Search

Seneca Valley virus 1 (SVV-1) has been associated with vesicular disease in swine, with clinical signs indistinguishable from those of other notifiable vesicular diseases such as foot-and-mouth disease. Rapid and accurate detection of SVV-1 is central to confirm the disease causing agent, and to initiate the implementation of control processes. The development of rapid, cost-effective diagnostic assays that can be used at the point of sample collection has been identified as a gap in preparedness for the control of SVV-1. This study describes the development and bench validation of two reverse transcription loop-mediated amplification (RT-LAMP) assays targeting the 5'-untranslated region (5'-UTR) and the VP3-1 region for the detection of SVV-1 that may be performed at the point of sample collection. Both assays were able to demonstrate amplification of all neat samples diluted 1/100 in negative pig epithelium tissue suspension within 8 min, when RNA was extracted prior to the RT-LAMP assay, and no amplification was observed for the other viruses tested. Simple sample preparation methods using lyophilized reagents were investigated, to negate the requirement for RNA extraction. Only a small delay in the time to amplification was observed for these lyophilized reagents, with a time from sample receipt to amplification achieved within 12 min. Although diagnostic validation is recommended, these RT-LAMP assays are highly sensitive and specific, with the potential to be a useful tool in the rapid diagnosis of SVV-1 in the field., (© 2018 Blackwell Verlag GmbH.)

We developed a new technique suitable for improved detection of low-copy dsRNA using modified oligonucleotides as primers in RT-qPCR. Insertion of G 8AE -clamp residues into primers significantly improves thermal stability of duplexes with RNA without decrease of hybridization selectivity. The applicability of modified primers is demonstrated for detection of low-copy Kemerovo virus dsRNA.

Aberrant DNA methylation of cell-free circulating DNA (cfDNA) has recently gained attention for its use as biomarker in cancer diagnosis, prognosis, and prediction of therapeutic response. Quantification of cfDNA methylation levels requires methods with high sensitivity and specificity due to low amounts of cfDNA available in plasma, high degradation of cfDNA, and/or contamination with genomic DNA. To date, several approaches for measuring cfDNA methylation have been established, including quantitative methylation-specific PCR (qMSP), which represents a simple, fast, and cost-effective technique that can be easily implemented into clinical practice. In this chapter, we provide a detailed protocol for SYBR Green qMSP analysis which is currently used in our laboratory for cfDNA methylation detection. Useful information regarding successful qMSP primers design are also provided.

Polymerase enzymes catalyze the replication of DNA by incorporating deoxynucleoside monophosphates (dNMPs) into a primer strand in a 5' to 3' direction. Monitoring kinetic aspects of this catalytic process provides mechanistic information regarding polymerase-mediated DNA synthesis and the influences of nucleobase structure. For example, a range of polymerases have different capacities to synthesize DNA depending on the structure of the inserted dNMP (natural or synthetic) and also depending on the templating DNA base (modified vs. unmodified). Under steady-state conditions, relative rates depend on the deoxynucleoside triphosphate (dNTP) residence times in the ternary (polymerase-DNA-dNTP) complex. This chapter describes a method to measure steady-state incorporation efficiencies by which polymerase enzymes insert dNMPs into primer-template (P/T) oligonucleotides. The method described involves the use of a primer oligonucleotide 5' radiolabeled with [γ- 32 P]ATP. Significant established applications of this experiment include studies regarding mechanisms of nucleotide misincorporation as a basis of chemically induced DNA mutation. Further, it can provide information important in various contexts ranging from biophysical to medical-based studies.

Cetacean morbillivirus (CeMV) has caused repeated epizootics and interepizootic fatalities in a variety of cetacean species worldwide. Recently, a novel CeMV strain (GD-CeMV) was linked to a mass die-off of Guiana dolphins (Sotalia guianensis) in Brazil. Southern right whales (SRWs; Eubalaena australis) migrate to the southern Brazilian coast during austral winter and spring (June through November) for breeding and calving. Because unexplained high calf mortality rates have recurrently been documented in SRWs, we hypothesized they could be infected with CeMV. We developed a novel real-time RT-PCR method based on SYBR ® GREEN for detection of CeMV and identified the virus in three out of five stranded SRWs from Santa Catarina state, Brazil. The partial sequences of the morbillivirus phosphoprotein gene suggest that the virus is similar to the GD-CeMV strain. Our results indicate CeMV can infect SRWs and should be considered in the differential aetiologic diagnosis of infectious diseases in this species. It also raises concern for potential conservation implications for this species in its main coastal breeding area off Southern Brazil., (© 2018 Blackwell Verlag GmbH.)

Loop-mediated isothermal amplification is a promising method in the area of nucleic acid detection. However, it suffers from a high rate of false-positive amplifications that largely restrict its application. In this study, we observed gold nanoparticles (AuNP) absorbing single-stranded DNA primers and interacting with Bst DNA polymerase via electrostatic adsorption. As a result of these interactions, the presence of the gold nanoparticles exerted a hot-start effect on the loop-mediated isothermal amplification system. Based on these results, we developed a novel AuNP-mediated nucleic acid isothermal amplification assay. This assay displays significantly enhanced specificity-the proportion of false positive decreased from 76% to 0% and from 100% to 0% for the detection of rotavirus and the β-actin gene, respectively, with the hot-start temperature of 48 °C. Moreover, our AuNP-based assay maintained good sensitivity and a satisfactory detection limit (1 × 10 3 copies/μL) compared with the conventional assay. This approach has the potential to solve the nonspecificity problem of loop-mediated isothermal amplification, thereby promoting its real-world application, particularly, in clinical settings., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Advanced methods for developing and applying responsive DNA nanodevices are of great interest. Herein, we report a stretchable DNAzyme that allows simple, multiplexed and sensitive fluorescent detection of antibodies. We find that rigid antibody can tightly stretch soft, antigen-labelled DNAzyme strand and disrupt the hybridization between DNAzyme and its substrate. Based on this finding, we develop a novel strategy to detect antibodies. Due to the robustness and high activity of DNAzyme, this assay can easily detect target as low as 1 ± 0.25 pM and achieve multiplexed detection by using a cocktail of DNAzymes. The proposed assay not only provides a new approach to readily measure antibody, but broadens the application of DNAzyme that is usually employed to detect metal ions or indirectly analyze biomolecules without the cumbersome design., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Huanglongbing (HLB) or citrus greening is highly destructive disease that is affecting the citrus industry worldwide and it has killed millions of citrus plants globally. HLB is caused by the phloem limited, Gram negative, non-culturable, alpha-proteobacterium, 'Candidatus Liberibacter asiaticus'. Currently, polymerase chain reaction (PCR) and real time PCR have been the gold standard techniques used for detection of 'Ca. L. asiaticus'. These diagnostic methods are expensive, require well equipped laboratories, not user-friendly and not suitable for on-site detection of the pathogen. In this study, a sensitive, reliable, quick and low cost recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) technique has been developed as a diagnostic tool for detection of 'Ca. L. asiaticus'. The assay was standardized by designing the specific primer pair and probe based on the conserved 16S rRNA gene of 'Ca. L. asiaticus'. The assay was optimized for temperature and reaction time by using purified DNA and crude plant extracts and the best HLB-RPA-LFA was achieved at the isothermal temperature of 38°C for 20 to 30 min. The efficacy and sensitivity of the assay was carried out by using field grown, HLB-infected, HLB-doubtful and healthy citrus cultivars including mandarin, sweet orange cv. mosambi, and acid lime. The HLB-RPA-LFA did not show cross-reactivity with other citrus pathogens and is simple, cost-effective, rapid, user-friendly and sensitive. Thus, the HLB-RPA-LFA method has great potential to provide an improved diagnostic tool for detection of 'Ca. L. asiaticus' for the farmers, nurserymen, disease surveyors, mobile plant pathology laboratories, bud-wood certification and quarantine programs., Competing Interests: The authors have declared that no competing interests exist.

Killer cell immunoglobulin-like receptor (KIR) genes encode cell surface molecules that recognize HLA molecules and modulate the activity of natural killer (NK) cells. KIR genes exhibit presence and absence polymorphism, which generates a variety of gene-content haplotypes in worldwide populations. KIR gene-content variation is implicated in many diseases and is also important for placentation and transplantation. Because of the complexity of KIR polymorphism, variation in this family is still mostly studied at the gene-content level, even with the advent of next-generation sequencing (NGS) methods. Gene-content determination is generally expensive and/or time-consuming. To overcome these difficulties, we developed a method based on multiplex polymerase chain reaction with specific sequence primers (PCR-SSP) followed by melting curve analysis that allows cost-effective, precise and fast generation of results. Our method was 100% concordant with a gel-based method and 99.9% concordant with presence and absence determination by NGS. The limit of detection for accurate typing was 30 ng of DNA (0.42 μM) with 260/230 and 260/280 ratios as low as 0.19 and of 0.44. In addition, we developed a user-friendly Java-based computational application called killerPeak that interprets the raw data generated by Viia7 or QuantStudio 7 quantitative PCR machines and reliably exports the final genotyping results in spreadsheet file format. The combination of a reliable method that requires low amount of DNA with an automated interpretation of results allows scaling the KIR genotyping in large cohorts with reduced turnaround time., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

DNA-damage tolerance protects cells via at least two sub-pathways regulated by proliferating cell nuclear antigen (PCNA) ubiquitination in eukaryotes: translesion DNA synthesis (TLS) and template switching (TS), which are stimulated by mono- and polyubiquitination, respectively. However, how cells choose between the two pathways remains unclear. The regulation of ubiquitin ligases catalyzing polyubiquitination, such as helicase-like transcription factor (HLTF), could play a role in the choice of pathway. Here, we demonstrate that the ligase activity of HLTF is stimulated by double-stranded DNA via HIRAN domain-dependent recruitment to stalled primer ends. Replication factor C (RFC) and PCNA located at primer ends, however, suppress en bloc polyubiquitination in the complex, redirecting toward sequential chain elongation. When PCNA in the complex is monoubiquitinated by RAD6-RAD18, the resulting ubiquitin moiety is immediately polyubiquitinated by coexisting HLTF, indicating a coupling reaction between mono- and polyubiquitination. By contrast, when PCNA was monoubiquitinated in the absence of HLTF, it was not polyubiquitinated by subsequently recruited HLTF unless all three-subunits of PCNA were monoubiquitinated, indicating that the uncoupling reaction specifically occurs on three-subunit-monoubiquitinated PCNA. We discuss the physiological relevance of the different modes of the polyubiquitination to the choice of cells between TLS and TS under different conditions.

Exosomes as nanosized biomarkers hold great potential for the diagnosis of cancer. However, the low concentration of cancer-derived exosomes present in biofluids makes early diagnosis strenuous. Here, we developed a fluorescent biosensing platform, namely a dual signal amplification, for the ultrasensitive detection of leukemia cell-derived exosomes. The protocol consists of three steps: first, leukemia-derived exosomes containing CD63 and nucleolin were captured by anti-CD63 antibody modified magnetic bead conjugates (MB-CD63); then, a DNA primer comprising a nucleolin-recognition aptamer (AS1411) was applied to bind the exosomes which further initiated a rolling circle amplification (RCA) reaction to generate many repeat sequences for hybridization with gold nanoparticle (GNP)-DNA-fluorescent dye (FAM) conjugates (GNP-DNA-FAM); finally, nicking endonuclease (Nb·BbvCI) assisted target recycling was introduced. As a result, FAM was released from GNP-DNA-FAM conjugates, transformed from the quenching state to the emission state and thus fluorescence signals continuously accumulated. With this dual signal amplification platform, as low as 1 × 102 particles per μL exosomes could be detected. Furthermore, we have successfully applied this method for the detection of exosomes in spiked serum samples, indicating a promising tool for clinical application.

Vibrio parahaemolyticus is a common and important pathogen that causes human gastroenteritis worldwide. A rapid, sensitive, and specific assay is urgently required for detection and differentiation of V. parahaemolyticus strains. We designed three sets of primers and probes using groEL and two virulence genes (tdh and trh) from V. parahaemolyticus, and developed a multiplex real-time PCR protocol. The sensitivity and specificity of the multiplex assay was evaluated by environmental and clinical specimens of V. parahaemolyticus. The multiplex PCR response system and annealing temperature were optimized. The detection limits of the multiplex real-time PCR were 10 4 and 10 5 CFU/mL (or CFU/g) in pure cultures and spiked oysters, respectively. The multiplex real-time PCR specifically detected and differentiated V. parahaemolyticus from 35 Vibrio strains and 11 other bacterial strains. Moreover, this method can detect and distinguish virulent from nonvirulent strains, with no cross-reactivity observed in the bacteria tested. This newly established multiplex real-time PCR assay offers rapid, specific, and reliable detection of the total and pathogenic V. parahaemolyticus strains, which is very useful during outbreaks and sporadic cases caused by V. parahaemolyticus infection.

Here we investigated the gene of a transforming growth factor (TGF)-β type I receptor-like molecule in Haemonchus contortus, a highly pathogenic and economically important parasitic nematode of small ruminants. Designated Hc-tgfbr1, this gene is transcribed in all developmental stages of H. contortus, and the encoded protein has glycine-serine rich and kinase domains characteristic of a TGF-β family type I receptor. Expression of a GFP reporter driven by the putative Hc-tgfbr1 promoter localised to two intestinal rings, the anterior-most intestinal ring (int ring I) and the posterior-most intestinal ring (int ring IX) in Caenorhabditis elegans in vivo. Heterologous genetic complementation using a plasmid construct containing Hc-tgfbr1 genomic DNA failed to rescue the function of Ce-daf-1 (a known TGF-β type I receptor gene) in a daf-1-deficient mutant strain of C. elegans. In addition, a TGF-β type I receptor inhibitor, galunisertib, and double-stranded RNA interference (RNAi) were employed to assess the function of Hc-tgfbr1 in the transition from exsheathed L3 (xL3) to the L4 of H. contortus in vitro, revealing that both galunisertib and Hc-tgfbr1-specific double-stranded RNA could retard L4 development. Taken together, these results provide evidence that Hc-tgfbr1 is involved in developmental processes in H. contortus in the transition from the free-living to the parasitic stage., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and utilizes specific DNA primers covalently linked to the antibodies. A hybridization step followed by DNA amplification with fluorescent probes permit visualization of spots of proximity by fluorescence microscopy. Since the development of PLA in 2002, it has been increasingly used to detect the interaction between two proteins with high sensitivity and specificity. It is a simple and sensitive technique to study protein-protein interaction in cells. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

Rapid and specific detection of single nucleotide polymorphisms (SNPs) related to drug resistance in infectious diseases is crucial for accurate prognostics, therapeutics and disease management at point-of-care. Here, we present a novel amplification method and provide universal guidelines for the detection of SNPs at isothermal conditions. This method, called USS-sbLAMP, consists of SNP-based loop-mediated isothermal amplification (sbLAMP) primers and unmodified self-stabilizing (USS) competitive primers that robustly delay or prevent unspecific amplification. Both sets of primers are incorporated into the same reaction mixture, but always targeting different alleles; one set specific to the wild type allele and the other to the mutant allele. The mechanism of action relies on thermodynamically favored hybridization of totally complementary primers, enabling allele-specific amplification. We successfully validate our method by detecting SNPs, C580Y and Y493H, in the Plasmodium falciparum kelch 13 gene that are responsible for resistance to artemisinin-based combination therapies currently used globally in the treatment of malaria. USS-sbLAMP primers can efficiently discriminate between SNPs with high sensitivity (limit of detection of 5 × 10 1 copies per reaction), efficiency, specificity and rapidness (<35 min) with the capability of quantitative measurements for point-of-care diagnosis, treatment guidance, and epidemiological reporting of drug-resistance.

Polyoxymetalates (POMs) ([SiW 11 O 39 {Sn(CH 2 ) 2 CO)}] 4- and [P 2 W 17 O 61 {Sn(CH 2 ) 2 CO)}] 6- ) were used to modify dideoxynucleotides (ddNTPs) through amide bond formation, and applied to the multiplexed detection of single nucleotide polymorphisms (SNPs) in an electrochemical primer extension reaction. Each gold electrode of an array was functionalised with a short single stranded thiolated DNA probe, specifically designed to extend with the POM-ddNTP at the SNP site to be interrogated. The system was applied to the simultaneous detection of 4 SNPs within a single stranded 103-mer model target generated using asymmetric PCR, highlighting the potential of POM-ddNTPs for targeted, multiplexed SNP detection. The four DNA bases were successfully labelled with both ([SiW 11 O 39 {Sn(CH 2 ) 2 CO)}] 4- and [P 2 W 17 O 61 {Sn(CH 2 ) 2 CO)}] 6- ), and [SiW 11 O 39 {Sn(CH 2 ) 2 CO)}] 4- demonstrated to be the more suitable due to its single oxidation peak, which provides an unequivocal signal. The POM-ddNTP enzymatically incorporated to the DNA anchored to the surface was visualised by AFM using gold coated mica. The developed assay has been demonstrated to be highly reproducible, simple to carry out and with very low non-specific background signals. Future work will focus on applying the developed platform to the detection of SNPs associated with rifampicin resistance in real samples from patients suffering from tuberculosis., (Copyright © 2018. Published by Elsevier B.V.)

Alkaline phosphatase (ALP) is an important diagnostic indicator for various human diseases including bone diseases, liver dysfunction, diabetes, breast and prostatic cancers. However, the conventional methods for ALP assay are usually cumbersome and time-consuming with low sensitivity. Here, we develop a new fluorescent method for ultrasensitive detection of ALP activity on the basis of primer dephosphorylation-initiated isothermal circular exponential amplification. We design two dual-functional hairpin probes (HP1 and HP2), which function as both the templates for exponential amplification reaction (EXPAR) and the generators for signal output. In the presence of ALP, the 3'-phosphorylated primer is dephosphorylated and subsequently hybridizes with the 3' protruding end of HP1 to initiate the first strand displacement amplification (SDA), producing trigger 1 and fluorescence signal. The released trigger 1 is complementary to the 3' protruding end of HP2 for the initiation of the second SDA, producing trigger 2 and fluorescence signal. Notably, trigger 2 is complementary to the 3' protruding end of HP1 and may subsequently initiate two consecutive SDAs, enabling circular EXPAR to generate an amplified fluorescence signal. This method exhibits high sensitivity with a detection limit of 2.0 × 10 -10 U μL -1 and a large dynamic range of 5 orders of magnitude from 1.0 × 10 -9 to 1.0 × 10 -4 U μL -1 , and it can measure ALP at the single-cell level. Importantly, this method can be applied for the measurement of kinetic parameters and the screening of potential inhibitors, providing a powerful tool for ALP-related biomedical research and clinical diagnosis.

Various additives can enhance the quality of PCR amplification, but these generally require considerable optimization to achieve peak performance. Here, we demonstrate that the use of thiol-modified primers can enhance both PCR sensitivity and yield. In experiments with V. parahaemolyticus genomic DNA, this primer modification enhances PCR sensitivity by more than 100-fold, with accompanying improvements in amplicon yield. Then, an artificial plasmid with the same primer binding regions and different internal amplification sequence was designed. The result showed that the amplification also be improved by using the same thiol-modified primers. It indicated the enhancement was not caused by the effect of the thiol-modified primers on the second structure of amplification sequence. Subsequent experiments demonstrate that the effects of this modification are potentially due to altered interaction between the primers and proteins in the reaction mixture. Amplification with thiol-modified primers was strongly inhibited by the presence of extraneous proteins relative to standard DNA primers, which indicates that thiol-modified primers may be inhibited due to interaction with these proteins. In contaminant-free reactions, however, the thiol-modified primers might interact more strongly with DNA polymerase, which could in turn improve PCR amplification.

Bartonella rochalimae is an emerging zoonotic pathogen present in the United States, South America, and Europe. The molecular detection of B. rochalimae frequently relies on polymerase chain reaction (PCR) assays that target the genus Bartonella coupled with DNA sequencing for species determination. However, the presence of other Bartonella spp. in the sample being tested may result in false-negative results for B. rochalimae , especially when Sanger sequencing is used. We developed a sensitive and specific quantitative PCR platform for B. rochalimae by targeting the intergenic transcribed spacer, gltA , and rpoB genes, which are recommended for subtyping characterization. This PCR platform achieved the limit of detection between five and 10 genomic equivalents per reaction and did not amplify DNA from other Bartonella species or selected hosts. This PCR platform is a fast and cost-effective option to be used in epidemiological evaluations of reservoirs and vectors and in detecting and quantifying B. rochalimae infection in humans.

A deoxyadenosine triphosphate (dATP) analogue for DNA labeling was synthesized with the 1-methylcyclopropene (1MCP) group at the 7-position of 7-deaza-2'-deoxyadenosine and applied for primer extension experiments. The real-time kinetic data reveals that this 1MCP-modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP-modified deoxyuridine triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site-specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although nucleic acid amplification test (NAT) is widely used for pathogen detection, rapid NAT systems that do not require special and expensive instruments must be developed in order to enable point of care (POC)-NATs, which contribute to early initiation of treatment. As a POC-NAT system, Kaneka DNA chromatography chip (KDCC), developed using DNA tag-bound primer through modified substance, was shown to be suitable for POC testing, due to the rapid detection time, simple procedures, and low manufacturing costs. However, owing to some modifications in primer, the detection performance and amplification speed were shown to be reduced when using KDCC, counteracting the increased speed of detection. To solve these issues and improve the speed of this NAT system, we investigated a better modification substance for KDCC. Here, azobenzene-modified primers were shown to have the highest amplification speed and detection performance in KDCC, of all modifications tested in this study, showing 10-100-fold lower detection limit but maintaining the same reaction time. Additionally, rapid herpes simplex virus detection system with azobenzene modified primers was developed. We believed that this breakthrough will contribute toward enabling greater utilization of POC-NATs for medical care, especially in developing countries and clinics., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Purpose: To develop a fast and inexpensive genotyping assay to identify the Mycobacterium tuberculosis complex (MTC) species most prevalent in human tuberculosis (TB), based on the thermal denaturation profiles of PCR products from mycobacterial 16S rDNA and three MTC genomic regions of difference (RD)., Methodology: Genotypes were determined by the presence and thermal denaturation profiles of the amplicons generated in the 'preliminary' PCR mixture (16S rDNA), followed by those of the simultaneous D1 (RD9+, RD1-) and D2 (RD4+, RD4-) PCR mixtures. The 16S rDNA profile identifies the genus Mycobacterium; the absence of any additional RD profile identifies Mycobacterium non-tuberculous (MNT) strains; additional RD4+ and RD9+ profiles without RD1- identify M. tuberculosis; an additional RD4+ profile per se identifies M. africanum; an additional RD4- profile per se identifies Mycobaterium bovis; additional RD1- and RD4- profiles identify M. bovis BCG., Results: Genotypes of a panel with 44 mycobacterial strains coincided in 16 MB and five non-MTC strains; in the remaining 23 MTC strains, 17 MTB and five MA concordant genotypes and one discordant MB genotype were resolved. The genotypes of 13 human and bovine MTC isolates coincided in all four MB and eight of the nine MTB isolates., Conclusion: Sensitivity, specificity and positive and negative predictive values of the method are 100 % for the genus Mycobacterium, which resolves MB, MTB and MA genotypes. Species/genotype agreement is 97.7 % for the panel and 92.3 % for the MTC isolates. This method may be advantageously used to identify the most prevalent MTC species in humans.

Microcystins, the lethal cyanotoxins from Microcystis aeruginosa, can inhibit the activity of protein phosphatase and promote liver tumors. Herein, a dual-modal split-type immunosensor was constructed to detect microcystin-LR (MC-LR), based on the photocurrent change of CdS/ZnO hollow nanorod arrays (HNRs) and the blue shift of the surface plasmon resonance peak from Au nanobipyramids@Ag. By using mesoporous silica nanospheres as the carrier to immobilize secondary antibody and DNA primer, a hybridization chain reaction was adopted to capture alkaline phosphatase, while its catalytic reaction product, ascorbic acid, exhibited dual functions. The detailed mechanism was investigated, showing that ascorbic acid can not only act as the electron donor to capture the holes in CdS/ZnO-HNRs, leading to the increase photocurrent, but also as the reductant to form silver shells on Au nanobipyramids, generating multiply vivid color variations and blue shifts. Compared with the traditional photoelectrochemical immunosensor or colorimetric method for MC-LR, a more accurate and reliable result can be obtained, due to different mechanisms and independent signal transduction. Therefore, this work can not only propose a new dual-modal immunosensor for MC-LR detection but also provide innovative inspiration for constructing sensitive, accurate, and visual analysis for toxins.

The paper describes a voltammetric method for the quantitation of the activity of telomerase extracted from cancer cells. A thiolated single-stranded telomerase substrate primer was firstly immobilized on a gold electrode. In the presence of a mixture of telomerase and deoxynucleotide triphosphates, the primer becomes elongated and contains repetitive nucleotide sequences (TTAGGG) n . After hybridization with blocker DNA, gold nanoparticles are added and captured by the elongated single-stranded DNA. This reduces the charge transfer resistance of the gold electrode. The telomerase activity is then quantified via differential pulse voltammetry, typically at 0.12 V (vs. SCE). The method is PCR-free, rapid, and convenient. It was applied to the detection of HeLa cells via the telomerase activity of lysed cells. The detection range was from 500 to 50,000 cells/mL and the detection limit was as low as 500 cells/mL. Graphical abstract A telomerase substrate (TS) primer is immobilized on a gold electrode as the sensing interface to detect the activity of telomerase extracted from cancer cells. Unmodified gold nanoparticles (AuNPs) are utilized which change the electrochemical responses.

Background: RB1 gene screening aids clinical management and genetic counselling in retinoblastoma families. Here we present epigenetic changes identified during routine molecular RB1 screening of tumor and blood samples. Complications in interpreting RB1 methylation are discussed., Materials and Methods: Screening for RB1 promoter hypermethylation was carried out by Methylation Specific PCR (MS-PCR) after bisulphite modification of DNA. The cohort consisted of 315 tumors, and 204 blood samples, from 497 retinoblastoma patients (22 patients had both blood and tumor screened)., Results: 11.4% of retinoblastoma tumors had promoter hypermethylation. It was not routinely detected in blood samples, or in tumors with two other oncogenic RB1 changes. One blood sample had promoter hypermethylation due to an X;13 translocation. One tumor had low level methylation as well as two other oncogenic changes. Histopathological analysis of a small subset of age-matched tumors was similar regardless of promoter hypermethylation status., Conclusions: Promoter hypermethylation was detected in 11.4% of the retinoblastoma tumors and should be tested for in routine RB1 screening programmes. Constitutional samples are not expected to display RB1 hypermethylation. In a small proportion of cases it may not be possible to use this somatic change in patient management.

The ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone was first observed in Japan in 2015, and in 2017, it was documented in Denmark, Canada, and Australia. Here, we describe a PCR for direct detection of the penA gene associated with this strain that can be used to enhance surveillance activities.

DNA Pol θ-mediated end joining (TMEJ) is a microhomology-based pathway for repairing double-strand breaks in eukaryotes. TMEJ is also a pathway for nonspecific integration of foreign DNAs into host genomes. DNA Pol θ shares structural homology with the high-fidelity replicases, and its polymerase domain (Polθ) has been shown to extend ssDNA without an apparent template. Using oligonucleotides with distinct sequences, we find that with Mg 2+ and physiological salt concentrations, human Polθ has no terminal transferase activity and requires a minimum of 2 bp and optimally 4 bp between a template/primer pair for DNA synthesis. Polθ can tolerate a mismatched base pair at the primer end but loses >90% activity when the mismatch is 2 bp upstream from the active site. Polθ is severely inhibited when the template strand has a 3' overhang within 3-4 bp from the active site. In line with its TMEJ function, Polθ has limited strand-displacement activity, and the efficiency and extent of primer extension are similar with or without a downstream duplex., Competing Interests: The authors declare no conflict of interest.

All known forms of life encode their genetic information in a sequence of bases of a genetic polymer and produce copies through replication. How this process started before polymerase enzymes had evolved is unclear. Enzyme-free copying of short stretches of DNA or RNA has been demonstrated using activated nucleotides, but not replication. We have developed a method for enzyme-free replication. It involves extension with reversible termination, enzyme-free ligation, and strand capture. We monitored nucleotide incorporation for a full helical turn of DNA, during both a first and a second round of copying, by using mass spectrometry. With all four bases (A/C/G/T), an "error catastrophe" occurred, with the correct sequence being "overwhelmed" by incorrect ones. When only C and G were used, approximately half of the daughter strands had the mass of the correct sequence after 20 copying steps. We conclude that enzyme-free replication is more likely to be successful with just the two strongly pairing bases than with all four bases of the genetic alphabet., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The success of widely used oligonucleotide-based experiments, ranging from PCR to microarray, strongly depends on an accurate design. The design process involves a number of steps, which use specific parameters to produce high quality oligonucleotides. Oli2go is an efficient, user friendly, fully automated multiplex oligonucleotide design tool, which performs primer and different hybridization probe designs as well as specificity and cross dimer checks in a single run. The main improvement to existing oligonucleotide design web-tools is that oli2go combines multiple steps in an all-in-one solution, where other web applications only accomplish parts of the whole design workflow. Especially, the oli2go specificity check is not only performed against a single species (e.g. mouse), but against bacteria, viruses, fungi, invertebrates, plants, protozoa, archaea and sequences from whole genome shotgun sequence projects and environmental samples, at once. This allows the design of highly specific oligonucleotides in multiplex applications, which is further assured by performing dimer checks not only on the primers themselves, but in an all-against-all fashion. The software is freely accessible to all users at http://oli2go.ait.ac.at/.

Research on anammox (anaerobic ammonium oxidizing) bacteria is important due to their biogeochemical and industrial application significance since the first discovery made over two decades ago. By coupling NH 4 + and NO 2 - biochemically to form N 2 gas, anammox bacteria contribute significantly to global marine and terrestrial nitrogen balance (responsible for 50, 9~40, and 4~37% of the nitrogen loss for marine, lakes, and paddy soil) and are also useful in energy-conserving nitrogen removal in wastewater treatment. PCR-based detection and quantification of anammox bacteria are an easy, essential, and widely accessible technique used ubiquitously for studying them in many environmental niches. In this article, we make a summary on practical applications of 16S rRNA and functional gene PCR primers, including hydrazine dehydrogenase (Hzo), nitrite reductase (NirS), hydrazine synthase (Hzs), and cytochrome c biogenesis proteins (Ccs) in detection of them. PCR primer performances in both practical applications and tests in silico are also presented for comparison. For detecting general and specific anammox bacterial groups, selection of appropriate PCR primers for different environmental samples and practical application guidance on choice of appropriate primer pairs for different purposes are also offered. This article provides practical information on selection and application of PCR technique in detection of anammox bacteria from the diverse environments to further promote convenient applications of this technique in research and other purposes.

The cellular replicative DNA polymerases cannot initiate DNA synthesis without a priming 3' OH. During DNA replication, this is supplied in the context of a short RNA primer molecule synthesized by DNA primase. The primase of archaea and eukaryotes, despite having varying subunit compositions, share sequence and structural homology. Intriguingly, archaeal primase has been demonstrated to possess the ability to synthesize DNA de novo, a property shared with the eukaryotic PrimPol enzymes. The dual RNA and DNA synthetic capabilities of the archaeal DNA primase have led to the proposal that there may be a sequential hand-off between these synthetic modes of primase. In the current work, we dissect the functional interplay between DNA and RNA synthetic modes of primase. In addition, we determine the key determinants that govern primer length definition by the archaeal primase. Our results indicate a primer measuring system that functions akin to a caliper., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

There has been an urgent need to quickly screen and isolate patients with viral infections from patients with similar symptoms at point-of-care. In this study, we introduce a new microfluidic method for detection of various viruses using rolling circle amplification (RCA) of pathogens on the surface of thousands of microbeads packed in microchannels. When a targeted pathogen meets the corresponding particular template, the DNAs are rapidly amplified into a specific dumbbell shape through the RCA process, forming a DNA hydrogel and blocking the flow path formed between the beads. Due to the significant increase in reaction surface area, the detection time was shortened to less than 15 min and the detection limit of various pathogens has been reached to 0.1 pM. By injecting the stained liquid, the existence of the target pathogens in a sample fluid can be determined with the naked eye. Furthermore, by integrating multi-channel design, simultaneous phenotyping of various infective pathogens (i.e., Ebola, Middle East respiratory syndrome (MERS), and others) in biological specimens can be performed at a point-of-care., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

The H6 subtype avian influenza viruses (AIVs) possess the capacity for zoonotic transmission from avian species to humans. Establishment of a specific, rapid and sensitive method to screen H6 AIVs is necessary. Based on the conserved domain of the matrix and H6 AIV hemagglutinin genes, two TaqMan minor-groove-binder probes and multiplex real-time RT-PCR primers were designed in this study. The multiplex real-time RT-PCR assay developed in this study had high specificity and repeatability and a detection limit of 30 copies per reaction. This rapid diagnostic method will be useful for clinical detection and surveillance of H6 AIVs in China.

Hand, foot and mouth disease (HFMD) is a serious public health problem, and coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) are two of the major causative pathogens, in addition to enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). A simple and rapid reverse transcription recombinase-aided amplification assay (RT-RAA) was developed for the detection of CVA10 and CVA6 in this study. The analytical sensitivity for detection of CVA10 and CVA6 at 95% probability by probit regression analysis was 35 copies per reaction and 38 copies per reaction, respectively, with 100% specificity. Compared with commercial RT-qPCR assays, when testing 455 fecal specimens, the kappa value of the RT-RAA assay for CVA10 and CVA6 was 0.920 (p < 0.001) and 0.952 (p < 0.001), respectively. Moreover, four samples that were positive for CVA10 and five that were positive for CVA6 by RT-RAA but negative by RT-qPCR were further determined to be true positives. These results demonstrate that the proposed RT-RAA assays are very valuable tools for the detection of CVA10 and CVA6 and have potential for use in resource-limited settings.

Background: Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder with an average age at onset of 40 years. It is a polyglutamine (polyQ) disorder that is caused by an increase in the number of CAG repeats in the huntingtin (HTT) gene. Genetic tests that accurately determine the number of CAG repeats are performed for confirmation of diagnosis, predictive testing of persons at genetic risk for inheriting HD, and prenatal testing. The aim of our study was to evaluate efficacy of triplet-primed polymerase chain reaction (TP-PCR) for routine diagnosis of HD in suspected cases from India., Methods: We evaluated a combination of CAG flanking PCR and triplet-primed PCR for estimation of CAG repeats in 503 cases with clinical suspicion of HD., Results: There were 250 cases (49.7%) that showed the presence of expanded alleles, with 241 (47.9%) being fully penetrant alleles and nine (1.8%) in the reduced penetrance category. There were seven juvenile cases with an age of onset of < 20 years, with the longest allele comprising 106 CAG repeats found in an 8-year-old male patient. The results demonstrated an inverse (R = - 0.67) relationship between CAG length and age at clinical onset., Conclusion: Our study on pan-Indian cases is one of the largest studies reported so far in India and focuses on the most accurate and comprehensive molecular diagnostic evaluation of HD.

The newly discovered Candidatus 'Methanoperedens nitroreducens' (M. nitroreducens), mediating nitrate-dependent anaerobic oxidation of methane, is an important microorganism in linking carbon and nitrogen cycles. In order to explore the diversity of M. nitroreducens-like archaea in various environmental niches with advanced high-throughput sequencing, new primers based on alpha subunit of methyl-coenzyme M reductase gene were designed. The PCR results demonstrated that the new primers could effectively detect M. nitroreducens-like archaea from an enrichment culture dominated by M. nitroreducens as well as samples collected from a natural freshwater lake and a full-scale wastewater treatment plant (WWTP). By high-throughput sequencing, more than 30,000 M. nitroreducens-like sequences were obtained. Phylogenetic analysis of these sequences along with published sequences showed that M. nitroreducens-like archaea could be divided into three sub-branches (named as Group A, Group B and Group C in this study). Clear geographical difference was observed, with Group A and Group B dominating samples in Queensland (Australia) and in European ecosystems, respectively. Further quantitative PCR revealed that the M. nitroreducens-like archaea were more abundant in WWTP than the freshwater lake. The study provided a large number of sequences for M. nitroreducens-like archaeal communities, thus expanded our understanding on the ecological diversity of M. nitroreducens-like archaea.

DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA-DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

Background: Single-nucleotide polymorphisms play an important role in the susceptibility of many diseases, evolutionary studies, and genetic mapping. The rs4958843 in IRGM promoter is associated with tuberculosis and Crohn's disease. As this SNP is not present in any of the restriction sites, PCR-RFLP is not possible. Therefore, we have developed artificial-RFLP method to genotype this SNP., Methods: We designed forward primer with mismatches that resulted in the creation of a restriction site for enzyme NheI in the amplicon. Control samples of known genotypes were obtained by sequencing. The amplified product for SNP rs4958843 was digested with NheI restriction enzyme and resolved on an agarose gel to know the genotypes of the samples., Results: Results of sequencing and A-RFLP were concordant. The developed method was applied to genotype this polymorphism in 100 samples from healthy individuals. The allelic frequencies of SNP rs4958843 were C (0.16) and T (0.84), while corresponding genotypic distribution was CC (2), CT (29), and TT (69)., Conclusion: The newly developed method is simple, easy, and cost-effective which could be used to genotype IRGM polymorphism -1161 C/T (rs4958843) in various populations in the replication studies and has its applicability in the clinical settings. The developed method was applied for genotyping samples from healthy individuals from North India. For the first time, we report the frequency of this polymorphism from this region., (© 2017 Wiley Periodicals, Inc.)

Background: Glyphosate resistance in Amaranthus palmeri, one of the most prevalent herbicide-resistant weeds in the USA, is attributable to amplification and increased expression of the gene encoding the target site of glyphosate, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The EPSPS gene and the surrounding 287 kilobases (kb) of amplified sequence are unique to glyphosate-resistant plants and termed the EPSPS cassette. It has only been sequenced in one A. palmeri population from Mississippi. This research compares EPSPS cassettes in seven resistant and five sensitive populations from geographically distant locations within the USA, including Mississippi, Arizona, Kansas, Maryland, Delaware and Georgia., Results: Polymerase chain reaction (PCR) products from 40 primer pairs specific to the cassette were similar in size and sequence in resistant populations. Several primer pairs failed to generate PCR products in sensitive populations. Regions of the cassette sequenced in the resistant populations were found to be nearly identical to those from Mississippi. Gene expression analysis showed that both EPSPS and another gene in the cassette, a reverse transcriptase, were elevated in all resistant populations tested relative to the sensitive populations., Conclusion: EPSPS cassettes from distant resistant populations were nearly homologous. Considering the complexity of the cassette, and the degree of similarity among some cassette sequences, the results are consistent with the hypothesis that glyphosate resistance probably evolved once and then rapidly spread across the USA. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this study, the co-infection of Torque teno sus virus (TTSuV) and porcine circovirus type 3 (PCV3) was reported. One hundred and ten of 132 (83.3%) PCV3-positive samples were co-infected with Torque teno sus virus 1 (TTSuV1). Ninety-four of 132 (71.2%) PCV3-positive samples were co-infected with Torque teno sus virus 2 (TTSuV2). Sixty-six of 132 (50.0%) of PCV3-positive samples were co-infected with both TTSuV1 and TTSuV2. There were no clinical signs of infection in pigs that were both PCV3-positive and PCV2-negative, in either multiparous sows or live-born infants. The high co-infection rate provides valuable information for the further study of the pathological correlation between PCV3 and TTSuVs., (© 2017 Blackwell Verlag GmbH.)

A novel RPA-IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 10 3 CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA-IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

Rabbit haemorrhagic disease virus (RHDV, or GI.1) is a calicivirus in the genus Lagovirus that has been widely utilized in Australia as a biological control agent for the management of overabundant wild European rabbit (Oryctolagus cuniculus) populations since 1996. Recently, two exotic incursions of pathogenic lagoviruses have been reported in Australia; GI.1a-Aus, previously called RHDVa-Aus, is a GI.1a virus detected in January 2014, and the novel lagovirus GI.2 (previously known as RHDV2). Furthermore, an additional GI.1a strain, GI.1a-K5 (also known as 08Q712), was released nationwide in March 2017 as a supplementary tool for wild rabbit management. To discriminate between these lagoviruses, a highly sensitive strain-specific multiplex RT-PCR assay was developed, which allows fast, cost-effective and sensitive detection of the four pathogenic lagoviruses currently known to be circulating in Australia. In addition, we developed a universal RT-qPCR assay to be used in conjunction with the multiplex assay that broadly detects all four viruses and facilitates quantification of viral RNA load in samples. These assays enable rapid detection, identification and quantification of pathogenic lagoviruses in the Australian context. Using these assays, a novel recombinant lagovirus was detected in rabbit tissue samples, which contained the non-structural genes of GI.1a-Aus and the structural genes of GI.2. This variant was also recovered from the liver of a European brown hare (Lepus europaeus). The impact of this novel recombinant on Australian wild lagomorph populations and its competitiveness in relation to circulating field strains, particularly GI.2, requires further studies., (© 2017 Blackwell Verlag GmbH.)

Real-time PCR (qPCR) is widely used in the life sciences. For quantifying DNA, a standard curve is required. Common methods for standard development are time consuming, costly, necessitate a specific skill set, and pose a contamination risk. Using a targeted synthetic oligonucleotide, such as a gBlocks ® Gene Fragment, overcomes these drawbacks and provides researchers an accurate and quick solution to standard development. Here, we demonstrate that using a gBlocks fragment as a standard provides comparable sensitivity, reliability, and assay performance to a purified amplicon standard.

Intoduction: Inherited cataract, opacification of the lens, is the most common worldwide cause of blindness in children. We aimed to identify the genetic cause of autosomal dominant (AD) posterior nuclear cataract in a four generation British family., Methods: Whole genome sequence (WGS) was performed on two affected and one unaffected individual of the family and further validated by direct sequencing. Haplotype analysis was performed via genotying., Results: A splice-site mutation c.2826-9G>A in the gene EPHA2, encoding EPH receptor A2 was identified and found to co-segregate with disease., Conclusions: We have identified a recurrent splice-site mutation c.2826-9G>A in EPHA2 causing isolated posterior nuclear cataract, providing evidence of further phenotypic heterogeneity associated with this variant.

A total of 27 seminal plasma samples from cattle-breeding farms or semen centres located in Minas Gerais, Brazil, previously negative by serological and tested positive for Brucella spp. with primer specific for the amplification of the gene virb5 by polymerase chain reaction (PCR) were analysed for the detection of Brucella abortus DNA by PCR. It was found that nine samples (33.33%) contained B. abortus B19 strain DNA, two (7.40%) contained B. abortus DNA and five (18.51%) contained both DNA. The larger number of samples with B. abortus B19 strain DNA would explained by the environmental contamination by vaccinated females with persistent excretion or some illegal vaccination process. It is first reported of male bovines detected with both DNA., (© 2017 Blackwell Verlag GmbH.)

Swap and extend: The autonomous synthesis of single-stranded DNA molecules of arbitrary size and sequence composition can easily be achieved by primer exchange reaction (PER) cascades, in which the sequential polymerase-mediated extension of DNA primers is guided by catalytic hairpins. This highlight illustrates the potential of this method for applications in DNA nanotechnology., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we obtained the whole genomes of three porcine bocaparvovirus (PBoV) strains (GD6, GD10, and GD23) by polymerase chain reaction. Sequence analysis showed that all three field strains belonged to PBoV group 3 (G3). The phylogenetic trees based on NS1, NP1, and VP1 differed to the extent that these PBoVs were potentially more closely related to bocaparvoviruses known to infect other animals than to other PBoVs. GD6, GD10, and GD23 all included the conserved sequences YLGPF and HDXXY, with known phospholipase A2 activity. Using recombination-detection software we identified a natural recombinant breakpoint in the NS1 region of PBoV G3. The results of this study will further the epidemiological characterization of PBoVs.