A novel and cost-efficient allele-specific PCR method for multiple SNP genotyping in a single run.

Cost-effective methods for DNA genotyping were needed because single nucleotide polymorphisms (SNPs) were essential biomarkers associated with many diseases. Allele-specific PCR (AS-PCR) has the advantages of mature instruments and high sensitivity. But conventional AS-PCR needs to multiply the number of reactions or primers for multiple targets, which complicates the operation and increases the cost. Herein, we proposed a novel AS-PCR method for multiple SNP genotyping in a single run. Wild-type allele-specific primer (WT primer) was designed for each target gene. The sample and WT primers only needed to undergo multiplexed AS-PCR once simultaneously. After AS-PCR, the concentration of remaining primers varied among the samples of each genotype combination, due to the different matching performance between template and WT primers. The remaining primers then triggered multiplexed molecular beacon-rolling circle amplification, and the molecular beacons labelled with different fluorescent dyes corresponded to different targets. The fluorescence ratios of the sample to the positive control were used as the genotyping indexes. This method was able to detect samples with concentrations as low as 10 fM. We successfully applied the method to the multiple genotyping of 23 hair root samples for ADH1B and ALDH2 genes, obtaining completely consistent results with sequencing. The reagent cost was 0.6 dollar for one sample, showing a good cost performance. This proposed approach had a great application prospect in simultaneously rapid and accurate genotyping of multi-SNPs, and provided a new method for personalized health management.

Fluorescence Biosensor for One-Step Simultaneous Detection of Mycobacterium tuberculosis Multidrug-Resistant Genes Using nanoCoTPyP and Double Quantum Dots.

The diagnosis of multidrug-resistant tuberculosis (MDR-TB) is crucial for the subsequent drug guidance to improve therapy and control the spread of this infectious disease. Herein, we developed a novel florescence biosensor for simultaneous detection of Mycobacterium tuberculosis (Mtb) multidrug-resistant genes (rpoB531 for rifampicin and katG315 for isoniazid) by using our synthesized nanocobalt 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine (nanoCoTPyP) and double quantum dots (QDs). Several nanoCoTPyPs with different charges and morphology were successfully prepared via the surfactant-assisted method and their quenching ability and restoring efficiency for DNA detection were systematically analyzed. It was found that spherical nanoCoTPyP with positive charge exhibited excellent quenching effect and sensing performance for the two DNAs' detection due to its affinity differences towards single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). ssDNA attached on QDs (QDs-ssDNA) was specifically hybridized with targets to form QDs-dsDNA, resulting in fluorescence recovery due to the disruption of the interactions between nanoCoTPyP and ssDNA. Two drug-resistant genes could be simultaneously quantified in a single run and relatively low limits of detection (LODs) were obtained (24 pM for T1 and 20 pM for T2). Furthermore, the accuracy and reliability of our method were verified by testing clinical samples. This simple and low-cost approach had great potential to be applied in clinical diagnosis of MDR-TB.

Determination of tuberculosis-related volatile organic biomarker methyl nicotinate in vapor using fluorescent assay based on quantum dots and cobalt-containing porphyrin nanosheets.

Methyl nicotinate (MN) is a representative and typical volatile organic marker of Mycobacterium tuberculosis, and the specific detection of MN in human breath facilitates non-invasive, rapid, and accurate epidemic screening of tuberculosis infection. Herein, we constructed a fluorescent assay consisted of CdTe quantum dots (QD) and cobalt-metalized tetrakis(4-carboxyphenyl) porphyrin (CoTCPP) nanosheets to determine methyl nicotinate (MN) in vapor samples. Red-emission QD (λex=370 nm, λem=658 nm) acts as signal switches whose fluorescence signals can be effectively quenched by CoTCPP nanosheets but restored in the presence of MN. The strategy relied on the distinct binding affinity of cobalt ion and MN. MN restored the fluorescence of QD quenched by CoTCPP in a concentration-dependent manner, which exhibited a well-linear relationship in the range 1-100 µM, and a limit of detection of 0.59 µM. The proposed platform showed sensitivity and selectivity to detect MN in vapor samples with satisfactory RSD below 3.33%. The method is cheap, simple, and relatively rapid (detected within 4 min), which suggests a potential in tuberculosis diagnosis in resource- and professional-lacked areas.

Allele-specific PCR with a novel data processing method based on difference value for single nucleotide polymorphism genotyping of ALDH2 gene.

Single nucleotide polymorphism (SNP) analysis based on allele-specific polymerase chain reaction (AS-PCR) is a relatively effective and economical method compared with other genotyping technologies such as DNA sequencing, DNA hybridization and isothermal amplification strategies. But AS-PCR is limited by its labor-intensive optimization of reaction parameters and time-consuming result assessment. In this study, we put forward a novel idea of data processing to address this problem. SNP analysis was accomplished by AS-PCR with endpoint electrochemical detection. For each sample, two separate reactions were run simultaneously with two sets of allele-specific primers (wild-type primers for W system and mutant primers for M system). We measured their redox current signals on screen-printed electrodes once AS-PCR finished and calculated the difference value of current signals between two systems to determine the genotyping result. Based on the difference value of fluorescent signals, real-time fluorescent PCR was used to study reaction parameters in AS-PCR. With screened parameters, we obtained the genotyping results within 50 min. 36 hair-root samples from volunteers were analyzed by our method and their genotypes of ALDH2 gene (encoding aldehyde dehydrogenase 2) were totally identical with data from commercialized sequencing. Our work first employed difference value between two reaction systems to differentiate allele and provided a novel idea of data processing in AS-PCR method. It is able to promote the quick analysis of SNP in the fields of health monitor, disease precaution, and personalized diagnosis and treatment.

Developmental Toxicity of Diethylnitrosamine in Zebrafish Embryos/Juveniles Related to Excessive Oxidative Stress.

Diethylnitrosamine (DEN) is present in food, water, and daily supplies and is regarded as a toxicant of carcinogenicity. The developmental toxicity of DEN has been rarely reported as yet. In this study, zebrafish were exposed to different concentrations of DEN at 6 h post-fertilization (hpf) to access embryonic toxicity of the compound. The results show that DEN resulted in negative effects of hatching rate, heartbeat, body length, and spontaneous movement. Deformities, including notochord malformation, pericardium edema, embryonic membrane turbidity, tail hypoplasia, yolk sac deformity, and growth retardation, happened during exposure period. Moreover, production of reactive oxygen species (ROS) significantly increased after DEN treatment. Then, alterations of the expression level of oxidative stress-related genes were observed in our results. To our knowledge, this is the first study concerning the effect of DEN on zebrafish. And from the information of our research, we speculated that development toxicity of DEN should be related to the excessive oxidative stress.

The protection of novel 2-arylethenylquinoline derivatives against impairment of associative learning memory induced by neural Aß in C. elegans Alzheimer's disease model.

Cerebral deposition of amyloid ß-peptide (Aß), a fundamental feature of Alzheimer's disease (AD), damages the neurocytes and impairs the cognition functions and associative learning memory of AD patients. A series of novel 2-arylethenylquinoline derivatives were synthesized and evaluated in our previous study, which inhibited Aß aggregation in vitro effectively at the concentration of 20 µmol/L and exhibited high antioxidant activity. In order to verify the capacity of anti-AD in vivo, the transgenic Caenorhabditis elegans (C. elegans) strain CL2355 expressing neural Aß was employed as the AD model to investigate the neuroprotective activity of seven high-potential compounds (4a1, 4a2, 4b1, 4b2, 4c1, 4c2, 4c3) selected from those derivatives. Learning memory associated chemotaxis assay was performed to evaluate the neural repairment capacity. The underlying mechanism was investigated by mRNA analysis of Aß gene and heat shock protein genes (hsp-16.1 and hsp-16.2) and Western blot of Aß. Our data indicated that among seven tested compound, 4b1 and 4c2 reduced Aß-induced stress, suppressed the expression of neural Aß monomers and toxic oligomers, and recovered the damaged associative learning memory in C. elegans AD model. These findings further confirmed their potentials to become valuable agents for AD therapy.

Aucklandia lappa DC. extract enhances gefitinib efficacy in gefitinib-resistance secondary epidermal growth factor receptor mutations.

ETHNOPHARMACOLOGICAL RELEVANCE: Aucklandia lappa DC. is a widely used medicinal plant in China, India and Pakistan for a long time. Previously, a number of different pharmacological experiments in vitro and in vivo have convincingly demonstrated the abilities of it to exhibit anticancer activities. Reynoutria japonica Houtt. has also been widely used as traditional Chinese medicinal plant. Previous studies have demonstrated that it is bioactive to exhibit anticancer activities. AIM OF THE STUDY: This study aims to investigate whether the extracts of Aucklandia lappa DC. and Reynoutria japonica Houtt. are capable of treating drug-resistant non-small cell lung cancer (NSCLC), providing support for novel usage beyond traditional uses. MATERIALS AND METHODS: Extracts combined with gefitinib have been tested taking the vulval development of transgenic C. elegans (jgIs25) as an effective and simple in vivo model system, evaluating their efficacy against acquired NSCLC. Synchronous larval 1 (L1) larvae were treated with extracts plus gefitinib and cultured to obtain mainly L4 larvae. The multivulva (Muv) phenotype was recorded at the adult stage. RESULTS: Our data showed that Aucklandia lappa DC. extract could significantly enhance the efficacy of gefitinib, suppressing the Muv phenotype of jgIs25. Meanwhile, it could also down-regulate the mRNA and protein expression of EGFR in jgIs25. Collectively, our results verified that the capability of Aucklandia lappa DC. to inhibit Muv phenotype may be based on the EGFR signaling pathway inhibition. CONCLUSION: We demonstrated that the co-administration of Aucklandia lappa DC. with gefitinib may provide an effective strategy for the therapy of EGFR inhibitor resistant NSCLCs.

Microfluidic platform integrated with worm-counting setup for assessing manganese toxicity.

We reported a new microfluidic system integrated with worm responders for evaluating the environmental manganese toxicity. The micro device consists of worm loading units, worm observing chambers, and a radial concentration gradient generator (CGG). Eight T-shape worm loading units of the micro device were used to load the exact number of worms into the corresponding eight chambers with the assistance of worm responders and doorsills. The worm responder, as a key component, was employed for performing automated worm-counting assay through electric impedance sensing. This label-free and non-invasive worm-counting technique was applied to the microsystem for the first time. In addition, the disk-shaped CGG can generate a range of stepwise concentrations of the appointed chemical automatically and simultaneously. Due to the scalable architecture of radial CGG, it has the potential to increase the throughput of the assay. Dopaminergic (DAergic) neurotoxicity of manganese on C. elegans was quantitatively assessed via the observation of green fluorescence protein-tagged DAergic neurons of the strain BZ555 on-chip. In addition, oxidative stress triggered by manganese was evaluated by the quantitative fluorescence intensity of the strain CL2166. By scoring the survival ratio and stroke frequency of worms, we characterized the dose- and time-dependent mobility defects of the manganese-exposed worms. Furthermore, we applied the microsystem to investigate the effect of natural antioxidants to protect manganese-induced toxicity.