Unique Primer Research Articles

Golden EGG, a simplified Golden Gate cloning system to assemble multiple fragments

The Golden Gate method is an efficient tool for seamless assembly of multiple DNA fragments, which uses Type IIS restriction endonucleases, cleaving the DNA outside of their recognition site to release DNA parts from PCR fragments or entry clones, thus allowing the design of overhangs for ligation at will. However, the construction of the entry clones requires the use of other restriction enzyme(s) or cloning techniques and different entry vectors for the individual overhangs. Here, we present a simplified Golden Gate cloning approach termed Golden EGG. It features (1) a single entry vector with a specific cloning site to host the DNA parts; (2) a unique primer design to create the restriction enzyme recognition site to release the fragments with the overhangs at will; (3) the use of a single Type IIS enzyme for the construction of both the entry and destination clones; (4) a specific temperature profile during the digestion-ligation reaction. Our user-friendly, streamlined method retains the key attributes of the Golden Gate technique, while offering the potential to generate compatible parts with any existing Golden Gate toolkit and to be accessible to a wide user base without the need for extensive acquisition of new vectors or expensive enzymes.

A DAMP-Based Assay for Rapid and Affordable Diagnosis of Bacterial Meningitis Agents: Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae.

The rapid and accurate diagnosis of meningitis is critical for preventing severe complications and fatalities. This study addresses the need for accessible diagnostics in the absence of specialized equipment by developing a novel diagnostic assay. The assay utilizes dual-priming isothermal amplification (DAMP) with unique internal primers to significantly reduce non-specificity. For fluorescence detection, the dye was selected among Brilliant Green, Thioflavin T, and dsGreen. Brilliant Green is preferred for this assay due to its availability, high fluorescence level, and optimal sample-to-background (S/B) ratio. The assay was developed for the detection of the primary causative agents of meningitis (Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae), and tested on clinical samples. The developed method demonstrated high specificity, no false positives, sensitivity comparable to that of loop-mediated isothermal amplification (LAMP), and a high S/B ratio. This versatile assay can be utilized as a standalone test or an integrated assay into point-of-care systems for rapid and reliable pathogen detection.

PUPpy: a primer design pipeline for substrain-level microbial detection and absolute quantification.

Profiling microbial communities at high resolution and with absolute quantification is essential to uncover hidden ecological interactions within microbial ecosystems. Nevertheless, achieving resolved and quantitative investigations has been elusive due to methodological limitations in distinguishing and quantifying highly related microbes. Here, we describe Phylogenetically Unique Primers in python (PUPpy), an automated computational pipeline to design taxon-specific primers within defined microbial communities. Taxon-specific primers can be used to selectively detect and quantify individual microbes and larger taxa within a microbial community. PUPpy achieves substrain-level specificity without the need for computationally intensive databases and prioritizes user-friendliness by enabling both terminal and graphical user interface applications. Altogether, PUPpy enables fast, inexpensive, and highly accurate perspectives into microbial ecosystems, supporting the characterization of bacterial communities in both in vitro and complex microbiota settings.

High throughput screening of mesenchymal stromal cell morphological response to inflammatory signals for bioreactor-based manufacturing of extracellular vesicles that modulate microglia

Due to their immunomodulatory function, mesenchymal stromal cells (MSCs) are a promising therapeutic with the potential to treat neuroinflammation associated with neurodegenerative diseases. This function is mediated by secreted extracellular vesicles (MSC-EVs). Despite established safety, MSC clinical translation has been unsuccessful due to inconsistent clinical outcomes resulting from functional heterogeneity. Current approaches to mitigate functional heterogeneity include ‘priming’ MSCs with inflammatory signals to enhance function. However, comprehensive evaluation of priming and its effects on MSC-EV function has not been performed. Furthermore, clinical translation of MSC-EV therapies requires significant manufacturing scale-up, yet few studies have investigated the effects of priming in bioreactors. As MSC morphology has been shown to predict their immunomodulatory function, we screened MSC morphological response to an array of priming signals and evaluated MSC-EV identity and potency in response to priming in flasks and bioreactors. We identified unique priming conditions corresponding to distinct morphologies. These conditions demonstrated a range of MSC-EV preparation quality and lipidome, allowing us to discover a novel MSC-EV manufacturing condition, as well as gain insight into potential mechanisms of MSC-EV microglia modulation. Our novel screening approach and application of priming to MSC-EV bioreactor manufacturing informs refinement of larger-scale manufacturing and enhancement of MSC-EV function.

A frontier-orbital view of the initial steps of lytic polysaccharide monooxygenase reactions.

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes that oxidatively cleave the strong C-H bonds in recalcitrant polysaccharide substrates, thereby playing a crucial role in biomass degradation. Recently, LPMOs have also been shown to be important for several pathogens. It is well established that the Cu(II) resting state of LPMOs is inactive, and the electronic structure of the active site needs to be altered to transform the enzyme into an active form. Whether this transformation occurs due to substrate binding or due to a unique priming reduction has remained speculative. Starting from four different crystal structures of the LPMO LsAA9A with well-defined oxidation states, we use a frontier molecular orbital approach to elucidate the initial steps of the LPMO reaction. We give an explanation for the requirement of the unique priming reduction and analyse electronic structure changes upon substrate binding. We further investigate how the presence of the substrate could facilitate an electron transfer from the copper active site to an H2O2 co-substrate. Our findings could help to control experimental LPMO reactions.

Integration of in-cassette lysis, purification, and lateral flow strips-based sensor for rapid and on-site detection of yak milk adulteration

Amplification-based nucleic acid testing has become a widely used method for food authenticity analysis. However, due to the need for specialized instrumentation, technical expertize, and trained operators, routine nucleic acid analysis can only be performed in a laboratory setting. For perishable products like yak milk, rapid and accurate detection of dairy components on-site is crucial for the automated management of the production process and improving the milk's quality at dairies. As a feasibility study, we developed an assay strategy for the simultaneous qualitative detection of yak and cow derived components in yak milk. We presented a handheld integrated cassette that combined rapid extraction and purification of dairy nucleic acids, loop-mediated isothermal amplification (LAMP), and visual detection of lateral flow strips (LFS). To enable visual detection of LAMP amplification products on LFS, we utilized a unique primer modification method that labeled the amplification terminator and single oligonucleotide sequence at the 5′ end of the loop primer. This modification avoids the use of antibodies and conventional fluorescein/biotin. The cassette allowed for DNA transfer, fluid drive, and liquid mixing by magnetic force and gravity alone, without the need for complex pumps and valves. The disposable cassette is easy to use, costs less than $1 to produce, has low instrument dependency, and is well suited to on-site detection. The platform can detect 0.1% of cow milk adulteration in yak milk within 50 min. This fully integrated "sample in-result out" detection procedure can also be extended to other detection areas.

Rapid and visual identification of HIV-1 using reverse transcription loop-mediated isothermal amplification integrated with a gold nanoparticle-based lateral flow assay platform.

Human immunodeficiency virus type one (HIV-1) infection remains a major public health problem worldwide. Early diagnosis of HIV-1 is crucial to treat and control this infection effectively. Here, for the first time, we reported a novel molecular diagnostic assay called reverse transcription loop-mediated isothermal amplification combined with a visual gold nanoparticle-based lateral flow assay (RT-LAMP-AuNPs-LFA), which we devised for rapid, specific, sensitive, and visual identification of HIV-1. The unique LAMP primers were successfully designed based on the pol gene from the major HIV-1 genotypes CRF01_AE, CRF07_BC, CRF08_BC, and subtype B, which are prevalent in China. The optimal HIV-1-RT-LAMP-AuNPs-LFA reaction conditions were determined to be 68°C for 35 min. The detection procedure, including crude genomic RNA isolation (approximately 5 min), RT-LAMP amplification (35 min), and visual result readout (<2 min), can be completed within 45 min. Our assay has a detection limit of 20 copies per test, and we did not observe any cross-reactivity with any other pathogen in our testing. Hence, our preliminary results indicated that the HIV-1-RT-LAMP-AuNPs-LFA assay can potentially serve as a useful point-of-care diagnostic tool for HIV-1 detection in a clinical setting.

Ancient mitochondrial genome diversity in South America: Contributions from Quebrada del Toro, Northwestern Argentina.

The objective of this study was to enhance our understanding of the population history in South America, specifically Northwestern Argentina, by analyzing complete ancient mitogenomes of individuals from the Ojo de Agua archeological site (970 BP) in Quebrada del Toro (Salta, Argentina). We analyzed teeth from four individuals from the site Ojo de Agua (970 ± 60 BP), located in Quebrada del Toro (Andean region of Northwestern Argentina). DNA extracts were converted to double-stranded DNA libraries and indexed using unique dual-indexing primer combinations. DNA libraries were then enriched for the complete mitochondrial genome, pooled at equimolar concentrations, and sequenced on an Illumina® MiSeq™ platform. Reads from high quality libraries were trimmed, merged, and then mapped to the revised Cambridge Reference Sequence. The aDNA damage patterns were assessed and contamination estimated. Finally, variants were called, filtered, and the consensus mitogenome was constructed and used for haplogroup assignment. We also compiled available mitogenome sequences from ancient and present-day populations from the Southcentral Andes and other surrounding regions in Argentina. Maximum Likelihood and Bayesian phylogenetic reconstructions were obtained using the generated dataset. We successfully obtained the complete mitogenome sequence from one individual with an average depth coverage of 102X. We discovered a novel haplotype that was assigned to haplogroup D1. Phylogenetic reconstructions suggests that this haplotype falls within the sister branches of the D1j lineage, forming a well-supported clade. The estimate TMRCA of this clade that includes D1j and its sister branches ranged between 12,535 and 18,669 ya. The sequence analyzed in this study represents the first ancient mitogenome from within the valley region in Northwestern Argentina. We found that a representative of a lineage highly associated with D1j was already present approximately 1000 BP in the region. Our results agree with the proposed origin of D1j in other regions north of Patagonia and independent of the Pacific coast fast migratory route, contrary to what was originally hypothesized. This study highlights the lack of information regarding pre-Hispanic genetic diversity and contributes to the knowledge about the peopling process in South America.

P95: Heart Lung Machine in a Box

Disclosure: There are no conflicts of interest while writing this ASAIO abstract Purpose of study: We propose a solution to the treatment of seriously ill cardiac arrest and pulmonary distressed patients. This can be done by significantly decreasing the current time intervals of when substantial cardiac and/or pulmonary assistance is started. Acute cardiac arrest patients should be treated within seconds to minutes, and patients in serious pulmonary distress should be treated within minutes to hours. This will hopefully help them avoid using a harmful ventilator. Methods: The key is to utilize the redesigned “Heart Lung Machine in a Box” immediately. Dr. Arnold Lande is the pioneer of the ECMO technology and has revised his original design. Its unique rapid vacuum-aided priming design assures that air embolisms will not occur. The device draws oxygen through an oxygenator rather than pushing it through like the current technologies Results: By design, the upgraded ECMO technology helps eliminate accidents, works through power failures, avoids too much pressure potentially coming in, and requires less supervision than the current ECMO technology. Unlike now, these patients will not have to be observed constantly due to the new advanced safety measures in place. Summary: This will significantly increase the number of patients that can be treated by the ECMO technology thus lowering the labor costs for the medical facility. The redesigned “Heart-Lung Machine in a Box” ECMO technology can be widely and inexpensively stocked everywhere from the shelf in an ambulance to the ICU.

The study of mutations of CDK4 and TP53 genes in selected breast lesions

Lesions are abnormal changes in tissue. The theories behind tumorigenesis can be genetic epigenetic, immune surveillance and monoclonal hypothesis, which led to genetic changes. The genetic change can be the inactivation of tumor suppressor genes or the activation of oncogenes in the cell cycle. This study investigated the presence of gene mutation in CDK4 and TP53 genes in breast lesions. Ten (10) formalin fixed paraffin embedded tissue block, which includes 5 cases of fibroadenoma and 5 cases of invasive ductal carcinoma from the pathological archives were used. The nuclei amplification technique was carried out on the formalin fixed paraffin embedded tissue blocks. DNA extraction involves lysing the cells and solubilizing DNA, which is followed by chemical or enzymatic methods to remove macromolecules, lipids, RNA or proteins. Thereafter, the DNA is amplified using reagents and heated (denaturing) and cooling (annealing) steps. PCR requires free nucleotide (dNTPs), template DNA to amplify and unique single-stranded DNA primers that bind upstream (5') and downstream (3') of the DNA region of the tissue blocks. The integrity of the DNA is assessed by loading approximately 100 ng per sample on a 1.5% agarose gel and the sizes of PCR products were estimated by comparison with the mobility of a 100bp molecular weight ladder that was run alongside the samples in the gel. However, the amplified fragment was purified to remove PCR reagents and thereafter it was sequenced. The sequence of single nucleotide polymorphism of each gene was analyzed, and the results were reported. In mutation was more in both genes compared to the sequence of FA and IDC. From this study, both genes showed segments of mutation which were either SNPs or functional mutation. It showed that both genes were modified from a proto-oncogene to an oncogene and the protein it produces is an oncoprotein and as such it favours the alteration of both genes led to cell proliferation. The data generated from this research has highlighted the importance of carrying out further studies in assessing both genes to understand their involvement in the etiology of carcinogenesis which will help in clinical information about breast cancer. In line with the findings, there is an urgent need to understand carcinogenesis which may serve in early diagnosis and understanding of breast conditions and further help prevent or delay the onset of chronic complications and reduce morbidity and mortality rates to improve and assure the health of individuals.

Abstract A23: Less is more: Ultra-low input sequencing allows for evaluation of stem cells as a predictor of post-transplant relapse in the myelodysplastic syndromes

Abstract Malignant hematopoietic stem cells (HSCs) initiate the myelodysplastic syndromes (MDS) and drive treatment failure through their resistance to all non-transplant therapies. As a result, allogeneic stem cell transplantation (allo-SCT) is the only curative therapy for MDS. However, relapse remains the leading cause of treatment failure after allo-SCT. Improved tools to predict relapse in this setting are necessary to allow for pre-emptive therapeutic interventions, which may be more effective when the burden of disease is still low. Given prior evidence that HSCs serve as the functional unit of resistance to non-transplant therapies, we asked whether relapse after transplant originates from MDS HSCs, and whether their persistence can predict relapse. To overcome the limiting number of HSCs available in clinical bone marrow (BM) samples, we adapted a workflow developed for assessment of cell-free DNA and utilized a hybridization capture-based targeted sequencing panel covering 260 myeloid malignancy-associated genes. Unique dual index primers (UDIs) were utilized to tag input DNA, improving PCR duplicate identification, which overcome issues with amplification bias typically faced by low-input sequencing. This ultimately allowed us to consistently capture evaluable read depths from as few as 10 sorted cells. We assembled an initial cohort of 21 patients who underwent allo-SCT for MDS or secondary acute myeloid leukemia. Inclusion criteria were follow-up time greater than 1.5 years (or relapse in this timeframe), BM sampling prior to Day+120 post-transplant and relapse, and biospecimens allowing for sorting of greater than 10 HSCs. We sequenced sorted CD34+CD38-CD45RA- HSCs and multipotent progenitors (MPPs), CD34+CD38- CD45RA+ lymphoid-primed multipotent progenitors (LMPPs), and CD34+CD38+ progenitors, as well as bulk BM. With a median follow-up of two years, 9 of the 21 patients relapsed, with an average time to relapse of 362 days. Detection of disease mutations in HSCs/MPPs in any BM at D+30-D+120 post-transplant was 100% specific and 88% sensitive for relapse, showing improvements in both metrics over bulk BM assessment (22% sensitive and 66% specific), despite far lower cell input and read depth. Average time from detection to relapse was 6.9 months. In patients destined to relapse, disease mutations are often present in HSCs at a variant allele frequency of greater than 45%, while simultaneously undetectable on high-depth sequencing of bulk cells (&amp;gt;400x coverage). While even higher depth error-corrected sequencing bulk BM could likely detect such mutations, it would require &amp;gt;20x the sequencing depth, and could not identify the involved cell populations. In conclusion, we show for the first time that relapse of MDS after allo-SCT is driven by failure to eradicate MDS HSCs. Detection of MDS HSCs early post-transplant is highly predictive for relapse and opens new doors for pre-emptive interventions to prevent relapse. These findings are being validated in an independent cohort of 46 patients and updated data will be presented at the meeting. Citation Format: Benjamin Kroger, Yi Huang, Prapti Patel, Aimaz Afrough, Gurbakhash Kaur, Larry Anderson, Farrukh Awan, Praveen Ramakrishnan, Kunal Verma, Russell Vittrup, Robert H Collins, Madhuri Vusirikala, Yazan F Madanat, David Sallman, Carmelo Gurnari, Jaroslaw Maciejewski, Roni Tamari, Stephen Chung. Less is more: Ultra-low input sequencing allows for evaluation of stem cells as a predictor of post-transplant relapse in the myelodysplastic syndromes [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A23.

Relationship Between the Prime-Counting Function and a Unique Prime Number Sequence

In mathematics, the prime counting function $\pi(x)$ is defined as the function yielding the number of primes less than or equal to a given number $x$. In this paper, we prove that the asymptotic limit of a summation operation performed on a unique subsequence of the prime numbers yields the prime number counting function $\pi(x)$ as $x$ approaches $\infty$. We also show that the prime number count $\pi(n)$ can be estimated with a notable degree of accuracy by performing the summation operation on the subsquence up to a limit $n$.

Visual and rapid identification of Chlamydia trachomatis and Neisseria gonorrhoeae using multiplex loop-mediated isothermal amplification and a gold nanoparticle-based lateral flow biosensor.

Sexually transmitted chlamydia and gonorrhea infections caused by the bacteria Chlamydia trachomatis and Neisseria gonorrhoeae remain a major public health concern worldwide, particularly in less developed nations. It is crucial to use a point of care (POC) diagnostic method that is quick, specific, sensitive, and user-friendly to treat and control these infections effectively. Here, a novel molecular diagnostic assay, combining multiplex loop-mediated isothermal amplification (mLAMP) with a visual gold nanoparticles-based lateral flow biosensor (AuNPs-LFB) was devised and used for highly specific, sensitive, rapid, visual, and easy identification of C. trachomatis and N. gonorrhoeae. Two unique independent primer pairs were successful designed against the ompA and orf1 genes of C. trachomatis and N. gonorrhoeae, respectively. The optimal mLAMP-AuNPs-LFB reaction conditions were determined to be 67°C for 35min. The detection procedure, involving crude genomic DNA extraction (~5 min), LAMP amplification (35min), and visual results interpretation (<2min), can be completed within 45min. Our assay has a detection limit of 50 copies per test, and we did not observe any cross-reactivity with any other bacteria in our testing. Hence, our mLAMP-AuNPs-LFB assay can potentially be used for POC testing to detect C. trachomatis and N. gonorrhoeae in clinical settings, particularly in underdeveloped regions.

Genome survey and genetic characterization of Acacia pachyceras O. Schwartz.

Acacia pachyceras O. Schwartz (Leguminoseae), a woody tree growing in Kuwait is critically endangered. High throughput genomic research is immediately needed to formulate effective conservation strategies for its rehabilitation. We therefore, performed a genome survey analysis of the species. Whole genome sequencing generated ~97 Gb of raw reads (92x coverage) with a per base quality score above Q30. The k-mer analysis (17 mer) revealed its genome to be 720Mb in size with an average guanine-cytosine (GC) ratio of 35%. The assembled genome was analyzed for repeat regions (45.4%-interspersed repeats; 9%-retroelements; 2%-DNA transposons). BUSCO assessment of completeness of genome identified 93% of assembly to be complete. Gene alignments in BRAKER2 yielded 34,374 transcripts corresponding to 33,650 genes. Average length of coding sequences and protein sequences were recorded as 1,027nts and 342aa, respectively. GMATA software filtered a total of 901,755 simple sequence repeats (SSRs) regions against which 11,181 unique primers were designed. A subset of 110 SSR primers were PCR validated and demonstrated for its application in genetic diversity analysis of Acacia. The SSR primers successfully amplified A. gerrardii seedlings DNA depicting cross transferability among species. The principal coordinate analysis and the split decomposition tree (bootstrapping runs of 1000 replicates) distributed the Acacia genotypes into two clusters. The flow cytometry analysis revealed the A. pachyceras genome to be polyploid (6x). The DNA content was predicted as 2.46 pg, 1.23 pg, and 0.41 pg corresponding to 2C DNA, 1C DNA and 1Cx DNA, respectively. The results provide a base for further high throughput genomic studies and molecular breeding for its conservation.

Description of novel capsule biosynthesis loci of Campylobacter jejuni clinical isolates from South and South-East Asia.

Campylobacter jejuni is a major cause of bacterial diarrhea worldwide and associated with numerous sequela, including Guillain-Barré Syndrome, inflammatory bowel disease, reactive arthritis, and irritable bowel syndrome. C. jejuni is unusual for an intestinal pathogen in its ability to coat its surface with a polysaccharide capsule (CPS). The genes responsible for the biosynthesis of the phase variable CPS is located in the hypervariable region of C. jejuni genome which has been used to develop multiplex PCR to classify CPS types based on the Penner serotypes. However, there still are non-typable CPS C. jejuni by the current multiplex PCR scheme. The application of the next generation sequencing and whole genome analysis software were used for the identification of novel capsule biosynthesis of C. jejuni isolates. Unique PCR primers were designed to identify these new capsule biosynthesis loci. The designed primers sets were combined in a new multiplex mix called epsilon. The unique sequences provide an additional information of the biosynthesis loci responsible for some of the common CPS sugars/residues such as heptose, deoxtyheptose and MeOPN among C. jejuni in this new group of CPS multiplex assay. This new primer complements the current C. jejuni multiplex capsule typing system and will help in identifying previously untypeable capsule locus of C. jejuni isolates.

An Optimized CoBRA Method for the Microfluidic Electrophoresis Detection of Breast Cancer Associated RASSF1 Methylation.

Although breast cancer screening assays exist, many are inaccessible and have high turnaround times, leaving a significant need for better alternatives. Hypermethylation of tumor suppressor genes is a common epigenetic marker of breast cancer. Methylation tends to occur most frequently in the promoter and first exon regions of genes. Preliminary screening tests are crucial for informing patients whether they should pursue more involved testing. We selected RASSF1, previously demonstrated to be aberrantly methylated in liquid biopsies from breast cancer patients, as our gene of interest. Using CoBRA as our method for methylation quantification, we designed unique primer sets that amplify a portion of the CpG island spanning the 5' end of the RASSF1 first exon. We integrated the CoBRA approach with a microfluidics-based electrophoresis quantification system (LabChip) and optimized the assay such that insightful results could be obtained without post-PCR purification or concentration, two steps traditionally included in CoBRA assays. Circumventing these steps resulted in a decreased turnaround time and mitigated the laboratory machinery and reagent requirements. Our streamlined technique has an estimated limit of detection of 9.1 ng/μL of input DNA and was able to quantify methylation with an average error of 4.3%.

ЕКСПРЕСІЯ ГЕНІВ МЕХАНОЧУТЛИВИХ РЕЦЕПТОРІВ У ДЕТРУЗОРІ СЕЧОВОГО МІХУРА ЩУРА

B a c k g r o u n d . Autonomic mechanosensitivity of the muscle layer (detrusor) of the urinary bladder is an actual problem that still remains understudied. It is known that a number of mechanosensitive channels are probably expressed, but to date, only the leading role of the mechanosensitive channel TREK-1 has been established. The aim of the study was to check the expression of mechanoreceptor genes of smooth muscle tissue (KCNK2 (TREK-1), TRPV4, Piezo1, and Piezo2) in the bladder detrusor. M e t h o d s . Unique primers were selected for the detection of the specified channel genes, as well as the reference gene GAPDH, using the BLAST software. Validation was performed using one-step PCR with reverse transcription on the CFX96 amplifier with fluorescence detection of the Fam sample. A matrix RNA sample was isolated from the muscle and urothelial layers of the urinary bladder and dorsal root ganglia (DRG) of healthy Wistar rats using a commercial nucleic acid extraction column kit (Biocorp, Ukraine). R e s u l t s . In our samples, Piezo1 and TREK-1 channels were shown to be expressed in the urothelial and muscle layers of the bladder, whereas TRPV4 was only expressed in the urothelium. Piezo2 gene expression was not detected. C o n c l u s i o n s . The selected sequences of primers for TREK-1, Piezo1, TRPV4, and the "housekeeping" gene GAPDH are effective for studying the expression of these genes in rats. The synthesized sequences correspond to the expected fragment sizes. The genes of three mechanosensitive channels are expressed in the urinary bladder: TREK-1, Piezo1, and TRPV4.

Point‐of‐Care Diagnostic Platforms for Loop‐Mediated Isothermal Amplification

The loop‐mediated isothermal amplification (LAMP) method is one of the Nucleic acid amplification tests (NAATs) that allows for the amplification of target regions without using a thermal cycle. With its unique primer design, LAMP ensures the rapid replication of the targeted DNA region with high specificity and high efficiency. LAMP technology is used for diagnostic purposes in pathogen detection due to its ease of use, low cost, and simplicity without requiring complex equipment. A wide range of LAMP diagnostic platforms have been developed for applications in bacteria, virus, and parasitic pathogen detection. Herein, the methodology of LAMP technology and its applications in pathogen detection and SNP genotyping and mutation detection are discussed. Point‐of‐care (PoC) LAMP platforms designed with the principles of microfluidic chip technology, including LAMP‐on‐a‐chip, paper‐based LAMP, and smartphone‐based LAMP applications have been elaborated. LAMP technology represents a fast, robust, and reliable diagnostic platform for point‐of‐care testing.

Identification of fish species through tRNA-based primer design

BackgroundThe correct establishment of the barcode classification system for fish can facilitate biotaxonomists to distinguish fish species, and it can help the government to verify the authenticity of the ingredients of fish products or identify unknown fish related samples. The Cytochrome c oxidation I (COI) gene sequence in the mitochondria of each species possesses unique characteristics, which has been widely used as barcodes in identifying species in recent years. Instead of using COI gene sequences for primer design, flanking tRNA segments of COI genes from 2618 complete fish mitochondrial genomes were analyzed to discover suitable primers for fish classification at taxonomic family level. The minimal number of primer sets is designed to effectively distinguish various clustered groups of fish species for identification applications. Sequence alignment analysis and cross tRNA segment comparisons were applied to check and ensure the primers for each cluster group are exclusive.ResultsTwo approaches were applied to improve primer design and re-cluster fish species. The results have shown that exclusive primers for 2618 fish species were successfully discovered through in silico analysis. In addition, we applied sequence alignment analysis to confirm that each pair of primers can successfully identify all collected fish species at the taxonomic family levels.ConclusionsThis study provided a practical strategy to discover unique primers for each fishery species and a comprehensive list of exclusive primers for extracting COI barcode sequences of all known fishery species. Various applications of verification of fish products or identification of unknown fish species could be effectively achieved.

Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data.

Advances in next-generation sequencing (NGS) have improved the resolution of T-cell receptor (TCR) repertoire analysis, and recent single-cell sequencing has made it possible to obtain information about TCR pairs. In our previous study, cytomegalovirus (CMV) pp65-specific T-cell response restricted by a single human leukocyte antigen (HLA) class I allotype was observed in an individual. Therefore, to effectively clone an antigen-specific TCR from these T cells, we developed a TCR cloning system that does not require a single cell level. First, we established the improved Jurkat reporter cell line, which was TCRαβ double knock-out and expressed CD8αβ molecules. Furthermore, functional TCRs were directly obtained by reverse TCR cloning using unique CDR3-specific PCR primers after bulk TCR sequencing of activation marker-positive CD8 T cells by NGS. A total of 15 TCRα and 14 TCRβ strands were successfully amplified by PCR from cDNA of 4-1BB-positive CD8 T cells restricted by HLA-A*02:01, HLA-A*02:06, HLA-B*07:02, and HLA-B*40:06. The panels with combinations of TCRα and TCRβ genes were investigated using Jurkat reporter cell line and artificial antigen-presenting cells (APCs). In two TCR pairs restricted by HLA-A*02:01, one TCR pair by HLA-A*02:06, four TCR pairs by HLA-B*07:02, and one TCR pair by HLA-B*40:06, their specificity and affinity were confirmed. The TCR pair of A*02:01/1-1 showed alloreactivity to HLA-A*02:06. The one TCR pair showed a higher response to the naturally processed antigen than that of the peptide pool. This reverse TCR cloning system will not only provide functional information to TCR repertoire analysis by NGS but also help in the development of TCR-T therapy.