Low Sensitivity of Real Time PCRs Targeting Retrotransposon Sequences for the Detection of Schistosoma japonicum Complex DNA in Human Serum

While hybridization probe-based real-time PCR assays targeting highly repetitive multi-copy genome sequences for the diagnosis of S. mansoni complex or S. haematobium complex from human serum are well established, reports on the evaluation of respective assays for the identification of S. japonicum complex DNA in human serum are scarce. Here, we assessed the potential use of the retrotransposon sequences SjR2 and SjCHGCS19 from S. japonicum, S. mekongi and S. malayensis for the diagnosis of Asian Schistosoma infections. Based on available S. japonicum sequences and newly provided S. mekongi and S. malayensis sequences, hybridization probe-based real-time PCRs targeting SjR2 and SjCHGCS19 of the S. japonicum complex were designed both as consensus primer assays as well as multi-primer assays for the coverage of multiple variants of the target sequences. The assays were established using plasmids and S. mekongi DNA. While the consensus primer assays failed to detect S. mekongi DNA in human serum samples, the multi-primer assays showed positive or borderline positive results but only in 9.8% (6/61) of serum samples from patients with confirmed S. mekongi infections. Some cross-reactions with samples positive for S. mansoni or S. haematobium were observed but with the SjCHGCS19-PCR only. In spite of the low sensitivity, the presented experience may guide future evaluations of S. japonicum-complex-specific PCRs from human serum.

Single Copy Oligonucleotide Fluorescence In Situ Hybridization Probe Design Platforms: Development, Application and Evaluation

Oligonucleotides fluorescence in situ hybridization (Oligo-FISH) is an emerging technology and is an important tool in research areas such as detection of chromosome variation, identification of allopolyploid, and deciphering of three-dimensional (3D) genome structures. Based on the demand for highly efficient oligo probes for oligo-FISH experiments, increasing numbers of tools have been developed for probe design in recent years. Obsolete oligonucleotide design tools have been adapted for oligo-FISH probe design because of their similar considerations. With the development of DNA sequencing and large-scale synthesis, novel tools have been designed to increase the specificity of designed oligo probes and enable genome-scale oligo probe design, which has greatly improved the application of single copy oligo-FISH. Despite this, few studies have introduced the development of the oligo-FISH probe design tools and their application in FISH experiments systematically. Besides, a comprehensive comparison and evaluation is lacking for the available tools. In this review, we provide an overview of the oligo-FISH probe design process, summarize the development and application of the available tools, evaluate several state-of-art tools, and eventually provide guidance for single copy oligo-FISH probe design.

Comparison of the Diagnostic Performance of qPCR, Sanger Sequencing, and Whole-Genome Sequencing in Determining Clarithromycin and Levofloxacin Resistance in Helicobacter pylori

Helicobacter pylori antibiotic resistance is increasing worldwide, emphasizing the urgent need for more rapid resistance detection prior to the administration of H. pylori eradication regimens. Macrolides and fluoroquinolones are widely used to treat H. pylori. In this study, we aimed to compare the diagnostic performance of A) 23SrDNA qPCR (with melting curve analysis) and an in-house developed gyrA qPCR followed by Sanger sequencing with a commercial IVD-marked hybridization probe assay (for 23SrDNA and gyrA) using 142 gastric biopsies (skipping culturing) and B) the same two qPCR for 23SrDNA and gyrA (including Sanger sequencing) with whole-genome sequencing (WGS) and phenotypic characterization of clarithromycin and levofloxacin resistance using 76 cultured isolates. The sensitivity of both qPCRs was 100% compared to that of the commercial IVD-marked hybridization probe assay for the detection of H. pylori in gastric biopsies (without resistance testing). The specificity of the qPCR gyrA followed by Sanger sequencing was 100%, indicating that the best sequence identity was always H. pylori. The results show good agreement between molecular tests, especially between qPCR (inclusive Sanger sequencing) and WGS. Discrepancies (concerning mutated or wild type of positive H. pylori gastric biopsies) were observed between Sanger sequencing of the gyrA gene and the corresponding commercial hybridization probe assay, mostly because the high sequence diversity of the gyrA gene even at positions adjacent to the relevant codons of 87 and 91 interfered with obtaining correct results from the hybridization probe assay. Interestingly, we found several mixed sequences, indicating mixed populations in the gastric biopsies (direct detection without culturing). There was a high percentage of both levofloxacin and clarithromycin resistance in gastric biopsies (both between 22% and 29%, direct detection in gastric biopsies). Therefore, we recommend analyzing both targets in parallel. We confirmed that phenotypic resistance is highly correlated with the associated mutations. We concluded that the two qPCR followed by Sanger sequencing of the gyrA gene is a fast, cost-effective and comprehensive method for resistance testing of H. pylori directly in gastric biopsies.

Novel Biosensing Strategies for the in Vivo Detection of microRNA

As a regulatory molecule of post-transcriptional gene expression, microRNA (miRNA) is a class of endogenous, non-coding small molecule RNAs. MiRNA detection is essential for biochemical research and clinical diagnostics but challenging due to its low abundance, small size, and sequence similarities. In this chapter, traditional methods of detecting miRNA like polymerase chain reaction (PCR), DNA microarray, and northern blotting are introduced briefly. These approaches are usually used to detect miRNA in vitro. Some novel strategies for sensing miRNAs in vivo, including hybridization probe assays, strand-displacement reaction (SDR), entropy-driven DNA catalysis (EDC), catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), DNAzyme-mediated assays, and CRISPR-mediated assays, are elaborated in detail. This chapter describes the principles and designs of these detection technologies and discusses their advantages as well as their shortcomings, providing guidelines for the further development of more sensitive and selective miRNA sensing strategies in vivo.

Chemocatalytic Amplification Probes Enable Transcriptionally-Regulated Au(I)-Catalysis in E. coli and Sensitive Detection of SARS-CoV-2 RNA Fragments

<div>The union of transition metal catalysis with native biochemistry presents a powerful opportunity</div><div>to perform abiotic reactions within complex biological systems.(1,2) However, several chemical</div><div>compatibility challenges associated with incorporating reactive metal centers into complex</div><div>biological environments have hindered efforts in this area, despite the many opportunities it may</div><div>present. More challenging than chemical compatibility is biocommunicative transition metal</div><div>catalysis, where the reactivity of the metal species is regulated by native biological stimuli, akin</div><div>to natural biocatalytic processes. Here we report a novel Au(I)-DNAzyme that is activated by short</div><div>nucleic acids in a highly sequence-specific manner and that is compatible with complex biological</div><div>matrices. The active Au(I)-DNAzyme catalyzes the formation of a fluorescent molecule with >10</div><div>turnovers. This functional allostery, resulting in chemocatalytic signal amplification, is competent</div><div>in complex biological settings, including within recombinant E. coli cells, where the catalytic</div><div>activity of the Au(I)-DNAzyme is regulated by transcription of an inducible plasmid. We further</div><div>demonstrate the potential of this transition metal oligonucleotide complex as a highly sensitive and</div><div>selective hybridization probe, permitting the detection of attomolar concentrations (ca. 60</div><div>molecules/µL) of SARS-CoV-2 RNA gene fragments in simulated biological matrices with ≥85%</div><div>accuracy. Notably, this sensitive detection platform avoids expensive and poorly-scalable</div><div>biochemical components (e.g. post-synthetically modified oligonucleotides or enzymes) and</div><div>utilizes small molecule fluorophores, inexpensive Au salts and oligonucleotides composed of</div><div>canonical bases. This discovery highlights promising opportunities to perform abiotic catalysis in</div><div>complex biological settings under transcriptional regulation, as well as a chemocatalytic strategy</div><div>for PCR-free, direct-detection of RNA and DNA.</div><div><br></div><p>The union of transition metal catalysis with native biochemistry presents a powerful opportunity to perform abiotic reactions within complex biological systems. However, several chemical compatibility challenges associated with incorporating reactive metal centers into complex biological environments have hindered efforts in this area, despite the many opportunities it may present. More challenging than chemical compatibility is biocommunicative transition metal catalysis, where the reactivity of the metal species is regulated by native biological stimuli, akin to natural biocatalytic processes. Here we report a novel Au(I)-DNAzyme that is activated by short nucleic acids in a highly sequence-specific manner and that is compatible with complex biological matrices. The active Au(I)-DNAzyme catalyzes the formation of a fluorescent molecule with >10 turnovers. This functional allostery, resulting in chemocatalytic signal amplification, is competent in complex biological settings, including within recombinant <i>E. coli </i>cells, where the catalytic activity of the Au(I)-DNAzyme is regulated by transcription of an inducible plasmid. We further demonstrate the potential of this transition metal oligonucleotide complex as a highly sensitive and selective hybridization probe, permitting the detection of attomolar concentrations (<i>ca.</i> 60 molecules/ L) of SARS-CoV-2 RNA gene fragments in simulated biological matrices with ≥85% accuracy. Notably, this sensitive detection platform avoids expensive and poorly-scalable biochemical components (e.g. post-synthetically modified oligonucleotides or enzymes) and utilizes small molecule fluorophores, inexpensive Au salts and oligonucleotides composed of canonical bases. This discovery highlights promising opportunities to perform abiotic catalysis in complex biological settings under transcriptional regulation, as well as a chemocatalytic strategy for PCR-free, direct-detection of RNA and DNA.</p>

Chemocatalytic Amplification Probes Enable Transcriptionally-Regulated Au(I)-Catalysis in E. coli and Sensitive Detection of SARS-CoV-2 RNA Fragments

<div>The union of transition metal catalysis with native biochemistry presents a powerful opportunity</div><div>to perform abiotic reactions within complex biological systems.(1,2) However, several chemical</div><div>compatibility challenges associated with incorporating reactive metal centers into complex</div><div>biological environments have hindered efforts in this area, despite the many opportunities it may</div><div>present. More challenging than chemical compatibility is biocommunicative transition metal</div><div>catalysis, where the reactivity of the metal species is regulated by native biological stimuli, akin</div><div>to natural biocatalytic processes. Here we report a novel Au(I)-DNAzyme that is activated by short</div><div>nucleic acids in a highly sequence-specific manner and that is compatible with complex biological</div><div>matrices. The active Au(I)-DNAzyme catalyzes the formation of a fluorescent molecule with >10</div><div>turnovers. This functional allostery, resulting in chemocatalytic signal amplification, is competent</div><div>in complex biological settings, including within recombinant E. coli cells, where the catalytic</div><div>activity of the Au(I)-DNAzyme is regulated by transcription of an inducible plasmid. We further</div><div>demonstrate the potential of this transition metal oligonucleotide complex as a highly sensitive and</div><div>selective hybridization probe, permitting the detection of attomolar concentrations (ca. 60</div><div>molecules/µL) of SARS-CoV-2 RNA gene fragments in simulated biological matrices with ≥85%</div><div>accuracy. Notably, this sensitive detection platform avoids expensive and poorly-scalable</div><div>biochemical components (e.g. post-synthetically modified oligonucleotides or enzymes) and</div><div>utilizes small molecule fluorophores, inexpensive Au salts and oligonucleotides composed of</div><div>canonical bases. This discovery highlights promising opportunities to perform abiotic catalysis in</div><div>complex biological settings under transcriptional regulation, as well as a chemocatalytic strategy</div><div>for PCR-free, direct-detection of RNA and DNA.</div><div><br></div><p>The union of transition metal catalysis with native biochemistry presents a powerful opportunity to perform abiotic reactions within complex biological systems. However, several chemical compatibility challenges associated with incorporating reactive metal centers into complex biological environments have hindered efforts in this area, despite the many opportunities it may present. More challenging than chemical compatibility is biocommunicative transition metal catalysis, where the reactivity of the metal species is regulated by native biological stimuli, akin to natural biocatalytic processes. Here we report a novel Au(I)-DNAzyme that is activated by short nucleic acids in a highly sequence-specific manner and that is compatible with complex biological matrices. The active Au(I)-DNAzyme catalyzes the formation of a fluorescent molecule with >10 turnovers. This functional allostery, resulting in chemocatalytic signal amplification, is competent in complex biological settings, including within recombinant <i>E. coli </i>cells, where the catalytic activity of the Au(I)-DNAzyme is regulated by transcription of an inducible plasmid. We further demonstrate the potential of this transition metal oligonucleotide complex as a highly sensitive and selective hybridization probe, permitting the detection of attomolar concentrations (<i>ca.</i> 60 molecules/ L) of SARS-CoV-2 RNA gene fragments in simulated biological matrices with ≥85% accuracy. Notably, this sensitive detection platform avoids expensive and poorly-scalable biochemical components (e.g. post-synthetically modified oligonucleotides or enzymes) and utilizes small molecule fluorophores, inexpensive Au salts and oligonucleotides composed of canonical bases. This discovery highlights promising opportunities to perform abiotic catalysis in complex biological settings under transcriptional regulation, as well as a chemocatalytic strategy for PCR-free, direct-detection of RNA and DNA.</p>

Chemocatalytic Amplification Probes Enable Transcriptionally-Regulated Au(I)-Catalysis in E. coli and Sensitive Detection of SARS-CoV-2 RNA Fragments

<div>The union of transition metal catalysis with native biochemistry presents a powerful opportunity</div><div>to perform abiotic reactions within complex biological systems.(1,2) However, several chemical</div><div>compatibility challenges associated with incorporating reactive metal centers into complex</div><div>biological environments have hindered efforts in this area, despite the many opportunities it may</div><div>present. More challenging than chemical compatibility is biocommunicative transition metal</div><div>catalysis, where the reactivity of the metal species is regulated by native biological stimuli, akin</div><div>to natural biocatalytic processes. Here we report a novel Au(I)-DNAzyme that is activated by short</div><div>nucleic acids in a highly sequence-specific manner and that is compatible with complex biological</div><div>matrices. The active Au(I)-DNAzyme catalyzes the formation of a fluorescent molecule with >10</div><div>turnovers. This functional allostery, resulting in chemocatalytic signal amplification, is competent</div><div>in complex biological settings, including within recombinant E. coli cells, where the catalytic</div><div>activity of the Au(I)-DNAzyme is regulated by transcription of an inducible plasmid. We further</div><div>demonstrate the potential of this transition metal oligonucleotide complex as a highly sensitive and</div><div>selective hybridization probe, permitting the detection of attomolar concentrations (ca. 60</div><div>molecules/µL) of SARS-CoV-2 RNA gene fragments in simulated biological matrices with ≥85%</div><div>accuracy. Notably, this sensitive detection platform avoids expensive and poorly-scalable</div><div>biochemical components (e.g. post-synthetically modified oligonucleotides or enzymes) and</div><div>utilizes small molecule fluorophores, inexpensive Au salts and oligonucleotides composed of</div><div>canonical bases. This discovery highlights promising opportunities to perform abiotic catalysis in</div><div>complex biological settings under transcriptional regulation, as well as a chemocatalytic strategy</div><div>for PCR-free, direct-detection of RNA and DNA.</div><div><br></div>

Efficient Sequencing, Assembly, and Annotation of Human KIR Haplotypes

The homology, recombination, variation, and repetitive elements in the natural killer-cell immunoglobulin-like receptor (KIR) region has made full haplotype DNA interpretation impossible without physical separation of chromosomes. Here, we present a new approach using long-read sequencing to efficiently capture, sequence, and assemble diploid human KIR haplotypes. Sequences for capture probe design were derived from public full-length gene and haplotype sequences. IDT xGen® Lockdown probes were used to capture 2-8 kb of sheared DNA fragments followed by sequencing on a PacBio Sequel. The sequences were error corrected, binned, and then assembled using the Canu assembler. The assembly was evaluated on 16 individuals (8 African American and 8 Europeans) from whom ground truth was known via long-range sequencing on fosmid-isolated chromosomes. Using only 18 capture probes, the results show that the assemblies cover 97% of the GenBank reference, are 99.97% concordant, and it takes only 1.8 contigs to cover 75% of the reference. We also report the first assembly of diploid KIR haplotypes from long-read WGS, including the first sequencing of cB05∼tB01, which pairs a KIR2DS2/KIR2DS3 fusion with the tB01 region. Our targeted hybridization probe capture and sequencing approach is the first of its kind to fully sequence and phase all diploid human KIR haplotypes, and it is efficient enough for population-scale studies and clinical use.