Rapid identification of diarrheagenic Escherichia coli based on barcoded magnetic bead hybridization

Diarrhea, as a global public health problem, causes a large number of infections and deaths every year. Although Escherichia coli (E. coli) is one of the normal flora microorganisms in the human intestinal tract, it has five pathogenic bacteria types that can cause human diarrhea, known as diarrheagenic E. coli. When people are infected, rapid and accurate diagnosis, along with timely treatment, are especially important. Here, we introduce a new method to identify and analyze a large number of pathogenic strains in E. coli by multiplex PCR and barcoded magnetic bead hybridization. Results show that the detection sensitivities of enterohemorrhagic E. coli, enterotoxigenic E. coli, enteropathogenic E. coli, enteroinvasive E. coli and enteroaggregative E. coli were 1.3 × 10³ CFU/mL, 2 × 10⁴ CFU/mL, 4 × 10⁴ CFU/mL, 7.2 × 10⁴ CFU/mL and 1.7 CFU/mL respectively. This method has strong specificity and high sensitivity and detects multiple target sequences in one experiment. Compared with other methods, BMB array has great application potential.     

Nanoliter droplet array for microRNA detection based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR

In this paper, a nanoliter droplet array based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR for quantification of microRNA was developed. By employing T4 RNA ligase 2 instead of T4 DNA ligase, we designed simplified stem-loop probes to perform microRNA-templated DNA ligation and reduced the non-specific ligation of T4 DNA ligase. SYBR green I dye was employed instead of TaqMan probes in present miniaturized real-time PCR systems. Specifically, we optimized the dosage of SYBR Green I dye in nanoliter droplet and verified the performance of this system by detecting synthetic mir-122 with a 6 logs dynamic range (from 1.5 × 10⁵ to 1.5 × 10¹⁰ copies). Linear relationship of the standard curve (R² = 0.9997) and high PCR amplification efficiency (96.83%) were obtained under the optimized conditions. We detected the expression of mir-122 across five mouse tissues and the result was consistent with that TaqMan microRNA assay. We think this miniaturized real-time PCR platform reduced the detection cost considerably, thus showing the great potential to quantitative biology.     

Pathogen identification using mass spectrometry in the clinical microbiology laboratory

The recent application of Matrix-assisted Laser Desorption/Ionization Time-of-Flight and Polymerase Chain Reaction Electrospray Ionization Quadrupole Time-of-Flight mass spectrometry approaches to microbial identification has initiated a revolution in the clinical microbiology lab. The commercial application of these technologies to pathogen identification has only begun in the last five years, and already new potentially life-saving applications of these technologies are rapidly identifying organisms that in the past have proven notoriously difficult to identify. In this review, we will provide a brief historical perspective on how these developments arose, describe why they are being successfully applied now and provide an overview of current approaches. Using examples involving clinical isolates of Staphylococcus aureus, a perspective on future use and developments of mass spectrometry in the identification of microbial organisms is provided.     

Multi-scale mapping algorithm for INI cancellation and a novel mixed-numerologies OFDM transceiver

OFDM with mixed-numerologies enhances the system flexibility effectively to meet the demands of diversified application scenarios. However, the coexistence of waveforms with different numerologies leads to serious inter-numerology interference (INI), and the corresponding relationship between the number of guard subcarriers and the power of INI needs to be considered for scheduling subcarriers. In this paper, we propose a multi-scale mapping (MSM) and INI cancellation (MSM-INIC) algorithm as well as the corresponding de-MSM algorithm for mixed-numerologies OFDM system. Based on the proposed algorithms, we provide a novel transceiver in the scenario of multi-path fading channel, in which subcarrier scheduling does not need to consider whether the guard band is allocated. In the proposed transmitter, an additional MSM-INIC module is employed to pre-compensate signal distortion for downlink, and in the receiver, a de-MSM module is applied to de-map the received signals for recovering the original numerologies. Furthermore, we reveal the inherent property of the mapped signals, and propose a low computational complexity de-MSM algorithm accordingly. Simulation results verify the superiority of the proposed transceiver in BER performance as well as spectrum efficiency even without any guard band.     

Full utilization of a mass spectrometer using on‐demand sharing with multiple LC units

The applicability of liquid chromatography–mass spectrometry (LC/MS) is often limited by throughput. The sharing of a mass spectrometer with multiple LCs significantly improves throughput; however, the reported systems have not been designed to fully utilize the MS duty cycle, and as a result to achieve maximum throughput. To fully utilize the mass spectrometer, the number of LC units that a MS will need to recruit is application dependent and could be significantly larger than the current commercial or published implementations. For the example of a single analyte, the number may approach the peak capacity to a first degree approximation. Here, the construction of a MS system that flexibly recruits any number of LC units demanded by the application is discussed, followed by the method to port a previously developed LC/MS method to the system to fully utilize a mass spectrometer. To demonstrate the performance and operation, a prototypical MS system of eight LC units was constructed. When 1‐min chromatographic separations were performed in parallel on the eight LCs of the system, the average LC/MS analysis time per sample was 10.5 s when applied to the analysis of samples in 384‐well plate format. This system has been successfully used to conduct large‐volume biochemical assays with the analysis of a variety of molecular entities in support of drug discovery efforts. Allowing the recruitment of the number of LC units appropriate for a given application, this system has the potential to be a plug‐and‐play system to fully utilize a mass spectrometer. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of PCR internal controls for DNA profiling with the AmpFℓSTR® SGM Plus® amplification kit

Forensic DNA profiling uses a series of commercial kits that co‐amplify several loci in one reaction; the products of the PCR are fluorescently labelled and analysed using CE. Before CE, an aliquot of the PCR is mixed with formamide and an internal lane size standard. Using the SGM Plus amplification kit, we have developed two internal non‐amplified controls of 80 bp and 380 bp that are labelled with ROX fluorescent dye and added to the PCR. Combined with two internal amplification controls of 90 bp and 410 bp, they provide additional controls for the PCR, electrokinetic injection, and CE and also function as an internal size standard.     

Development of multiplex PCR system with 15 X-STR loci and genetic analysis in three nationality populations from China

The aim of this study is to develop a new multiplex PCR system that simultaneously amplifies the 15 X-chromosome short tandem repeats (X-STRs) loci in the same PCR reaction, and to obtain the 15 X-STR loci database in three nationality populations from China. This multiplex system includes DXS7133, DXS6801, DXS981, DXS6809, DXS7424, DXS6789, DXS9898, DXS7132, GATA165B12, DXS101, DXS10075, DXS6800, GATA31E08, DXS10074, and DXS10079, which were successfully analyzed on 1251 DNA samples (670 males and 581 females) from Guangdong Han population, Xinjiang Uigur and Kazakh. The allele frequencies and mutation rates of the 15 loci were investigated, and the allele frequency distribution among different populations was compared. A total of 6-17 alleles for each locus were observed and altogether 170 alleles for all the selected loci were found. Thirteen cases with mutation of the above loci were detected in 11,850 meioses. Pairwise comparisons of the allele frequencies distribution showed significant differences in most loci among different populations. The results indicate that this multiplex system may provide high polymorphism information for kinship testing and relationship investigations, and it is necessary to gain allele frequency and mutation rate of different population for forensic application.     

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.     

Assessment of Genetic Stability in Traditional Ginger Cultivated in Manipur,India Based on Molecular and Chemical Markers

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyze the genetic stability of ten local cultivars collected from nine districts of Manipur, India with the released ginger variety Nadia. A total of 15 RAPD and 8 ISSR primers resulted in 107 and 53 distinct and reproducible bands, respectively. A lack of polymorphism revealed the genetic stability among the local cultivars. Unlike molecular markers, analysis of essential oil composition by gas chromatography–mass spectrometry (GC-MS) revealed variation among 11 clones. Among eight major constituents obtained by GC-MS technique, cinnamyl acetate was found only in IBSD/Z-41d cultivars, whereas, in IBSD/Z-41o no trace of trans-geraniol was observed. Moreover, concentration of 6-gingerol determined by high-performance liquid chromatographic (HPLC) method shows that IBSD/Z-41r contains the highest and IBSD/Z-41i contains the lowest gingerol percentage.