Amplificação isotérmica mediada por loop para detecção de patógenos de plantas

Disease control is crucial to minimize potential losses in agriculture and thereby maintain high crop yield. However, for its effectiveness, the pathogen must be detected early and correctly in the production fields. Different methods of diagnosis can be used, from those based on symptoms to molecular tests. Loop-mediated isothermal amplification (LAMP) is a molecular technique that has been widely used in several biological fields, due to the ease with which it can be applied. The reaction can be carried out in a single thermal condition, due to the use of Bst DNA polymerase, isolated from the bacterium Bacillus stearothermophilus, which has high displacement activity. LAMP is a highly exponential amplification method that produces the target DNA in amounts 109 -1010 times between 45 and 60 minutes at 60-65°C. Its advantages are the visualization of results directly with the naked eye and the fact that it does not need sophisticated equipment for its application. In phytopathology, the technique has been gaining prominence in the detection of fungi, viruses, bacteria, nematodes and phytoplasmas, as well as in the monitoring of fungicide-resistant fungi. LAMP can benefit agriculture so that early, accurate and sensitive diagnostics can be carried out in the fields of cultivation and minimize losses caused by diseases. In this review, we present and discuss LAMP tests, developed for plant pathogens detection, which can be useful for researchers who wish to use the technique in their research area

Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus

A two-stage isothermal amplification method, which consists of a first-stage basic recombinase polymerase amplification (RPA) and a second-stage fluorescence loop-mediated isothermal amplification (LAMP), as well as a microfluidic-chip-based portable system, were developed in this study; these enabled parallel detection of multiplex targets in real time in around one hour, with high sensitivity and specificity, without cross-contamination. The consumption of the sample and the reagent was 2.1 μL and 10.6 μL per reaction for RPA and LAMP, respectively. The lowest detection limit (LOD) was about 10 copies. The clinical amplification of about 40 nasopharyngeal swab samples, containing 17 SARS-CoV-2 (severe acute respiratory syndrome coronavirus) and 23 measles viruses (MV), were parallel tested by using the microfluidic chip. Both clinical specificity and sensitivity were 100% for MV, and the clinical specificity and sensitivity were 94.12% and 95.83% for SARS-CoV-2, respectively. This two-stage isothermal amplification method based on the microfluidic chip format offers a convenient, clinically parallel molecular diagnostic method, which can identify different nucleic acid samples simultaneously and in a timely manner, and with a low cost of the reaction reagent. It is especially suitable for resource-limited areas and point-of-care testing (POCT).

A robust approach for industrial small-object detection using an improved faster regional convolutional neural network

With the increasing pace in the industrial sector, the need for a smart environment is also increasing and the production of industrial products in terms of quality always matters. There is a strong burden on the industrial environment to continue to reduce impulsive downtime, concert deprivation, and safety risks, which needs an efficient solution to detect and improve potential obligations as soon as possible. The systems working in industrial environments for generating industrial products are very fast and generate products rapidly, sometimes leading to faulty products. Therefore, this problem needs to be solved efficiently. Considering this problem in terms of faulty small-object detection, this study proposed an improved faster regional convolutional neural network-based model to detect the faults in the product images. We introduced a novel data-augmentation method along with a bi-cubic interpolation-based feature amplification method. A center loss is also introduced in the loss function to decrease the inter-class similarity issue. The experimental results show that the proposed improved model achieved better classification accuracy for detecting our small faulty objects. The proposed model performs better than the state-of-the-art methods.

Rapid identification of pathogens associated with ventilator-associated pneumonia by Nanopore sequencing

Background Aetiology detection is crucial in the diagnosis and treatment of ventilator-associated pneumonia (VAP). However, the detection method needs improvement. In this study, we used Nanopore sequencing to build a quick detection protocol and compared the efficiency of different methods for detecting 7 VAP pathogens. Methods The endotracheal aspirate (ETA) of 83 patients with suspected VAP from Peking University Third Hospital (PUTH) was collected, saponins were used to deplete host genomes, and PCR- or non-PCR-amplified library construction methods were used and compared. Sequence was performed with MinION equipment and local data analysis methods were used for sequencing and data analysis. Results Saponin depletion effectively removed 11 of 12 human genomes, while most pathogenic bacterial genome results showed no significant difference except for S. pneumoniae. Moreover, the average sequence time decreased from 19.6 h to 3.62 h. The non-PCR amplification method and PCR amplification method for library build has a similar average sensitivity (85.8% vs. 86.35%), but the non-PCR amplification method has a better average specificity (100% VS 91.15%), and required less time. The whole method takes 5–6 h from ETA extraction to pathogen classification. After analysing the 7 pathogens enrolled in our study, the average sensitivity of metagenomic sequencing was approximately 2.4 times higher than that of clinical culture (89.15% vs. 37.77%), and the average specificity was 98.8%. Conclusions Using saponins to remove the human genome and a non-PCR amplification method to build libraries can be used for the identification of pathogens in the ETA of VAP patients within 6 h by MinION, which provides a new approach for the rapid identification of pathogens in clinical departments.

Improved direct lysis PCR amplification method of microalgal culture for sequencing and species identification

Molecular approach plays important role in species identification for microalgae which involves sequencing of specific DNA barcode present in the genome. This approach involved preparation of template DNA for polymerase chain reaction (PCR) which is time consuming and requires large amounts of algal cells. Microalgal direct PCR have been used frequently for species identification, which simplified the DNA isolation procedure. However, the recent attempts to amplify the rbcL gene of microalga using the previously reported protocol led to poor repeatability. In this study, Nannochloropsis gaditana NIES-2587 was cultured in f/2 liquid medium. The culture growth was estimated on optical density value and the lysis process was improved using gradual temperature procedure during the PCR process. The same culture was extracted using manual DNA extraction method for comparison. The DNA obtained from both methods were amplified using RbclN primer pair to amplify 1486 bp partial sequence of Nannochloropsis rbcL gene, followed by the sequencing of the PCR product. Molecular identification based on the sequence result and BLAST analysis indicated that direct PCR and manual DNA extraction methods successfully produced high sequences result and confirmed the identity of microalgae species into N. gaditana strain CCMP527 with a genetic similarity of >99%.

Sensitive Colorimetric Detection of Interleukin-6 via Lateral Flow Assay Incorporated Silver Amplification Method

Interleukin-6 (IL-6) is a pro/anti-inflammatory cytokine, the quantitative detection of which has been extensively considered for diagnosis of inflammatory associated diseases. However, there has not yet been a reliable, low-cost, and user-friendly platform developed for point-of-care (POC) detection of IL-6, which will eliminate the conventional costly, time-consuming, and complex assays. In this work, we developed a lateral flow assay for colorimetric detection of IL-6, using anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs) as the detection probes. Silver amplification technique was incorporated with the newly developed assay in order to enhance the obtained colorimetric signals, allowing sensitive detection of IL-6 in human serum in the desired physiological ranges (i.e., 5–1000 pg/mL). A limit of detection of 5 pg/mL could be achieved for IL-6 detection in serum with the amplification step which was not achievable in the standard assay. The corresponding specificity and reproducibility tests were all preformed to confirm the reliability of this assay for quantitative measurement of IL-6 in a POC manner.