Identification of Sequence Variants in Genetic Disease-Causing Genes Using Targeted Next-Generation Sequencing

Identification of gene variants plays an important role in research on and diagnosis of genetic diseases. A combination of enrichment of targeted genes and next-generation sequencing (targeted DNA-HiSeq) results in both high efficiency and low cost for targeted sequencing of genes of interest.To identify mutations associated with genetic diseases, we designed an array-based gene chip to capture all of the exons of 193 genes involved in 103 genetic diseases. To evaluate this technology, we selected 7 samples from seven patients with six different genetic diseases resulting from six disease-causing genes and 100 samples from normal human adults as controls. The data obtained showed that on average, 99.14% of 3,382 exons with more than 30-fold coverage were successfully detected using Targeted DNA-HiSeq technology, and we found six known variants in four disease-causing genes and two novel mutations in two other disease-causing genes (the STS gene for XLI and the FBN1 gene for MFS) as well as one exon deletion mutation in the DMD gene. These results were confirmed in their entirety using either the Sanger sequencing method or real-time PCR.Targeted DNA-HiSeq combines next-generation sequencing with the capture of sequences from a relevant subset of high-interest genes. This method was tested by capturing sequences from a DNA library through hybridization to oligonucleotide probes specific for genetic disorder-related genes and was found to show high selectivity, improve the detection of mutations, enabling the discovery of novel variants, and provide additional indel data. Thus, targeted DNA-HiSeq can be used to analyze the gene variant profiles of monogenic diseases with high sensitivity, fidelity, throughput and speed

Poloxamer 407/188 Binary Thermosensitive Gel as a Moxidectin Delivery System: In Vitro Release and In Vivo Evaluation

Moxidectin (MXD) is an antiparasitic drug used extensively in veterinary clinics. In this study, to develop a new formulation of MXD, a thermosensitive gel of MXD (MXD-TG) was prepared based on poloxamer 407/188. Furthermore, the gelation temperature, the stability, in vitro release kinetics and in vivo pharmacokinetics of MXD-TG were evaluated. The results showed that the gelation temperature was approximately 27 °C. MXD-TG was physically stable and can be released continuously for more than 96 h in vitro. The Korsmeyer–Peppas model provided the best fit to the release kinetics, and the release mechanism followed a diffusive erosion style. MXD-TG was released persistently for over 70 days in sheep. Part of pharmacokinetic parameters had a difference in female and male sheep (p < 0.05). It was concluded that MXD-TG had a good stability, and its release followed the characteristics of a diffusive erosion style in vitro and a sustained release pattern in vivo

Prevalence and characteristics of extended-spectrum β-lactamase and plasmid-mediated fluoroquinolone resistance genes in Escherichia coli isolated from chickens in Anhui province, China.

The aim of this study was to characterize the prevalence of extended-spectrum β-lactamase (ESBL) genes and plasmid-mediated fluoroquinolone resistance (PMQR) determinants in 202 Escherichia coli isolates from chickens in Anhui Province, China, and to determine whether ESBL and PMQR genes co-localized in the isolates. Antimicrobial susceptibility for 12 antimicrobials was determined by broth microdilution. Polymerase chain reactions (PCRs), DNA sequencing, and pulsed field gel electrophoresis (PFGE) were employed to characterize the molecular basis for β-lactam and fluoroquinolone resistance. High rates of antimicrobial resistance were observed, 147 out of the 202 (72.8%) isolates were resistant to at least 6 antimicrobial agents and 28 (13.9%) of the isolates were resistant to at least 10 antimicrobials. The prevalence of blaCTX-M, blaTEM-1 and blaTEM-206 genes was 19.8%, 24.3% and 11.9%, respectively. Seventy-five out of the 202 (37.1%) isolates possessed a plasmid-mediated quinolone resistance determinant in the form of qnrS (n = 21); this determinant occurred occasionally in combination with aac(6')-1b-cr (n = 65). Coexistence of ESBL and/or PMQR genes was identified in 31 of the isolates. Two E. coli isolates carried blaTEM-1, blaCTX-M and qnrS, while two others carried blaCTX-M, qnrS and aac(6')-1b-cr. In addition, blaTEM-1, qnrS and aac(6')-1b-cr were co-located in two other E. coli isolates. PFGE analysis showed that these isolates were not clonally related and were genetically diverse. To the best of our knowledge, this study is the first to describe detection of TEM-206-producing E. coli in farmed chickens, and the presence of blaTEM-206, qnrS and aac(6')-1b-cr in one of the isolates

Comparison of the <i>Xba</i>I-PFGE patterns of 49 <i>Escherichia coli</i> isolates from chicken feces from chicken farms in the Anhui Province of China.

<p>Comparison of the <i>Xba</i>I-PFGE patterns of 49 <i>Escherichia coli</i> isolates from chicken feces from chicken farms in the Anhui Province of China.</p

Coexistence of ESBL and/or PMQR genes.

<p>Coexistence of ESBL and/or PMQR genes.</p

Resistance profiles of 202 <i>Escherichia coli</i> isolates from four chicken farms to 12 antimicrobials.

<p>Note: one to twelve in the X axis represents resistance to one antimicrobial to twelve antimicrobials. Y axis represents the rates of the isolates resistance to one antimicrobial to twelve antimicrobials.</p

Resistance rates of <i>Escherichia coli</i> isolates from four chicken farms to 12 antimicrobials.

<p>Abbreviations: AMX: amoxicillin, CRO: ceftriaxome, CTF: ceftiofur, AMI: amikacin, GEN: gentamicin, APR: apramycin, DC: doxycycline, OTC: oxytetracycline, FFC: florfenicol, ERO: enrofloxacin, OFX: ofloxacin, LOM: lomefloxacin.</p