Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis

Objective This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay （MCMRT-PCR） for the simultaneous detection of six common foodborne pathogenic bacteria （diarrhoeagenic Escherichia coli, Salmonella, and 5higella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2）. Methods A two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System {Applied Biosystems, USA）. Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. Results The detection limit of optimized MCMRT-PCR assay was 3.9x102 CFU/mLfor S. aureus, 4.4x102 CFU/mL for L. monocytogenes, 3.0x102 CFU/mL for Salmonella, 2.5x102 CFU/mL for Shigella, 2.1x102 CFU/mL for V. parahaemolyticus, and 1.2x102 CFU/mL for E. coll. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 10s CFU/mL. Conclusion A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention （CDC）.

High Resolution Melting Curve Analysis for Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis Clinical Isolates

Background: Pyrazinamide (PZA) is one of the most important drugs for tuberculosis (TB) treatment, however, its susceptibility is not routinely tested. High-resolution melting (HRM) curve analysis has been widely used for many applications. In this study, HRM assay was developed and evaluated for the detection of PZA resistance in Mycobacterium tuberculosis clinical isolates. Methods: Ninety five M. tuberculosis clinical isolates with different susceptibility patterns to anti-TB drugs were used to evaluate this assay. Isolates were phenotypically (Bactec MGIT 960) and genotypically (HRM and pncA gene sequencing) analysed for PZA resistance. Results: Bactec MGIT 960 analysis revealed that 29 of the 95 M. tuberculosis isolates were PZA resistant. In comparison to the Bactec MGIT 960, HRM showed a sensitivity of 47.7% and specificity of 74.6%, and the overall agreement between the two methods was 68.4%. Based on DNA sequencing, a correlation of 0.67 (significant at p-value pncA mutations was observed. PZA resistance was strongly associated with multi-drug resistant (MDR)-TB as it was shown in 79.3% of the MDR isolates included in the study. Conclusion: HRM is simple and useful for screening clinical M. tuberculosis isolates for PZA resistance, however, further modifications to improve its performance are required.

Stability and melting behavior of boron phosphide under high pressure

Boron phosphide(BP)has gained significant research attention due to its unique photoelectric and mechanical properties.In this work,we investigated the stability of BP under high pressure using x-ray diffraction and scanning electron microscope.The phase diagram of BP was explored in both B-rich and P-rich environments,revealing crucial insight into its behavior at 5.0 GPa.Additionally,we measured the melting curve of BP from 8.0 GPa to 15.0 GPa.Our findings indicate that the stability of BP under high pressure is improved within B-rich and P-rich environments.Furthermore,we report a remarkable observation of melting curve frustration at 10.0 GPa.This study will enhance our understanding of stability of BP under high pressure,shedding light on its potential application in semiconductor,thermal,and light-transmitting devices.

Ab Initio Two-Phase Molecular Dynamics on the Melting Curve of SiO_2

Ab initio two-phase molecular dynamics simulations were performed on silica at pressures of 20-160 GPa and temperatures of 2 500-6 000 K to examine its solid-liquid phase boundary. Results indicate a melting temperature （Tin） of 5 900 K at 135 GPa. This is 1 100 K higher than the temperature considered for the core-mantle boundary （CMB） of about 3 800 K. The calculated melting temperature is fairly consistent with classical MD （molecular dynamics） simulations. For liquid silica, the O-O coordination number is found to be 12 along the Tm and is almost unchanged with increasing pressure. The self-diffusion coefficients of O and Si atoms are determined to be 1.3×10^-9-3.3×10^-9 m2/s, and the viscosity is 0.02-0.03 Pa＇s along the Tin. We find that these transport properties depend less on pressure in the wide range up of more than 135 GPa. The eutectic temperatures in the MgO-SiO2 systems were evaluated and found to be 700 K higher than the CMB temperature, though they would decrease considerably in more realistic mantle compositions.

Simulation of Melting Behavior of the MgSi03 Perovskite Under Lower Mantle Conditions

The melting curve of MgSiO3 perovskite was simulated using molecular dynamics method combining with the effective pair potentials under the lower mantle conditions. It was shown that the state equation simulated for MgSiO3 perovskite is very successful in reproducing accurately the experimental data over a wide range of pressure. The pressure dependence of the simulated melting temperature of MgSiO3 perovskite is in agreement with the recent experimental data. The melting curve simulated for MgSiO3 is very steep at pressures below 60 GPa first, then it becomes smooth with increasing pressure. At the core mantle boundary pressure 135 GPa, MgSiO3 perovskite melts at 6500 K, which is significantly lower than that of the extrapolations of the experimental data from Zerr and Boehler.

Fusion Equation of Metals

An expression of initial slope of melting curve of pure metals was obtained as follows: (dT_m/dP)_o=T_(mo)/c, where c=1.09 (N_(at))^(5/3)z^(-1/3), the unit of c is GPa, N_(at) is the atomic concentra- tion (in 10^(28) m^(-3)), z is the valence, T_(mo) is the melting temperature (in k) of metal under one atmosphere. The calculated results for thirty-one metals agree well with experiments. It has also been proved that by using the free electron model of melting, the fusion equation of metals is Simon equation (T_m/T_(mo))q=1+(p/d). Two parameters q and d, which have to fit with experiments in Simon's empirical equation, now can be predicted theoretically, e.g. for Mg, giving q=1.56, d=7.88GPa, the calculated melting curve in a fairly wide pressure range (0～60GPa) is shown to be close to the experimental one.

Melting Characteristics of Iron Ore Fine During Sintering Process

The whole melting curve of iron ore during sintering process was obtained,and the melting characteristics of iron ore were defined and explained. The whole melting process of mixture,mixed by iron ore and CaO reagent at basicity of 2.0 and 4.0,respectively,was observed using a SiC heating furnace with camcorder unit,and the melting curves of mixture that were relative height vs temperature curves were obtained. Besides,the melting characteristics of iron ore during sintering were introduced through defining some points in the melting curves,such as liquid forming temperature,inflexion temperature and flowing temperature,and the meaning of different shapes of the melting curves was clarified.

A Novel 2-dimensional Multiplex qPCR Assay for Single-Tube Detection of Nine Human Herpesviruses

Human herpesviruses are double-stranded DNA viruses that are classified into nine species.More than 90%of adults are ever infected with one or more herpesviruses.The symptoms of infection with different herpesviruses are diverse ranging from mild or asymptomatic infections to deadly diseases such as aggressive lymphomas and sarcomas.Timely and accurate detection of herpesvirus infection is critical for clinical management and treatment.In this study,we established a single-tube nonuple qPCR assay for detection of all nine herpesviruses using a 2-D multiplex qPCR method with a house-keeping gene as the internal control.The novel assay can detect and distinguish different herpesviruses with 30 to 300 copies per 25µL single-tube reaction,and does not cross-react with 20 other human viruses,including DNA and RNA viruses.The robustness of the novel assay was evaluated using 170 clinical samples.The novel assay showed a high consistency(100%)with the single qPCR assay for HHVs detection.The features of simple,rapid,high sensitivity,specificity,and low cost make this assay a high potential to be widely used in clinical diagnosis and patient treatment.

Development of two different pharmacogenomics detection methods for second-generation antipsychotic drugs quetiapine and aripiprazole

Objective:Second-generation antipsychotics are widely used in mental illness,but the treatment effects and side effects are affected by single nucleotide polymorphisms(SNPs)of related genes.Quetiapine and aripiprazole are two frequently used secondgeneration antipsychotic drugs.The aim of this study was to develop two different SNP detection methods for four SNP alleles associated with the pharmacokinetics of quetiapine and aripiprazole,based on high-resolution melting(HRM)and multicolor melting curve assay(MMCA)respectively.Methods:Whole genome DNA samples were obtained from 240 healthy people(107 females and 133 males)without genetic diseases.HRM methods were established using four kinds of specific primers and a saturated fluorescent dye.Each SNP allele with their own primers was detected in a single reaction.In the MMCA method,a multiplex polymerase chain reaction with 4 different-colored fluorescent probes was established to detect four SNP alleles in a single reaction.All experimental protocols were approved by the Ethics Committee of the Shanghai Children’s Medical Center,China(SCMC-201015)on November 22,2010.Results:Two detection methods for the pharmacogenomics of quetiapine and aripiprazole,based on HRM and MMCA respectively,were established in this study.The single-target HRM method can be completed in 96 minutes,whereas the quadruplex MMCA method takes 133 minutes.It was found that the results of HRM and MMCA for the four SNP alleles had 100%coincidence with Sanger sequencing in the 240 samples.Conclusion:This study developed two methods for the detection of four pharmacogenomic SNP alleles that correlated with quetiapine and aripiprazole.Both methods are rapid,cost-saving,highly accurate and potentially facilitate rational use of second-generation antipsychotics for clinical medication.

Validation studies of the Para DNA^(■) Intelligence System with artificial evidence items

Short tandem repeat(STR)profiling is one of the mostly used systems for forensic applications.In certain circumstances,STR profiling is time-consuming and costly,which potentially leads to delays in criminal investigations.LGC(Laboratory of the Government Chemist,UK)Forensics has developed a robust STR profiling platform called the ParaDNAVR Intelligence Test System which can provide early tactical intelligence and aid investigators in making informed decisions on sample prioritization for detection.Here,we validated the ParaDNA^(■) intelligence test for its application in forensic cases using a range of mock evidence items following guidelines set by the Scientific Working Group on DNA Analysis Methods(SWGDAM).Specifically,we tested the sensitivity and accuracy of the ParaDNA intelligence test,as well as the success rates for detecting mock samples and for use in case scenarios.Our findings demonstrate that the ParaDNA intelligence test generates useful DNA profiles,especially for samples such as blood,saliva,and semen that contain ample DNA,indicating the benefits of including ParaDNA as a prior step in forensic STR profiling pipelines.