Two-step PCR Research Articles

The discovery of multidrug resistant Staphylococcus aureus harboring novel SaRI isolated from retail foods

Food-borne Staphylococcus aureus has shown multidrug resistance. According to our previous studies, most S. aureus isolated from retail foods in China exhibited resistance to a variety of antimicrobials. Further Genome Wide Association Study (GWAS) revealed that a novel gene cluster, containing erythromycin-resistant genes, mef (D)- msr (F), existed in 19 isolates. In this study, we aim to investigate the distribution and transmission of this novel gene cluster in these isolates and to further characterize it. As a result, the msr (F)-positive S. aureus isolates mainly belong to ST7 (n = 14), sporadically to ST14 (n = 4), and ST88 (n = 1) most of which showed multidrug resistance (MDR) phenotype. Different virulence factors (VFs) and antimicrobial resistance genes (ARGs) were identified by genomic mining in msr (F)-positive strains. In addition, core genome phylogenetic analysis revealed that most of the msr (F)-positive strains (14/19) were closely related despite different isolation time. Two types of antibiotic resistance islands, SaRI msr -I (15.1 kb) and SaRI msr -II (16–17 kb) carrying mef (D)- msr (F), were identified in their chromosomes. Importantly, the novel SaRI msr -I was inserted into rpsI gene which encoded 30S ribosomal protein S9 on the chromosome. SaRI msr -II was also inserted into rpsI but four ORF frames downstream of rpsI were absent. Within the SaRI msr -I and -II, an operon structure consisting of ARGs, mef (D) and msr (F), and a site-specific integrase gene int were identified. Finally, the excision and cyclization of the SaRI msr and operon mef (D)- msr (F) were verified by two-step PCR. This study is the first report of MDR S. aureus harboring novel SaRI msr isolated from retail foods, indicating that this novel SaRI msr harboring mef (D)- msr (F) is distributed in food-related S. aureus in China and is one of the determinants for erythromycin resistance. The occurrence and propagation of similar mef (D)- msr (F)-carrying SaRI msr from S. aureus need further investigation. • SaRI msr carrying mef (D)- msr (F) operon emerged in MDR S. aureus for the first time. • SaRI msr was novel structure, and both SaRI msr and mef (D)- msr (F) operon were active. • The novel SaRI msr were discovered via Genome Wide Association Study (GWAS). • This novel SaRI msr harbouring mef (D)- msr (F) in DMR S. aureus should be underscored.

Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia

SUMMARYActivated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato+ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP+ tdTomato+ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1β release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders.

Use of a Novel Detection Tool to Survey Orthohantaviruses in Wild-Caught Rodent Populations.

Orthohantaviruses are negative-stranded RNA viruses with trisegmented genomes that can cause severe disease in humans and are carried by several host reservoirs throughout the world. Old World orthohantaviruses are primarily located throughout Europe and Asia, causing hemorrhagic fever with renal syndrome, and New World orthohantaviruses are found in North, Central, and South America, causing hantavirus cardiopulmonary syndrome (HCPS). In the United States, Sin Nombre orthohantavirus (SNV) is the primary cause of HCPS with a fatality rate of ~36%. The primary SNV host reservoir is thought to be the North American deer mouse, Peromyscus maniculatus. However, it has been shown that other species of Peromyscus can carry different orthohantaviruses. Few studies have systemically surveyed which orthohantaviruses may exist in wild-caught rodents or monitored spillover events into additional rodent reservoirs. A method for the rapid detection of orthohantaviruses is needed to screen large collections of rodent samples. Here, we report a pan-orthohantavirus, two-step reverse-transcription quantitative real-time PCR (RT-qPCR) tool designed to detect both Old and New World pathogenic orthohantavirus sequences of the S segment of the genome and validated them using plasmids and authentic viruses. We then performed a screening of wild-caught rodents and identified orthohantaviruses in lung tissue, and we confirmed the findings by Sanger sequencing. Furthermore, we identified new rodent reservoirs that have not been previously reported as orthohantavirus carriers. This novel tool can be used for the efficient and rapid detection of various orthohantaviruses, while uncovering potential new orthohantaviruses and host reservoirs that may otherwise go undetected.

High-resolution melting analysis after nested PCR for the detection of SARS-CoV-2 spike protein G339D and D796Y variations

High-resolution melting (HRM) analysis was performed to detect G339D and D796Y variations in the SARS-CoV-2 Omicron variant spike protein. We employed two-step PCR consisting of the first RT–PCR and the second nested PCR to prepare the amplicon for HRM analysis. The melting temperatures (Tm) of the amplicon from the cDNA of the Omicron variant receptor binding domain (RBD) were 73.1 °C (G339D variation) and 75.1 °C (D796Y variation), respectively. These Tm values were clearly distinct from those of SARS-CoV-2 isolate Wuhan-Hu-1. HRM analysis after the two-step PCR was conducted on Omicron variant-positive specimens. The HRM curve and Tm value obtained with the Omicron variant-positive specimen were coincident with those of the amplicon from cDNA of the Omicron variant RBD. Our study demonstrates the utility of HRM analysis after two-step PCR for the detection of mutations in SARS-CoV-2 gene.

Genetic specificity of the white-headed ukrainian breed according to the BоLA-DRB3 gene

The study of the genetic specificity of local breeds is a promising direction in the context of preserving the biodiversity of cattle breeds in the world. The purpose of research was to analyze the allelic polymorphism of the BoLA-DRB3 gene of the White-Headed Ukrainian breed. The study was carried out with blood samples from 49 animals (11 bulls and 38 cows) of this breed. The allelic spectrum of the BoLA-DRB3 gene was detected based on two-step PCR (primers HLO-30, HLO-31 and HLO-32). Restriction was performed with endonucleases RsaI, HaeIII, BstYI. Restriction fragments were separated by electrophoresis in 9-12 % polyacrylamide gel. According to the test results, 29 alleles were detected. Seven alleles (*03, *11, *13, *15, *22, *23 and *24) were determined with a frequency of over 5 %, that was 65.3 % of the allele pool of the breed. The most common was allele *24 with a frequency of 15.3%. In the experimental sample, 37 genotypes were identified. The predominant variant was *11/*24 (16.2%). A slight excess of heterozygotes was detected (Fis = – 0.035). White-Headed Ukrainian breed is characterized by a significant level of differentiation (or specificity) according to the BoLA-DRB3 gene (Wright fixation index He = 0.959, Shannon-Wiener index H` = 2.93), that confirms the thesis of the important role of local breeds in preserving the biodiversity of genetic resources of cattle.

The effect of single-walled carbon nanotubes on DNA replication and the shape of DNA strands

Single-walled carbon nanotubes (SWNTs) can be thought of as a vector of medicines or other molecules. Furthermore DNA molecules work as a dispersing agent preventing SWNTs bundling. So it is necessary to fully understand the effect of SWNTs on DNA replication. SWNTs can protect DNA strands under high temperature though denature experiment. SWNTs can increase the yield of PCR products by two-step PCR and improve the reaction efficiency of DNA polymerase in a short extension time. The rpsL fidelity test showed that the error rate of PCR products in the control group was 5.3×-10 −6, the error rate in the experimental group was only 2.4×- 10 −6.

PTT-quant: a new method for direct identification and absolute quantification of premature transcription termination events, following the example of bacterial riboswitches

Regulation of gene expression by premature termination of transcription has been well described in all domains of life, including metazoans, yeast, plants, and bacteria. Although methods for identification of such regulatory events by sequencing are available, the focused biochemical studies of the mechanism are hampered by lack of highly sensitive and accurate experimental methods. Here, we propose a new method for absolute quantification of premature transcription termination events, PTT-quant. It is based on highly sensitive two-step digital droplet PCR protocol, coupled with normalized cDNA synthesis attained by site-specific pre-cleavage of investigated transcripts with RNase H. As a consequence, our method enables the reliable and sensitive quantification of both, prematurely terminated and full-length transcripts. By application of our method to investigation of transcriptional riboswitches in Bacillus subtilis, we were able to precisely measure the dynamics of S-adenosylmethionine (SAM) riboswitch induction, which turned to be ~ 23% higher in comparison the results obtained without cDNA synthesis normalization.Key points• A novel method for quantification of premature transcription termination events was established.• PTT-quant measures absolute concentration of full-length and terminated transcripts.• RNase H and the digital droplet PCR technique is used in PTT-quant.

A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha.

Simple SummaryAs a result of increasing demand for the pleiotropic cytokine TNF-α, recombinant human TNF-α protein with appropriate bioactivities was produced in several heterologous in vivo expression systems. While in vivo expression of this cytokine is laborious and lengthy, cell-free or in vitro expression system has the benefits of speed, simplicity, flexibility, focus of all the system energy on target protein synthesis alone, besides high soluble and functional protein yield. Therefore, we employed and optimized an E. coli-based cell-free system for the first time to express recombinant human TNF-α. Our findings revealed that cell-free expression system can be an alternative platform for producing soluble and functionally active recombinant TNF-α with a yield of 390 µg/mL in only 2 h at a temperature of 40 °C for further research and clinical trials.Cell-free (in vitro) expression is a robust alternative platform to the cell-based (in vivo) system for recombinant protein production. Tumor necrosis factor-alpha (TNF-α) is an effective pro-inflammatory cytokine with pleiotropic effects. The aim of the current study was de novo optimized expression of soluble and active human TNF-α by an in vitro method in an E. coli-based cell-free protein synthesis (CFPS) system and its biological activity evaluation. The codon-optimized synthetic human TNF-α gene was constructed by a two-step PCR, cloned into pET101/D-TOPO vector and then expressed by the E. coli CFPS system. Cell-free expression of the soluble protein was optimized using a response surface methodology (RSM). The anticancer activity of purified human TNF-α was assessed against three human cancer cell lines: Caco-2, HepG-2 and MCF-7. Data from RSM revealed that the lowest value (7.2 µg/mL) of cell-free production of recombinant human TNF-α (rhTNF-α) was obtained at a certain incubation time (6 h) and incubation temperature (20 °C), while the highest value (350 µg/mL) was recorded at 4 h and 35 °C. This rhTNF-α showed a significant anticancer potency. Our findings suggest a cell-free expression system as an alternative platform for producing soluble and functionally active recombinant TNF-α for further research and clinical trials.

Angiotensin-Converting Enzyme Genotype-Specific Immune Response Contributes to the Susceptibility of COVID-19: A Nested Case-Control Study.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has resulted in a global pandemic. Methodology: We used a two-step polymerase chain reaction to detect the ACE genotype and ELISA kits to detect the cytokine factor. We also used proteomics to identify the immune pathway related to the ACE protein expression. Result: In this study, we found that the angiotensin-converting enzyme (ACE) deletion polymorphism was associated with the susceptibility to COVID-19 in a risk-dependent manner among the Chinese population. D/D genotype distributions were higher in the COVID-19 disease group than in the control group (D/D odds ratio is 3.87 for mild (p value < 0.0001), 2.59 for moderate (p value = 0.0002), and 4.05 for severe symptoms (p value < 0.0001), logic regression analysis. Moreover, genotype-specific cytokine storms and immune responses were found enriched in patients with the ACE deletion polymorphism, suggesting the contribution to the susceptibility to COVID-19. Finally, we identified the immune pathway such as the complement system related to the ACE protein expression of patients by lung and plasma proteomics. Conclusion: Our results demonstrated that it is very important to consider gene polymorphisms in the population to discover a host-based COVID-19 vaccine and drug design for preventive and precision medicine.

Sequence Variations of 31 Υ-Chromosomal Short Tandem Repeats Analyzed by Massively Parallel Sequencing in Three U.S. Population Groups and Korean Population.

BackgroundRapidly mutating (RM) Y-chromosomal short tandem repeats (Y-STRs) have been demonstrated to increase the possibility of distinguishing between male relatives due to a higher mutation rate than conventional Y-STRs. Massively parallel sequencing (MPS) can be useful for forensic DNA typing as it allows the detection of sequence variants of many forensic markers. Here, we present sequence variations of 31 Y-STRs including nine RM Y-STRs (DYF387S1, DYF399S1, DYF404S1, DYS449, DYS518, DYS570, DYS576, DYS612, and DYS627), their frequencies, distribution, and the gain in the number of alleles using MPS.MethodsWe constructed a multiplex MPS assay capable of simultaneously amplifying 32 Y-chromosomal markers, producing amplicons ranging from 85–274 bp. Barcoded libraries from 220 unrelated males from four populations—African Americans, Caucasians, Hispanics, and Koreans—were generated via two-step polymerase chain reaction and sequenced on a MiSeq system. Genotype concordance between the capillary electrophoresis (CE) and MPS method and sequence variation of Y-STRs were investigated.ResultsIn total, 195 alleles were increased by MPS compared to CE-based alleles (261 to 456). The DYS518 marker showed the largest increase due to repeat region variation (a 3.69-fold increase). The highest increase in the number of alleles due to single nucleotide polymorphisms in the flanking region was found in DYF399S1. RM Y-STRs had more diverse sequences than conventional Y-STRs. Furthermore, null alleles were observed in DYS576 due to primer-binding site mutation, and allele drop-outs in DYS449 resulted from low marker coverage of less than the threshold.ConclusionThe results suggest that the expanded and discriminative MPS assay could provide more genetic information for Y-STRs, especially for RM Y-STRs, and could advance male individualization. Compiling sequence-based Y-STR data for worldwide populations would facilitate the application of MPS in the field of forensic genetics and could be applicable in solving male-related forensic cases.

Tokat ili Bağlarda Grapevine leafroll-associated virus -1,2,3,4'ün yaygınlığı ve Genetik Değişkenliği

Grapevine leafroll disease (GLRD) is one of the most important viral diseases of grapevine worldwide. The aim of this study was to investigate the prevelance of Grapevine leafroll associated virus-1 (GLRaV-1), Grapevine leafroll-associated virus-2 (GLRaV-2), Grapevine leafroll-associated virus-3 (GLRaV-3), Grapevine leafroll-associated virus 4 (GLRaV-4), and Grapevine leafroll-associated virus-9 (GLRaV-9) in Tokat province of Turkey. In 2018, leaf samples and one-year-old shoots of plants, showing virus-like symptoms, were collected from different vineyards in Central, Erbaa, Niksar, and Pazar districts of Tokat province. Totally 197 samples were obtained and tested molecularly using two-step polymerase chain reaction (PCR) by using virus-specific primers for GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4) and GLRaV-9. At least 106 (53.80%) samples were found to be infected showed single or multiple infections. The 103 (52.28%) samples resulted in positive with GLRaV-1 while the infection rates were %5.18 for GLRaV-3 with 10 samples, %2.53 for GLRaV-4 with 5 samples, %1.03 for GLRaV-2 and GLRaV-9 with 2 samples, respectively. The amplified PCR products of GLRaV-1, GLRaV-2 and GLRaV-3 were sequenced, aligned with MEGAX, and analyzed in BLAST for multiple comparisons of other isolates in the GenBank. Sequence analysis of viruses showed 94-96 % , 96-99 % and, 99-100 % nucleotide identity with references isolates in the GenBank for GLRaV-1, GLRaV-2, and UO3-3 isolate of GLRaV-3 respectively.

Predicción molecular de serotipos de Streptococcus suis aislados de granjas porcinas en México

Infections caused by Streptococcus suis (S. suis) pose a problem for the pig industry worldwide. Pigs often carry multiple serotypes of S. suis in the upper respiratory tract, where S. suis is frequently isolated from. The clinical diagnosis of the infection is presumptive and is generally based on clinical signs, the age of the animal and macroscopic lesions. In the laboratory, identification of S. suis is performed biochemically, and then, serotyping is performed with antisera to determine the serotype, but these tests can be inconclusive. To date, there are few studies that have documented the presence and diversity of S. suis serotypes in Mexico. In the present study, it was characterized S. suis strains from Mexican pig farms using molecular approaches; samples were first processed by PCR of the gdh gene to detect S. suis. Positive samples were then subjected to a two-step multiplex PCR (cps PCR) to detect and characterize each strain; the first step consisted of a grouping PCR and the second step consisted of a typing PCR. The serotypes detected in the pig farming areas of Mexico included 1/2, 2, 3, 5, 7, 8, 9, 17, and 23. These findings are important for the characterization of serotypes present in Mexico and for outbreak prevention.

Pneumococcal Serotype Identification by Capsular Sequence Typing (CST): A Modified Novel Approach for Serotyping Directly in Clinical Samples.

As almost 60–70% of Invasive Pneumococcal Disease (IPD) is identified by nonculture methods in Greece, serotyping is of high importance for the better monitoring of pneumococcal serotypes due to the availability of conjugate vaccines. The aim of the study was the modification and direct application of the Capsular Sequence Typing (CST) assay in clinical samples in order to serotype Streptococcus pneumoniae culture-negative, Polymerase Chain Reaction (PCR_-positive samples, followed by CST group specific single-tube PCR assays. A two-step PCR modified assay was applied on a total of 306 samples (such as CSF, blood, pleural and middle ear fluids, isolates) obtained from 283 patients with IPD. The overall performance permits a rapid, accurate and cost-effective method for nonculture pneumococcal serotyping. As the management of IPD is closely related to the continuous monitoring of pneumococcal serotypes, the proposed approach proved to be a valuable tool for the typing and epidemiological monitoring of S. pneumoniae, for the evaluation of the overall impact of vaccination programs in the era of pneumococcal conjugate vaccines, in order to initiate the appropriate vaccination strategy.

Identification of a Novel Geminivirus in Fraxinus rhynchophylla in Korea

Fraxinus rhynchophylla, common name ash, belongs to the family Oleaceae and is found in China, Korea, North America, the Indian subcontinent, and eastern Russia. It has been used as a traditional herbal medicine in Korea and various parts of the world due to its chemical constituents. During a field survey in March 2019, mild vein thickening (almost negligible) was observed in a few ash trees. High-throughput sequencing of libraries of total DNA from ash trees, rolling-circle amplification (RCA), and polymerase chain reaction (PCR) allowed the identification of a Fraxinus symptomless virus. This virus has five confirmed open reading frames along with a possible sixth open reading frame that encodes the movement protein and is almost 2.7 kb in size, with a nonanucleotide and stem loop structure identical to begomoviruses. In terms of its size and structure, this virus strongly resembles begomoviruses, but does not show any significant sequence identity with them. To confirm movement of the virus within the trees, different parts of infected trees were examined, and viral movement was successfully observed. No satellite molecules or DNA B were identified. Two-step PCR confirmed the virion and complementary strands during replication in both freshly collected infected samples of ash tree and Nicotiana benthamiana samples agro-inoculated with infectious clones. This taxon is so distantly grouped from other known geminiviruses that it likely represents a new geminivirus genus.

Rapid and Sensitive Detection of Salmonella spp. Using CRISPR-Cas13a Combined With Recombinase Polymerase Amplification.

Salmonella spp. is one of the most common foodborne disease-causing pathogens that can cause severe diseases in very low infectious doses. Rapid and sensitive detecting Salmonella spp. is advantageous to the control of its spread. In this study, a conserved short fragment of the Salmonella invA gene was selected and used to design primers and specific crRNA (CRISPR RNA) for establishing a one-tube and two-step reaction system for Salmonella spp. detection, by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a) cleavage. The established one-tube RPA-Cas13a method can complete the detection within 20 min and the two-step RPA-Cas13a method detection time within 45 min. The designed primers were highly specific to Salmonella spp. and had no cross-reaction with the other nine diarrheal bacteria. The one-tube RPA-Cas13a could detect the Salmonella genome with the limit of 102 copies, which was the same as real-time polymerase chain reaction (PCR), but less sensitive than two-step RPA-Cas13a (100 copies). The detection results of one-tube or two-step RPA-Cas13a and real-time PCR were highly consistent in clinical samples. One-tube RPA-Cas13a developed in this study provides a simple, rapid, and specific detection method for Salmonella spp. While two-step assay was more sensitive and suitable for samples at low abundance.

Short Communication: Latent Detection of Downy Mildew (Peronospora pisi) in Bioassays against Pisum sativum

Downy mildew of peas is caused by the obligate parasite Peronospora pisi, which occurs sporadically throughout temperate pea-growing regions across the world. To screen pea lines against this biotrophic pathogen, a suitable and reproducible in vitro method using living plant material is required. Field screening can be influenced by environmental factors, thus giving variable results. The aim of this study was to develop a method that could reliably be used to screen pea cultivars against P. pisi in a laboratory setting. A range of bioassays were used to test various methods of inoculation, utilizing sporangia and naturally infested soil. Latent infection was achieved by planting seeds in soil collected from a site with a known history of P. pisi infection and directly inoculating young pea plants with sporangia. Out of the 108 plants which survived the experimental period, only two plants expressed visible signs of disease; however, through a two-step nested PCR process we detected latent infection in 24 plants. This research highlights the importance of considering the presence of latent infection when screening pea lines against downy mildew.

Improved high throughput protocol for targeting eukaryotic symbionts in metazoan and eDNA samples.

Eukaryote symbionts of animals are major drivers of ecosystems not only because of their diversity and host interactions from variable pathogenicity but also through different key roles such as commensalism and to different types of interdependence. However, molecular investigations of metazoan eukaryomes require minimising coamplification of homologous host genes. In this study we (1) identified a previously published “antimetazoan” reverse primer to theoretically enable amplification of a wider range of microeukaryotic symbionts, including more evolutionarily divergent sequence types, (2) evaluated in silico several antimetazoan primer combinations, and (3) optimised the application of the best performing primer pair for high throughput sequencing (HTS) by comparing one‐step and two‐step PCR amplification approaches, testing different annealing temperatures and evaluating the taxonomic profiles produced by HTS and data analysis. The primer combination 574*F – UNonMet_DB tested in silico showed the largest diversity of nonmetazoan sequence types in the SILVA database and was also the shortest available primer combination for broadly‐targeting antimetazoan amplification across the 18S rRNA gene V4 region. We demonstrate that the one‐step PCR approach used for library preparation produces significantly lower proportions of metazoan reads, and a more comprehensive coverage of host‐associated microeukaryote reads than the two‐step approach. Using higher PCR annealing temperatures further increased the proportion of nonmetazoan reads in all sample types tested. The resulting V4 region amplicons were taxonomically informative even when only the forward read is analysed. This region also revealed a diversity of known and putatively parasitic lineages and a wider diversity of host‐associated eukaryotes.

Identification of lncRNA NR_028138.1 as a biomarker and construction of a ceRNA network for bipolar disorder

The pathogenesis of bipolar disorder (BD), a chronic mood disorder, is largely unknown. Noncoding RNAs play important roles in the pathogenesis of BD. However, little is known about the correlations of long noncoding RNAs (lncRNAs) with BD. Illumina high-throughput sequencing in BD patients and normal controls was used to identify differentially expressed (DE) genes. Two-step real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate DE-RNAs in the first cohort (50 BD and 50 control subjects). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and lncRNA-mRNA coexpression and lncRNA-microRNA (miRNA)-messenger RNA (mRNA) competing endogenous RNA (ceRNA) network analyses were used to predict the functions of DE-RNAs. Receiver operating characteristic (ROC) curve analysis and logistic regression were applied to evaluate diagnostic performance in an additional testing group (80 BD and 66 control subjects). A total of 576 significantly DE-lncRNAs and 262 DE-mRNAs were identified in BD patients, and 95 lncRNA-miRNA-mRNA interactions were used to construct a ceRNA regulatory network. Analysis of the first cohort showed that six RNAs (NR_028138.1, TCONS_00018621, TCONS_00002186, TNF, PID1, and SDK1) were differentially expressed in the BD group (P < 0.01). NR_028138.1 was used to establish a BD diagnostic model (area under the ROC curve 0.923, P < 0.004, 95% CI: 0.830–0.999). Verification in the second cohort revealed uniformly significant differences in NR_028138.1 (P < 0.0001). This study constructed a ceRNA regulatory network and provided a hypothesis for the pathogenesis of BD. NR_028138.1 was identified as a central element involved in the transcriptional regulation in BD and a potential biomarker.

Role of circulating microRNA-21 and microRNA-215 in the diagnosis of hepatitis C related hepatocellular carcinoma.

Several micro ribonucleic acids (miRNAs) are deregulated in hepatocellular carcinoma (HCC). Others are linked to clinical pathological features of HCC. The goal of this study was to investigate whether miRNA-21 and miRNA-215 gene expression could be used as a non-invasive diagnostic tool to diagnose HCC. The gene expression of mature miRNA -21 and miRNA -215 in serum was analysed retrospectively using singleplex TaqMan two-step stem-loop quantitative real-time reverse-transcription PCR in 40 patients with HCC, 40 with chronic hepatitis C virus (HCV) with cirrhosis and 40 apparently healthy controls. Expression of miRNA -21 was significantly more down regulated in patients with HCC than in those with non-cirrhotic HCV (P = 0.007; odds ratio = 5; 95% confidence interval 1.6-15.4). The receiver operating curve analysis of the ability of miRNA-21 expression to discriminate between HCC and non-cirrhotic HCV revealed an area under the curve of 0.712 with 70% sensitivity and 68% specificity at a cut-off of ≤ 1.4468. Thus, the expression level of miRNA -21 could discriminate HCC from non-cirrhotic HCV. Significant positive correlation was observed between expression levels of microRNA-21 and miRNA -215 (r = 0.783, p < 0.001), but no association was observed between expression level of miR-215 and HCC or chronic HCV (p = 0.474). MiRNA-21 may be a useful, non-invasive tool for diagnosing HCC. Non-cirrhotic HCV patients have five times the risk of developing HCC when the miRNA -21 level ≤ 1.4468.

Screening for gene doping transgenes in horses via the use of massively parallel sequencing.

Throughout the history of horse racing, doping techniques to suppress or enhance performance have expanded to match the technology available. The next frontier in doping, both in the equine and human sports areas, is predicted to be genetic manipulation; either by prohibited use of genome editing, or gene therapy via transgenes. By using massively-parallel sequencing via a two-step PCR method we can screen for multiple doping targets at once in pooled primer sets. This method has the advantages of high scalability through combinational indexing, and the use of reference standards with altered sequences as controls. Custom software produces transgene-specific amplicons from any Ensembl-annotated genome to facilitate rapid assay design. Additional scripts batch-process FASTQ data from experiments, automatically quality-filtering sequences and assigning hits based on discriminatory motifs. We report here our experiences in establishing the workflow with an initial 31 transgene and vector feature targets. To evaluate the sensitivity of parallel sequencing in a real-world setting, we performed an intramuscular (IM) administration of a control rAAV vector into two horses and compared the detection sensitivity between parallel sequencing and real-time qPCR. Vector was detected by all assays on both methods up to 79 h post-administration, becoming sporadic after 96 h.