Development of amplification-based technologies for enrichment of nucleic acids with difficult sequences or low-abundance point mutations

The aim of this study was to utilize molecular biology knowledge to develop novel amplification-based technologies, which enable enrichment of challenging nucleic acid targets to a level sufficient for detection, including those with extremely long, GC-rich and/or repetitive sequences and low-abundance single nucleotide RNA variants in the excess of alternative variants. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to generate a novel amplification technology (HPE-PCR), which temporarily destabilize secondary structures, in order to enhance DNA polymerase extension over GC-rich sequences. Different GC rich target sequences in the human genome, extremely long Fragile X GGC repeats and Myotonic Dystrophy type 1 CTG repeats have been used as models to develop and validate this novel technology. In order to detect a low-abundance RNA variant, we devised a novel technology using a competitive Extendable Blocking Probe (ExBP) in the reverse transcription reaction for allele-specific priming with superior selectivity. In ExBP-reverse transcription, the mismatch priming site on the alternative variant is blocked by a perfectly matched ExBP. This initiates formation of a stable cDNA-RNA hybrid that completely blocks false cross-priming by the target specific primer. In experimental models, the ExBP-based reverse transcription assay allowed for detection of multiple mutation types on different genes in at least 1000-fold excess of wildtype RNA and detection was linear over a 4 log dynamic range. This technique not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression. In conclusion, we have established HPE-PCR and ExBP-RT techniques to enable enrichment of different nucleic acid templates that are currently challenging to detect by PCR, such as long, GC rich and/or repetitive sequences as well as low-abundance point mutations in a vast excess of wildtype alleles. These techniques expand the capacities of current PCR technology; provide versatile and convenient research tools and open many new possibilities for its applications in molecular diagnostics.Yleistajuinen tiivistelmä ei saatavilla toistaiseks

Effects of hormone and fertilizers on early flower induction of Dendrobium anosmum hybrid seedlings under ex vitro condition

Early flowering of new orchids is important to save time for selecting valuable flowers and artificial induction of flowering is a critical consideration in the orchid production industry. In this study, a new Dendrobium anosmum hybrid was generated by cross-breeding between D. anosmum ‘Chau Nhu’ and D. anosmum ‘Di Linh’. The ancestors and hybrid seedlings from in-vitro culture were trained in the net house and their growth and flowering were evaluated under ex vivo conditions with specific fertilizers and hormones. The results suggest that the hybrid plants grew better than their parents in terms of stem height, stem diameter, and leaf number. Growth hormones were applied to stimulate early flowering in matured hybrids and it was discovered that ‘Keiki pro’, a commercial hormone product, produced the best results, with a flowering rate of 66.67% after two applications. Hybrid flowers varied in width from 36.36% (3.0-6.0 cm) to 63.64 % (more than 6.0 cm) from ancestral width in medium-sized and large-sized flowers, respectively. Also, the hybrid flower colours was mostly a combination of pink/violet (75C) and purple/pink (68A), which is different from their parents. Importantly, the dorsal sepal, petal colours, and shape of hybrid flowers varied significantly among individual hybrids, between hybrids and their progenitors. Some mutations in the lips and columns of the novel hybrid flowers were also visualized. Hence, the D. anosmum hybrid seedlings successfully induced flowers after a year of culture under optimal hormones and fertilizers conditions. The results can serve as a critical reference for the early flowering of the orchid seedlings

A novel ontology framework supporting model-based tourism recommender

In this paper, we present a tourism recommender framework based on the cooperation of ontological knowledge base and supervised learning models. Specifically, a new tourism ontology, which not only captures domain knowledge but also specifies knowledge entities in numerical vector space, is presented. The recommendation making process enables machine learning models to work directly with the ontological knowledge base from training step to deployment step. This knowledge base can work well with classification models (e.g., k-nearest neighbours, support vector machines, or naıve bayes). A prototype of the framework is developed and experimental results confirm the feasibility of the proposed framework. © 2021, Institute of Advanced Engineering and Science. All rights reserved

AMRViz enables seamless genomics analysis and visualization of antimicrobial resistance

We have developed AMRViz, a toolkit for analyzing, visualizing, and managing bacterial genomics samples. The toolkit is bundled with the current best practice analysis pipeline allowing researchers to perform comprehensive analysis of a collection of samples directly from raw sequencing data with a single command line. The analysis results in a report showing the genome structure, genome annotations, antibiotic resistance and virulence profile for each sample. The pan-genome of all samples of the collection is analyzed to identify core- and accessory-genes. Phylogenies of the whole genome as well as all gene clusters are also generated. The toolkit provides a web-based visualization dashboard allowing researchers to interactively examine various aspects of the analysis results. Availability: AMRViz is implemented in Python and NodeJS, and is publicly available under open source MIT license at https://github.com/amromics/amrviz

Efficient inference of large prokaryotic pangenomes with PanTA

Pangenome inference is an indispensable step in bacterial genomics, yet its scalability poses a challenge due to the rapid growth of genomic collections. This paper presents PanTA, a software package designed for constructing pangenomes of large bacterial datasets, showing unprecedented efficiency levels multiple times higher than existing tools. PanTA introduces a novel mechanism to construct the pangenome progressively without rebuilding the accumulated collection from scratch. The progressive mode is shown to consume orders of magnitude less computational resources than existing solutions in managing growing datasets. The software is open source and is publicly available at https://github.com/amromics/panta and at 10.6084/m9.figshare.23724705

Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

Peer reviewe

AMRomics: a scalable workflow to analyze large microbial genome collections

Whole genome analysis for microbial genomics is critical to studying and monitoring antimicrobial resistance strains. The exponential growth of microbial sequencing data necessitates a fast and scalable computational pipeline to generate the desired outputs in a timely and cost-effective manner. Recent methods have been implemented to integrate individual genomes into large collections of specific bacterial populations and are widely employed for systematic genomic surveillance. However, they do not scale well when the population expands and turnaround time remains the main issue for this type of analysis. Here, we introduce AMRomics, an optimized microbial genomics pipeline that can work efficiently with big datasets. We use different bacterial data collections to compare AMRomics against competitive tools and show that our pipeline can generate similar results of interest but with better performance. The software is open source and is publicly available at https://github.com/amromics/amromics under an MIT license

PHÁT HIỆN VI KHUẨN PASTEURELLA MULTOCIDA GÂY BỆNH TỤ HUYẾT TRÙNG Ở CỪU PHAN RANG BẰNG KỸ THUẬT PCR

Pasteurellosis is an infectious disease causing severe damage to livestock and poultry since it leads to animals’ death, including sheep of all ages. In this study, we developed a sensitive, specific and accurate PCR method for detecting P. multocida in Phan Rang sheep using FKMT1/RKMT1 primers targeted to the KMT1 gene of P. multocida. The PCR-based method was used to detect the KMT1 gene from 104 copies of the KMT1-bearing plasmid. The method’s specificity was demonstrated by the successful amplification of KMT1 from the mixture containing other DNA of Gram-negative bacteria, such as E. coli or M. haemolytica. In particular, with the present method, we successfully detected P. multocida with crude DNA obtained from P. multocida or nasal swabs of pasteurellosis-suspected sheep samples treated in a TE buffer containing 0.1% TritonX-100. This is the first report on using a PCR-based method for detecting P. multocida from Phan Rang sheep, and it can serve as the basis for an effective procedure to diagnose pasteurellosis in sheep.Tụ huyết trùng là một bệnh truyền nhiễm có thể gây thiệt hại nghiêm trọng cho chăn nuôi gia súc, gia cầm nếu không được chẩn đoán và điều trị kịp thời. Vì vậy, trong nghiên cứu này chúng tôi đã phát triển phương pháp PCR có độ nhạy, độ đặc hiệu và độ chính xác cao để phát hiện sự có mặt của vi khuẩn P. multocida, một trong những tác nhân gây bệnh tụ huyết trùng trên cừu Phan Rang. Gen KMT1 của vi khuẩn P. multocida được nhân lên bằng cặp mồi đặc hiệu FKMT1/RKMT1 ở nồng độ khoảng 104 bản sao của plasmid mang gen đích. Phản ứng PCR không bị ảnh hưởng khi có mặt DNA của một số vi khuẩn Gram âm khác như E. coli hay M. haemolytica. Đặc biệt, với phương pháp PCR, chúng tôi đã phát hiện sự có mặt của vi khuẩn P. multocida từ mẫu DNA thô thu được bằng xử lý mẫu vi khuẩn/mẫu dịch ngoáy mũi của cừu nghi nhiễm bệnh trong đệm TE chứa 0,1% TritonX-100 mà không cần tinh sạch DNA tổng số. Đây là nghiên cứu đầu tiên công bố một phương pháp xác định sự có mặt của P. multocida trên cừu Phan Rang và là cơ sở để xây dựng quy trình chẩn đoán hiệu quả nhằm góp phần kiểm soát bệnh tụ huyết trùng trên đối tượng này

NGHIÊN CỨU CHẨN ĐOÁN PASTEURELLA MULTOCIDA VÀ MANNHEIMIA HAEMOLYTICA BẰNG KỸ THUẬT MULTIPLEX PCR

The multiplex PCR assay has been used to diagnose different diseases in livestock in Vietnam; however, this method has not been well-applied to sheep diseases. This study developed a multiplex PCR assay to detect the two main agents, Pasteurella multocida and Mannheimia haemolytica, causing Pasteurellosis in sheep. By using two pairs of specific primers, with the optimum concentration of each primer being 5 pM, this method successfully amplified the kmt1 gene from P. multocida and the gcp gene from M. haemolytica in the same reaction containing DNA of P. multocida and M. haemolytica at the lowest concentration of 0.1 ng each. Moreover, this multiplex PCR reaction is not affected by the artificial presence of some other bacterial DNA. In particular, this multiplex PCR showed 100% confidence in a stability test on a small scale with 20 raw DNA samples obtained from direct heat treatment or enrichment before heat treatment of 10 field samples. This is the first study in Vietnam on diagnosing P. multocida and M. haemolytica by multiplex PCR. This method is valuable for detecting P. multocida and M. haemolytica in sheep and serves as a reference for detecting these bacteria in other household animal.Ở Việt Nam, kỹ thuật multiplex PCR đã được sử dụng để chẩn đoán nhiều bệnh trên vật nuôi nhưng kỹ thuật này hiện nay chưa được áp dụng để chẩn đoán bệnh trên cừu. Trong nghiên cứu này, chúng tôi đã phát triển một kỹ thuật multiplex PCR để phát hiện đồng thời hai tác nhân chính gây tụ huyết trùng trên cừu Phan Rang là Pasteurella multocida và Mannheimia haemolytica. Bằng cách sử dụng hai cặp mồi đặc hiệu, với nồng độ tối ưu của mỗi mồi là 5 pM, phương pháp này đã nhân thành công đoạn gen kmt1 từ P. multocida và gcp từ M. haemolytica trong cùng một phản ứng chứa hỗn hợp DNA tổng số của hai vi khuẩn này ở nồng độ thấp nhất là 0,1 ng mỗi loại. Thêm vào đó, phản ứng multiplex PCR này không bị ảnh hưởng bởi sự có mặt nhân tạo của một số DNA từ vi khuẩn khác. Đặc biệt, phương pháp multiplex PCR này có độ ổn định đạt 100% khi thử nghiệm trên quy mô nhỏ với 20 mẫu DNA thô thu được từ phương pháp xử lý nhiệt trực tiếp hay xử lý nhiệt sau khi làm giàu 10 mẫu thực địa. Đây là nghiên cứu đầu tiên ở Việt Nam về việc chẩn đoán tụ huyết trùng trên cừu bằng kỹ thuật multiplex PCR, kỹ thuật không chỉ có giá trị để phát hiện vi khuẩn P. multocida và M. haemolytica ở cừu mà còn có vai trò tham khảo để chẩn đoán những vi khuẩn này trong các đối tượng vật nuôi khác

Clinical Epidemiology Characteristics and Antibiotic Resistance Associated with Urinary Tract Infections Caused by E. coli

Introduction. In individuals with urinary tract infections, Escherichia coli (E. coli) is an ubiquitous causative agent and antibiotic resistance is on the rise throughout the world. Therefore, early diagnosis and appropriate choice of antimicrobials are essential. The purpose of our study is to describe some of the clinical and epidemiological characteristics and the laboratory test results of children treated in our hospital for urinary tract infections caused by E. coli. Methods. The study included 128 patients from 2 months to 15 years of age with urinary tract infections caused by E. coli and treated at the Haiphong Children’s Hospital during the periods of 2011–2013 and 2018–2020. Results. During the two study periods, 57 and 71 cases, respectively, were included. The most common clinical symptom was fever in 40 and 46 cases, respectively. The proportion of E. coli’s resistance to ampicillin increased from 85.3% in 2011–2013 to 97.1% in 2018–2020. In 2011–2013, 70.5% of E. coli isolates were resistant to cotrimoxazole, which increased to 81.4% during 2018–2020. During both periods, E. coli was highly sensitive to amikacin, at 87% and 95.5%, respectively. In 2018–2020, carbapenems (meropenem and imipenem) and piperacillin were also effective against E. coli. Conclusion. Our study revealed that high fever was the most prevalent clinical characteristic in urinary tract infections caused by E. coli in children and E. coli was mostly resistant to ampicillin, nalidixic acid, and cotrimoxazole but was highly sensitive to ciprofloxacin, amikacin, piperacillin, meropenem, and imipenem