Double-Tube Multiplex TaqMan Real-Time PCR for the Detection of Eight Animal-Derived Dairy Ingredients.

Milk and dairy products represent important sources of nutrition in our daily lives. The identification of species within dairy products holds importance for monitoring food adulteration and ensuring traceability. This study presented a method that integrated double-tube and duplex real-time polymerase chain reaction (PCR) with multiplex TaqMan probes to enable the high-throughput detection of animal-derived ingredients in milk and dairy products. The detection system utilized one pair of universal primers, two pairs of specific primers, and eight animal-derived specific probes for cow, buffalo, goat, sheep, camel, yak, horse, and donkey. These components were optimized within a double-tube and four-probe PCR multiplex system. The developed double-tube detection system could simultaneously identify the above eight targets with a detection limit of 10-0.1 pg/µL. Validation using simulated adulterated milk samples demonstrated a detection limit of 0.1%. The primary advantage of this method lies in the simplification of the multiplex quantitative real-time PCR (qPCR) system through the use of universal primers. This method provides an efficient approach for detecting ingredients in dairy products, providing powerful technical support for market supervision.

LC-MS/MS method for simultaneous estimation of raloxifene, cladrin in rat plasma: application in pharmacokinetic studies.

Aim: A newer LC-MS/MS method was developed and validated for the simultaneous quantification of raloxifene (RL) and cladrin (CL). Methodology: Both drugs were resolved in RP-18 (4.6 × 50 mm, 5 µ) Xbridge Shield column using acetonitrile and 0.1% aqueous solution of formic acid (FA) (70:30% v/v) as mobile phase by using biological matrices in female Sprague-Dawley rats using-MS/MS. Results: The developed method was found to be linear over the concentration ranges of 1-600 ng/ml, and lower limit of quantification was 1 ng/ml for RL and CL, respectively. Pharmacokinetic results of RL+CL showed Cmax = 4.23 ± 0.61, 26.97 ± 1.14 ng/ml, at Tmax(h) 5.5 ± 1.00 and 3.5 ± 1.00, respectively. Conclusion: Pharmacokinetic study results will be useful in the future for the combined delivery of RL and CL for osteoporosis treatment.

Accurate molecular identification of different meat adulterations without carryover contaminations on a microarray chip PCR-directed microfluidic lateral flow strip device.

Meat adulteration-based food fraud has recently become one of the global major economical, illegal, religious, and public health concerns. In this work, we developed a microarray chip polymerase chain reaction (PCR)-directed microfluidic lateral flow strip (LFS) device that facilitates the accurate and simultaneous identification of beef adulterated with chicken, duck, and pork, especially in processed beef products. To realize this goal, four pairs of amplification primers were designed and applied for specifically amplifying genomic DNA extracted from mixed meat powders in microarray chip. With the prominent advantage of this device lies in the flexible combination and integration of sample loading, detection, and reporting in microstructures, all the DNA amplicons can be individually visualized on the LFS unit, leading to the appearance of test lines (TC line, TD line, TP line, or TB line) as well as the control line (C line) for the species identification and quantification in beef products. Based on this new method, the adulterants were successfully distinguished and identified in mixtures down to 0.01% (wt.%) while the carryover aerogel contamination in routine molecular diagnostic laboratories was effectively avoided. The practicability, accuracy, and reliability of the device were further confirmed by using real-time PCR as a gold standard control on the successful identification of 50 processed ground meat samples sourced from local markets. The method and device proposed herein could be a useful tool for on-site identification of food authentication.

Simultaneous identification of groundwater pollution source and important hydrogeological parameters considering the noise uncertainty of observational data.

Groundwater pollution identification is an inverse problem. When solving the inverse problem using regular methods such as simulation-optimization or stochastic statistical approaches, requires repeatedly calling the simulation model for forward calculations, which is a time-consuming process. Currently, the problem is often solved by building a surrogate model for the simulation model. However, the surrogate model is only an intermediate step in regular methods, such as the simulation-optimization method that also require the creation and solution of an optimization model with the minimum objective function, which adds complexity and time to the inversion task and presents an obstacle to achieving fast inversion. In the present study, the extreme gradient boosting (XGBoost) method and the back propagation neural network (BPNN) method were used to directly establish the mapping relationships between the output and input of the simulation model, which could directly obtain the inversion results of the variables to be identified (pollution sources release histories and hydraulic conductivities) based on actual observational data for fast inversion. In addition, to consider the uncertainty of observation data noise, the inversion accuracy of the two machine learning methods was compared, and the method with higher precision was selected for the uncertainty analysis. The results indicated that both the BPNN and XGBoost methods could perform inversion tasks well, with a mean absolute percentage error (MAPE) of 4.15% and 1.39%, respectively. Using the BPNN, with better accuracy for uncertainty analysis, when the maximum probabilistic density value was selected as the inversion result, the MAPE was 2.13%. We obtained the inversion results under different confidence levels and decision makers of groundwater pollution prevention and control can choose different inversion results according to their needs.

Rapid and simultaneous visual screening of SARS-CoV-2 and influenza virufses with customized isothermal amplification integrated lateral flow strip.

Due to the similar clinical symptoms of influenza (Flu) and coronavirus disease 2019 (COVID-19), there is a looming infection threat of concurrent Flu viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we introduce a customized isothermal amplification integrated lateral flow strip (LFS) that is capable performing duplex reverse transcription-recombinase polymerase amplification (RT-RPA) and colorimetric LFS in a sequential manner. With customized amplification primer sets targeted to SARS-CoV-2 (opening reading frame 1a/b and nucleoprotein genes) and Flu viruses (Flu A and Flu B), the platform allows the rapid and simultaneous visual screening of SARS-CoV-2 and Flu viruses (Flu A and Flu B) without cross reactivity, false positives, and false negatives. Moreover, it maximally eases the detection, reduces the detection time (1 h), and improves the assay performance to detect as low as 10 copies of the viral RNA. Its clinical application is powerfully demonstrated with 100% accuracy for evaluating 15 SARS-CoV-2-positive clinical samples, 10 Flu viruses-positive clinical samples, and 5 negative clinical samples, which were pre-confirmed by standard qRT-PCR. We envision this portable device can meet the increasing need of online monitoring the serious infectious diseases that substantially affects health care systems worldwide.

Capture, Detection, and Simultaneous Identification of Rare Circulating Tumor Cells Based on a Rhodamine 6G-Loaded Metal-Organic Framework.

Circulating tumor cells (CTCs) play a key role in the development of tumor metastasis. It will be a big step forward for CTC application as a reliable clinical liquid biopsy marker to be able to identify the captured CTCs while achieving a high capture efficiency within one analytical system. Herein, in this work, a stimuli-responsive and rhodamine 6G (Rho 6G)-entrapped fluorescent metal-organic framework (MOF) probe, named MOF-Rho 6G-DNA, was designed to capture, detect, and subsequently identify CTCs from blood samples of cancer patients. The probe was fabricated by modifying the epithelial cell adhesion molecule (EpCAM) hairpin DNA aptamer with Rho 6G enclosed and an Arm-DNA-attached UiO-66-NH2 MOF by sequence complementation. CTCs could be captured by the EpCAM hairpin DNA on the probe; as a result, Rho 6G loaded in the probe was released, and the number of CTCs was positively related to the concentration of released Rho 6G. An excellent correlation of fluorescence intensities with CTC numbers was obtained from 2 to 500 cells/mL. More importantly, the MOF-Rho 6G-DNA probe simultaneously realized rapid identification of the captured cells within 30 min by only relying on the residue Rho 6G in the MOF cavity. The captured target cells can be conveniently released from the probe using the complementary DNA sequence. These performance features of the probe were further verified by blood samples from patients of various types of tumor.

Simultaneous identification of Fritillariae cirrhosae bulbus and its common adulterants in one reaction by multiplex ligation-dependent probe amplification and high-resolution melting curve assay.

Fritillariae cirrhosae bulbus, a well-known and precious medicinal and edible herb in China, causes remarkable effects on swelling and relieving cough, with fewer side effects than other congeneric medicine. It has been subject to various cheaper congeneric adulteration because of its high price and limited production. In this paper, a rapid, high throughput, sensitive and efficient technique was described for simultaneous identification of F. cirrhosae bulbus and its common adulterants by employing multiplex ligation-dependent probe amplification coupled with high-resolution melting (MLPA-HRM) curve assay in their internal transcribed spacer 1 (ITS1) regions. This assay was highly sensitive with a detection limit of 0.19 ng genomic DNA, and highly specific with no cross-reaction with common adulterants. Mixed sample analysis showed as low as 10% adulteration can be detected from F. cirrhosae bulbus in one MLPA-HRM reaction. Overall, the method described in this paper is well suited for detecting adulteration in F. cirrhosae bulbus.

Simultaneous identification of groundwater contamination source and aquifer parameters with a new weighted-average wavelet variable-threshold denoising method.

This paper first proposed a parallel heuristic search strategy for simultaneous identification of groundwater contamination source and aquifer parameters. As identification results are influenced by many factors, such as noisy contamination concentration data, data denoising is necessary. The existing wavelet threshold denoising method has unavoidable shortcomings; therefore, this paper first proposed a new weighted-average wavelet variable-threshold denoising (WWVD) method to improve the denoising effect for concentration data, which further enhanced the subsequent identification accuracy. However, frequent calls to the simulation model could produce high computational cost during likelihood calculation. Hence, single surrogate model of the simulation model was developed to reduce cost; however, it presented limitation. Thus, this paper first developed a differential evolution-tabu search (DE-TS) hybrid algorithm to construct an optimal ensemble surrogate model, which assembled Gaussian process, kernel extreme learning machine, and support vector regression. The first proposed DE-TS algorithm also improved the approximation accuracy of surrogate model to simulation model. This paper first proposed and implemented a parallel heuristic search iterative process for simultaneous identification, and the identification results were obtained when the iteration process terminated. The accuracy and efficiency of these newly proposed approaches were tested through a hypothetical case. Results showed that the WWVD method not only improved the denoising effect for concentration data but also enhanced the subsequent identification accuracy. The OES model using DE-TS hybrid algorithm improved the approximation accuracy of surrogate model to simulation model, and the parallel heuristic search strategy is helpful for simultaneous identification of groundwater contamination source and aquifer parameters.

Control of a two-DOF parallel robot with unknown parameters using a novel robust adaptive approach.

Model-based methods lose their performance in confronting with model uncertainties and disturbances. Accordingly, some degrees of adaptation to the involved conditions are required. In this paper, a novel robust adaptive scheme is proposed which guarantees the simultaneous identification and control of a system in the presence of external disturbances. Thereafter, the suggested algorithm is implemented on a 2-DOf spherical parallel robot as a stabilizer device. By identifying unknown parameters of Jacobian matrix, the relative identification error is obtained as 0.0207. Applying external excitations to the base, the ratio of end-effector to base orientation is acquired as 0.091, demonstrating proper stabilization in comparison with other two well-known methods. The proposed structure also reveals a reliable performance in tracking desired paths for the end-effector Euler angles.

Multiplex Ligation-Dependent Probe Amplification for Simultaneous Identification of Bungarus multicinctus and Its Common Adulterants in a Single Assay.

Bungarus multicinctus, an important traditional Chinese medicine, possesses remarkable medicinal activities, while lots of adulterants from other species were misused as B. multicinctus for its large demand and resource starvation. In order to accurately identify B. multicinctus and its common adulterants such as Sinonatrix annularis, Xenochrophis flavipunctatus, Deinagkistrodon acutus, and Naja atra, a simultaneous identification method was designed with multiplex ligation-dependent probe amplification (MLPA) analysis. Five species-specific MLPA probe-couples for B. multicinctus and its common adulterants were designed based on the universal primer amplified COI sequences, which can specifically detect the five species with no mutual interference, and sensitivity analysis showed as less as 5% B. multicinctus or 8.75% adulterants in the mixed samples can be identified in a MLPA assay, especially, the relative quantity of the adulterants can be also inferred based on the MLPA peak area values. Moreover, the results of the present study confirmed the effectiveness of this technique in terms of simultaneous identification of B. multicinctus and its common adulterants in an assay, which has great potential for ensuring the safety of this commercially valuable snake species.

Simultaneous Identification of 13 Foodborne Pathogens by Using Capillary Electrophoresis-Single Strand Conformation Polymorphism Coupled with Multiplex Ligation-Dependent Probe Amplification and Its Application in Foods.

Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) coupled with stuffer-free multiplex ligation-dependent probe amplification (MLPA) was developed to identify 13 species of foodborne pathogens simultaneously. Species-specific MLPA probes were designed for nine of these species. These probes were targeted to the groEL, glyA, MMS, tuf, inv, ipaH, nuc, vvh, and 16S rRNA genes, which corresponded to Bacillus cereus, Campylobacter coli, Cronobacter sakazakii, Enterococcus spp., Salmonella spp., Shigella spp., Staphylococcus aureus, Vibrio vulnificus, and Yersinia enterocolitica, respectively. MLPA probes that had been previously developed by our laboratory were used for the other four species (Campylobacter jejuni, Clostridium perfringens, Escherichia coli O157:H7, and Listeria monocytogenes). The CE-SSCP method was optimized to identify all 13 foodborne microbes simultaneously in a single electrogram, in which 50-500 pg genomic DNA was detected per microbe. Twelve species were detected from animal-derived food samples (specifically, milk and sliced ham) that had been artificially inoculated with 12 of the foodborne pathogens, excluding V. vulnificus, which is not usually associated with animal foods. The method developed here could be used as an early warning system for outbreaks of foodborne diseases associated with animal-derived foods in the food industry.

Comparative analysis of male and female populations on prevalence and antibiotic resistance of Mycoplasma hominis in China, 2005-2014.

The aim of this study was to estimate the prevalence and antimicrobial resistance rate of Mycoplasma hominis among male and female populations. A total of 67921 individuals were examined. All samples were isolated from patients at an outpatient clinic from January 2005 to December 2014. Species identification and antibiotic susceptibility testing were performed using Mycoplasma IST2. In this study, 523 (0.8%) and 4625 (6.8%) cultures, respectively, were positive for single M. hominis identification and simultaneous identification of both M. hominis and Ureaplasma spp. The results showed that both single and simultaneous identification were more frequent in the female than the male population. Moreover, the highest positive rates of single M. hominis identification were observed in 56-60-year-old males and 61-65-year-old females, and the rates of simultaneous identification were high in males aged >65 years and females aged 15-20 years. Among the seven antibiotics assessed, tetracycline, josamycin, doxycycline and pristinamycin were the most effective, whilst clarithromycin, ciprofloxacin and ofloxacin displayed extremely high resistance rates. Different antimicrobial susceptibility rates were observed between the two sexes, and the resistance rates to ofloxacin showed a significant difference (P<0.05). In conclusion, this study demonstrates that the prevalence of M. hominis varied significantly between the two sexes in the past 10 years and that the optimal antimicrobial therapy strategy should be directed by local susceptibility testing.