry, is promising with this optimized PCR approach.

Concentration and Distribution of Oligochaeta Worms in the Waters of Kejapanan, Pasuruan, Indonesia Polluted by Mercury Waste using DNA Barcode

Graphical Abstract Higlight Research Based on AAS test, results showed that the St2 sample has the highest concentration of mercury compared to other locations. St2 samples are samples taken right at the pollutant source. The results showed that the samples consist of the Nadidae family with two species, namely Limnodrilus hoffmeisteri and Branchiura sowerbyi. The COI gene that was successfully amplified had a length of approximately 700 bp using a 3000 bp DNA ladder as a comparison. Based on the results of the SEM-EDX test, the worm samples contained several elements. The majority of them are organic except Al, Si, and Ti. Aluminium (Al), Silicon (Si), and Titanium (Ti) are metals that are used by organisms. Abstract Physiological monitoring of mercury waste contamination can be carried out using the biota around the waters. This study aims to identify concentration of Hg and the types of worms in the waters of Kejapanan, Pasuruan, East Java with a molecular approach. Target gene amplification was carried out using the mitochondrial genome COI barcode primer. Analysis of molecular identification was performed with DNA analysis and phylogenetic, similarity, DNA sequence variation, genetic distance, and the BOLD System. The concentration Hg was analyzed using AAS and the distribution of mercury in the worms was analyzed using SEM Edax Mapping. The results showed that the pollutant source area (St2 sample) has the highest concentration of mercury compared to other locations. The results of molecular identification indicate the formation of two clusters. The amplified samples produced DNA bands according to the target (600-700 bp), and the process was continued with morphological-based-key identification. The results showed that they consist of the family Nadidae with two species, namely Limnodrilus hoffmeisteri and Branchiura sowerbyi. A DNA length of 709 bp as well as nucleotide composition. BLAST results showed that species L. hoffmeisteri and B. sowerbyi had similarity indexes of 99% and 86%, respectively. Based on the research results, it was found that there was an accumulation of mercury exposure in worms in polluted areas. For this reason, the results of this study can provide a novelty that worms can be used as biomonitoring of water pollution using the barcode data.

Effect of DNA Template Concentration on Standard Polymerase Chain Reaction

The polymerase chain reaction (PCR) was an essential fundamental procedure widely used in molecular biology to amplify specific deoxyribonucleic acid (DNA) sequences. To investigate the efficiency and specificity of PCR using DNA extracted from human blood cells as sample, several concentrations of DNA templates were used to obtain accurate and reproducible results during this optimisation process. The principal objective was to identify which concentration was the best for amplifying the DNA target sequence and study the effects of the scale of concentrations to standard PCR. For this study, a series of PCR were conducted using various concentrations of DNA template ranging from 1 ng/μL to 100 ng/μL per reaction under optimized conditions of primer concentrations, annealing temperature and extension time. The results demonstrated that DNA template concentration significantly influenced the efficiency and specificity of PCR as the intensity of the target gene amplification band was increased on the agarose gel electrophoresis under ultraviolet light indicating the PCR product yield. Formation of primer-dimers became more prominent as the DNA template concentration reduced resulting in non-specific amplification. The concentrations ranging from 10 ng/μL to 70 ng/μL were the most optimal reactions profiting a remarkable feat of target gene amplification while concentrations less than 1 ng/μL produced none and non-specific outcomes retaining little amount of target gene amplification. In conclusion, our study highlights the pivotal role of DNA template concentration optimises PCR to increase reliability and reproducibility, extending our understanding of genetic analysis, diagnostic, and forensic sciences.

Research Note: Real-time fluorescence-based recombinase-aided amplification for rapid detection of Mycoplasma synoviae

Mycoplasma synoviae (MS) is an essential pathogenic mycoplasma in poultry worldwide, posing a serious threat to the poultry industry's health. Timely detection is imperative for early diagnosis, prevention, and control of MS infection. Current laboratory methods for MS detection are generally complicated, time-consuming, and require sophisticated equipment. Therefore, a simple and rapid method is urgently needed. This study developed a novel real-time fluorescence-based recombinase-aided amplification (RF-RAA) technique for detecting MS nucleic acids, enabling target gene amplification within 20 min at 39°C. The RF-RAA outcomes are interpretable in two modalities: real-time fluorescence monitoring employing a temperature-controlled fluorescence detector or direct visual inspection facilitated by a portable blue light transilluminator. This method exhibits robust specificity, demonstrating no cross-reactivity with various common poultry pathogens, and achieves high sensitivity, detecting as low as 10 copies/μL for the standard plasmid. Seventy-one clinical samples of chicken throat swabs were detected by RF-RAA and real-time fluorescence quantitative polymerase chain reaction (qPCR) methods. The diagnostic coincidence rates of qPCR with RF-RAA (fluorescence monitoring) and RF-RAA (visual observation) were determined to be 100% and 97.2% (69/71), respectively. In conclusion, the RF-RAA method developed in this study provides a rapid and visually observable approach for MS detection, offering a novel technique to diagnosing MS infection, especially in resource-limited settings.

Construction of PCR-SERS Method for Detection of Vibrio parahaemolyticus.

A paper-based surface enhancement of a Raman scattering substrate consisting of silver-nanowires stacked on glass-fiber filter paper was prepared. At the same time, the DNA-embedding molecule Eva Green was introduced as a signaling molecule for surface-enhanced Raman scattering (SERS) detection. Polymerase chain reaction (PCR) was used to amplify target genes and the method was developed into a rapid molecular diagnostic system. The total detection time of the developed detection method was 40 min, including 30 min of PCR amplification and 10 min of SERS measurement. After 30 PCR cycles, bacterial DNA with an initial concentration of 20 fg/μL and a bacterial suspension with an initial concentration of 7.2 × 101 CFUs/mL could be detected. When the enrichment culture time was 4 h, target bacteria with an initial contamination inoculation volume of 1.5 CFUs/mL could be detected in artificially contaminated samples. The method is fast and highly sensitive, and has not been applied to the detection of V. parahaemolyticus.

A Foldable Thermoplastic Microdevice Integrating Isothermal Amplification and Schiff-Reaction-Based Colorimetric Assay for the Detection of Infectious Pathogens

In this study, we introduce a plastic-based foldable microdevice that integrates loop-mediated isothermal amplification (LAMP) and a colorimetric assay based on the Schiff reaction to detect the genes of infectious bacteria. The device comprises two sides: a sample zone containing amplification chambers and a detection zone for the colorimetric assay. The detection zone contains poly(methyl methacrylate) structures for transferring the colorimetric reagent-soaked glass micro-fiber paper into the sample chambers. Specific genes of Staphylococcus aureus (S. aureus) and Streptococcus pneumoniae (S. pneumoniae), the most common bacterial infection causes, were amplified by LAMP assay. The S. aureus gene was detected up to 10 fg/μL and the S. pneumoniae gene up to 0.1 pg/μL. The amplified target genes were visually identified using a colorimetric assay with Schiff’s reagent, which showed clear color discrimination through a reaction with aldehyde groups derived from the DNA in the amplicons. The introduced method, integrating amplification and detection processes in a single device, is expected to be utilized in point-of-care testing analysis for the simple and rapid detection of infectious pathogens.

Large T antigen mediated target gene replication improves site-specific recombination efficiency.

With advantages of high-fidelity, monoclonality and large cargo capacity, site-specific recombination (SSR) holds great promises for precise genomic modifications. However, broad applications of SSR have been hurdled by low integration efficiency, and the amount of donor DNA available in nucleus for SSR presents as a limiting factor. Inspired by the DNA replication mechanisms observed in double-stranded DNA virus SV40, we hypothesized that expression of SV40 large T antigen (TAg) can increase the copy number of the donor plasmid bearing an SV40 origin, and in consequence promote recombination events. This hypothesis was tested with dual recombinase-mediated cassette exchange (RMCE) in suspension 293F cells. Results showed that TAg co-transfection significantly enhanced SSR in polyclonal cells. In the monoclonal cell line carrying a single landing pad at an identified genomic locus, 12% RMCE efficiency was achieved, and such improvement was indeed correlated with donor plasmid amplification. The developed TAg facilitated RMCE (T-RMCE) was exploited for the construction of large libraries of >107 diversity, from which GFP variants with enhanced fluorescence were isolated. We expect the underlying principle of target gene amplification can be applicable to other SSR processes and gene editing approaches in general for directed evolution and large-scale genomic screening in mammalian cells.

Nanozyme-assisted amplification-free CRISPR/Cas system realizes visual detection.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated) system has proven to be a powerful tool for nucleic acid detection due to its inherent advantages of effective nucleic acid identification and editing capabilities, and is therefore known as the next-generation of molecular diagnostic technology. However, the detection technologies based on CRISPR/Cas systems require preamplification of target analytes; that is, target gene amplification steps through isothermal amplification or PCR before detection to increase target analyte concentrations. This creates a number of testing limitations, such as extended testing time and the need for more sophisticated testing instruments. To overcome the above limitations, various amplification-free assay strategies based on CRISPR/Cas systems have been explored as alternatives, which omit the preamplification step to increase the concentrations of the target analytes. Nanozymes play a pivotal role in enhancing the sensitivity of CRISPR-based detection, enabling visual and rapid CRISPR assays. The utilization of nanozyme exceptional enzyme-like catalytic activity holds great promise for signal amplification in both electrochemical and optical domains, encompassing strategies for electrochemical signal sensors and colorimetric signal sensors. Rather than relying on converting a single detection target analyte into multiple analytes, these methods focus on signal amplification, the main mechanism of which involves the ability to form a large number of reporter molecules or to improve the performance of the sensor. This exploitation of nanozymes for signal amplification results in the heightened sensitivity and accuracy of detection outcomes. In addition to the strategies that improve sensor performance through the application of nanozymes, additional methods are needed to achieve visual signal amplification strategies without preamplification processes. Herein, we review the strategies for improving CRISPR/Cas systems that do not require preamplification, providing a simple, intuitive and preamplification-free CRISPR/Cas system detection platform by improving in-system one-step amplification programs, or enhancing nanozyme-mediated signal amplification strategies.

Detection Sequencing NDM and blaOXA Genes in Metallo-$\beta$-Lactamase Producing Klebsiella Pneumoniae

The prevalence of Carbapenem-resistance in Enterobacteriaceae is rising worldwide and poses a treatment challenge for healthcare facilities. This study aims to detect a sequence of New Delhi metallo- -lactamase (NDM) and bla OXA-48 gene in metalo-beta-lactamase-producing Klebsiella pneumoniae isolated from a few hospitals in the city of Baghdad. 74 Klebsiella pneumoniae were isolated from 320 clinical samples that were routinely submitted to the diagnostic laboratory of the chosen hospital. These samples included sputum, tissue swabs, folly tips, urine, ear swabs, bloodstream, pus, wound swabs, endotracheal tubes, and body fluids. In this study, 74 isolates were examined for expression of carbapenemase by phenotyping method. The modified Hodge Test (MHT) is utilized for phenotypic detection. MHT was positive for 24 carbapenem-resistant Klebsiella pneumoniae. Genotyping was accomplished by using polymerase chain reaction (PCR) to amplify target genes by employing particular primers. The NDM gene was detected in 19/24 isolates, and the blaOXA-48 gene was detected in 5/24 isolates. The presence of bla NDM and the dangerously high frequency of MBL in the Klebsiella pneumoniae strain are both concerning, and it has been demonstrated to be challenging to treat and inhibit infection.

Promoter from Chinese hamster elongation factor-1a gene and Epstein-Barr virus terminal repeats concatemer fragment maintain stable high-level expression of recombinant proteins.

The Chinese hamster ovary (CHO) cell line is the main host for the high-titer production of therapeutic and diagnostic proteins in the biopharmaceutical industry. In most cases, plasmids for efficient protein expression in CHO cells are based on the cytomegalovirus (CMV) promoter. The autologous Chinese hamster eukaryotic translation elongation factor 1α (EEF1A1) promoter is a viable alternative to the CMV promoter in industrial applications. The EEF1A1 promoter and its surrounding DNA regions proved to be effective at maintaining high-level and stable expression of recombinant proteins in CHO cells. EEF1A1-based plasmids' large size can lead to low transfection efficiency and hamper target gene amplification. We hypothesized that an efficient EEF1A1-based expression vector with a long terminal repeat fragment from the Epstein-Barr virus (EBVTR) could be truncated without affecting promoter strength or the long-term stability of target gene expression. We made a series of deletions in the downstream flanking region of the EEF1A1 gene, and then in its upstream flanking region. The resulting plasmids, which coded for the enhanced green fluorescent protein (eGFP), were tested for the level of eGFP expression in the populations of stably transfected CHO DG44 cells and the stability of eGFP expression in the long-term culture in the absence of selection agents. It was shown that in the presence of the EBVTR fragment, the entire downstream flanking region of the EEF1A1 gene could be excluded from the plasmid vector. Shortening of the upstream flanking region of the EEF1A1 gene to a length of 2.5 kbp also had no significant effect on the level of eGFP expression or long-term stability. The EBVTR fragment significantly increased expression stability for both the CMV and EEF1A1 promoter-based plasmids, and the expression level drop during the two-month culture was more significant for both CMV promoter-based plasmids. Target protein expression stability for the truncated plasmid, based on the EEF1A1 gene and EBVTR fragment, is sufficient for common biopharmaceutical applications, making these plasmid vectors a viable alternative to conventional CMV promoter-based vectors.

A smartphone-controllable molecular diagnostic platform for SARS-CoV-2 detection by reverse-transcription recombinase polymerase amplification

We constructed a portable molecular diagnostic microsystem, enabling the whole processes of genetic analysis for SARS-CoV-2. The proposed platform was a miniaturized size and was comprised of three main parts: a microfluidic device with a 3D-printed solution loading cartridge, an operating system including a tailor-made heater board, a syringe pump and an optical fluorescence detector, and a smartphone with a customized app for the remote and automatic control. The microfluidic device was designed to control the complicated fluidic flow through only the passive valves, and could perform the RNA extraction from the viral lysate via glass membranes and the target gene amplification by reverse-transcription recombinase polymerase amplification (RT-RPA). The 3D-printed cartridge allowed the facile chemical solution storage and loaded a designated solution into the microfluidic device. A smartphone with a browser-based app could control the on-and-off status of solenoid valves, the workflow of a syringe pump to automatically introduce solutions on a chip, the heater board to supply the constant temperature for RT-RPA, and the periodic activation of the Pi camera and a 488 nm laser to measure the fluorescence signal in the reaction chambers that showed the amplification profiles on the smartphone display in real-time. Using the proposed smartphone-controllable diagnostic platform, the N gene and S gene of SARS-CoV-2 were successfully detected at a limit-of-detection (LOD) of 10 copies/μL within 1 hr in a sample-to-answer-out format. Ten out of 22 clinical samples were successfully analyzed for SARS-CoV-2, demonstrating the practical applications for the on-site diagnostics of patient samples.

Molecular epidemiological study on rubella virus circulating in Yunnan Province during 2011-2021

Objective: To understand the genotype distribution and transmission pattern of rubella virus (RuV) circulating in Yunnan Province. Methods: Throat swab samples were collected from rubella outbreaks and sporadic cases in nine prefectures/cities of Yunnan Province from 2011 to 2021. Virus isolation, amplification of target genes and sequence determination were performed on the RuV-positive samples. The genotypes and lineages of Yunnan strains were determined by comparing them with the reference strains, and further phylogenetic analysis was performed with Yunnan strains and strains circulating in other provinces of China during the same period. Results: RuV circulating in Yunnan province during 2011-2021 showed significant genetic diversity, and three lineages, 1E-L1, 2B-L1 and 1E-L2, were detected. Two lineage-switches were also identified, including the conversion of 1E-L1 to 2B-L1 between 2012 and 2013, and the replacement of 2B-L1 to 1E-L2 after 2018. The time of the switches was basically consistent with the outbreak in Yunnan province in 2012 and the time of the rubella reemergence and epidemic between 2018 and 2019. The amino acid sequence of RuV virus strains in Yunnan province was highly conserved, and no important functional regions were changed. Conclusions: The transmission pattern of RuV in Yunnan province is generally consistent with the epidemic trend of RuV in other provinces of China.

Ultra-sensitive photoelectrochemical biosensor for determination of African swine fever virus based on surface plasmon resonance

Sensitive and specific detection of African swine fever virus (ASFV) is crucial for agricultural production and economic development due to the mortality and infectivity. In this study, a bismuth induced enhanced photoelectrochemical (PEC) biosensor based on in-situ loop mediated isothermal amplification (LAMP) was constructed using deposited bismuth nanoparticles loaded bismuth oxycarbonate (Bi/(BiO)2CO3) as photoactive material, using primers designed according to LAMP as recognition elements, and using in-situ LAMP to achieve nucleic acid amplification of target genes. As the Bi induced surface plasmon resonance (SPR) effect, enhanced light captures and effective electron hole separation, it could effectively enhance the photoelectric activity, so the prepared Bi/(BiO)2CO3 nanohybrid had higher photocurrent intensity and good stability. The constructed PEC biosensor has realized the detection of ASFV in real samples with good sensitivity, specificity and repeatability. In the range from 1.0 × 10−13 to 1.0 × 10−7 g/L, the photoelectric current decreased with the increase of the concentration of ASFV, and the detection limit was 3.0 × 10−14 g/L (about 0.048 copies/μL). Combining the advantages of LAMP with the excellent performance of PEC, it provides a simple, economical and efficient method for nucleic acid diagnosis, and also provides a new idea for biosensor detection.

Base Substitution Patterns in Partial of the Cytochrome C Oxidase Unit I (COI) mtDNA Genes in the Red Junglefowl (Gallus sp.) of North Sulawesi and some Gallus sp. accessions.

Partial of the cytochrome C oxidase unit I (COI) mtDNA genes of the red jungle fowl (Gallus sp.) were isolated and sequenced to examine the base substitution pattern compared to several accessions taken from GenBank. Samples were collected from several areas in North Sulawesi in the form of muscle tissue and prepared with 95% alcohol, and stored at temperatures below 500 C. Total DNA was isolated using the innuPREP DNA micro kit with a modified protocol. The target gene amplification used the primers BirdR1 and BirdF1, and the amplicons were sequenced at the Singapore FirstBase laboratory. Target gene sequences have been blasted through the nucleotide blast program provided by the National Center for Biotechnology Information (NCBI)—sequence analysis using MEGA5 and DnaSPv5 software. Target gene sequences were aligned using the Clustal-W program on MEGA5. The amplified target gene sequence length was 490 bp. The blast results showed that the target gene sequence was 98% identical to the COI gene sequence from several Gallus-gallus accessions from GenBank. The results of multiple alignments between the sequences of some of the genes examined showed the presence of polymorphic sites (S). The number of polymorphic sites (S) is 11, while the conserved sequence (C) is 386/397 = 0.977. The number of haplotypes (h) is 5, and the diversity of haplotypes (Hd) is 0.703. The overall genetic distance average was 0.008. The polymorphic sites and base substitution events occurred at sites number 145 (A↔G), 241 (G↔C), 268 (C↔A), 295 (C↔A), 348 (T↔C), 381(A) ↔T), 384 (T↔C), 390 (T↔A), 393 (C↔T), 394 (T↔C) and 395 (C↔T). The substitution pattern at these polymorphic sites consists of substitution transitions and transversions. The ratio of transition substitution and transversion events (Ts/Tv) = 1.4056. The substitution events at these sites are located at the bases in the first and third positions of the codon. Substitution events at these polymorphic sites partially cause the exchange of amino acids. The exchange of amino acids occurs at codons 49, 81, 90, 99 and 130.

Presence of SARS-CoV-2 in urban effluents in south-east Buenos Aires, Argentina, May 2020 to March 2022.

To implement and evaluate the use of wastewater sampling for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in two coastal districts of Buenos Aires Province, Argentina. In General Pueyrredon district, 400 mL of wastewater samples were taken with an automatic sampler for 24 hours, while in Pinamar district, 20 L in total (2.2 L at 20-minute intervals) were taken. Samples were collected once a week. The samples were concentrated based on flocculation using polyaluminum chloride. RNA purification and target gene amplification and detection were performed using reverse transcription polymerase chain reaction for clinical diagnosis of human nasopharyngeal swabs. In both districts, the presence of SARS-CoV-2 was detected in wastewater. In General Pueyrredon, SARS-CoV-2 was detected in epidemiological week 28, 2020, which was 20 days before the start of an increase in coronavirus virus disease 2019 (COVID-19) cases in the first wave (epidemiological week 31) and 9 weeks before the maximum number of laboratory-confirmed COVID-19 cases was recorded. In Pinamar district, the virus genome was detected in epidemiological week 51, 2020 but it was not possible to carry out the sampling again until epidemiological week 4, 2022, when viral circulation was again detected. It was possible to detect SARS-CoV-2 virus genome in wastewater, demonstrating the usefulness of the application of wastewater epidemiology for long-term SARS-CoV-2 detection and monitoring.

Detection of Periodontal Pathogens Based on an Integrated Continuous Flow PCR and Capillary Electrophoresis Microfluidic Chip

Continuous-flow PCR (CF-PCR) can realize rapid DNA amplification because of the high temperature variation rate. However, off-line detection methods for PCR may induce cross contamination. To overcome this problem, we herein fabricated an integrated CF-PCR and electrophoresis microfluidic chip. The optimal voltage applied in the electrophoresis part of the microfluidic chip was achieved by simulation in COMSOL. Coating the inside wall of the microchannel can inhibit electroosmotic flow and improve the resolution for DNA fragments. The temperature distribution of the serpentine part can meet the PCR and has no obvious suppressive effect on sample separation. Finally, we have performed the amplification of target genes for Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola and detected the corresponding PCR products in the microfluidic chip within 11 min. Such work provides a new method for the rapid detection of bacteria.

High-quality single amplicon sequencing method for illumina MiSeq platform using pool of ‘N’ (0–10) spacer-linked target specific primers without PhiX spike-in

BackgroundIllumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms, we developed a high throughput single amplicon sequencing method by introducing ‘N’ (0–10) spacers in target gene amplification primers that are pooled for simple handling.ResultWe evaluated the efficiency of ‘N’ (0–10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of ‘N’ (0–10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software,“MetReTrim”, to trim the ‘N’ (0–10) spacers from the raw reads (https://github.com/Mohak91/MetReTrim). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS™ microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We observed no difference between the communities represented by our method and standard illumina V3-V4 primer method.ConclusionThis method eliminates the need for PhiX spike-in for single amplicon sequencing on illumina MiSeq platform. This allows for sequencing of more number of samples in a run and a reduction in the overall cost. Given that Illumina sequencing works on SBS chemistry irrespective of the platform (such as HiSeq, MiSeq, NextSeq, NovaSeq, etc.) we propose that this strategy of using ‘N’ (0–10) spacer-linked primer design can be adopted for generating high-quality single locus amplicon sequencing in a high throughput manner across the illumina platform subject to further validation.

Lower fluidic resistance of double-layer droplet continuous flow PCR microfluidic chip for rapid detection of bacteria

BackgroundRapid diagnosis of harmful microorganisms demonstrated its great importance for social health. Continuous flow PCR (CF-PCR) can realize rapid amplification of target genes by placing the microfluidic chip on heaters with different temperature. However, bubbles and evaporation always arise from heating, which makes the amplification not stable. Water-in-oil droplets running in CF-PCR microfluidic chip with uniform height takes long time because of the high resistance induced by long meandering microchannel. To overcome those drawbacks, we proposed a double-layer droplet CF-PCR microfluidic chip to reduce the fluidic resistance, and meanwhile nanoliter droplets were generated to minimize the bubbles and evaporation. ResultsExperiments showed that (1) fluidic resistance could be reduced with the increase of the height of the serpentine microchannel if the height of the T-junction part was certain. (2) Running speed, the size and the number of generated droplets were positively correlated with the cross-sectional area of the T-junction and water pressure. (3) Droplet fusion happened at higher water pressure if other experimental conditions were the same. (4) 0.032 nL droplet was created if the cross-sectional area of T-junction and water pressure were 1600 μm2 (40 × 40 μm) and 7 kPa, respectively. Finally, we successfully amplified the target genes of Porphyromonas gingivalis within 11′16″ and observed the fluorescence from droplets. Significance and noveltySuch a microfluidic chip can effectively reduce the high resistance induced by long meandering microchannel, and greatly save time required for droplets CF-PCR. It offers a new way for the rapid detection of bacterial.

Evaluation of Legionella pneumophila SGUT Serotypes Isolated from Bath Water Using a Multiplex-PCR Serotyping Assay.

Legionella pneumophila, the primary causative agent of Legionnaires' disease, is classified into at least 15 serogroups (SGs). Before genotyping, serotyping is first performed to limit the sources of L. pneumophila infections that caused an outbreak. In addition to conventional assays using monoclonal or polyclonal antisera, serotyping using multiplex polymerase chain reaction (M-PCR) was recently developed for L. pneumophila. In this study, we applied the M-PCR system to 41 strains that remained to be SGUT (untypable) by slide agglutination tests among the 220 L. pneumophila strains isolated from bath water in Kobe City during 2016-2020, to determine SG-genotypes (SGg) by PCR amplification of the specific target gene of the SGs. Among the 41 SGUT strains, SGg4/10/14 was the most predominant (24/41, 58.5%), followed by SGg1 (7/41, 17.1%). Seven strains, except for the strains determined as SGg1, were identified as belonging to a single SGg by M-PCR serotyping (SGg5 [3/41, 7.3%], SGg8 [3/41, 7.3%], and SGg7 [1/41, 2.4%]). Furthermore, we found that the seven strains identified as SGg1 harbored particular genotypes. In conclusion, the M-PCR serotyping assay will be helpful for investigating the distribution of L. pneumophila in environmental and clinical settings.