African
swine fever (ASF) is one of the most severe diseases of
pigs. In this study, a CRISPR-Cas12a (also known as Cpf1) system coupled
with nucleic acid amplification was optimized for the detection of
ASF virus (ASFV). Two novel single-stranded DNA-fluorophore-quencher
(ssDNA-FQ) reporters were developed to increase the brightness of
the fluorescent signal for the visualization of nucleic acid detection.
The CRISPR-Cas12a system was used to simultaneously cleave the polymerase
chain reaction (PCR) or loop-mediated isothermal amplification (LAMP)
amplicons and the newly developed ssDNA-FQ reporter, resulting in
fluorescence that could be easily detected in multiple platforms,
especially on cheap and portable blue or UV light transilluminators.
This specific cleavage with fluorescence reveals the presence of the
amplicon and confirms its identity, thereby preventing false-positive
test results from nonspecific amplicons. This method is also uninterfered
by the presence of large amounts of irrelevant background DNA and
displays no cross-reactivity with other porcine DNA or RNA viruses.
When coupled with LAMP, the Cas12a platform can detect a plasmid containing
p72 with as few as 2 copies/μL reaction. Our results indicate
that the CRISPR-Cas12a enhanced fluorescence assay coupled with nucleic
acid amplification is robust, convenient, specific, confirmatory,
affordable, and potentially adaptable for ASF diagnosis.
Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it
could build new capacities for molecular point-of-care testing (POCT). In this study, we
evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher
(ssDNA-FQ) reporters for use with clusters of regularly spaced short palindrome repeats
(CRISPR)/Cas12a-based visual colorimetric assays. Among them, nine ssDNA-FQ reporters
were found to be suitable for direct visual colorimetric detection, with especially very
strong performance using ROX-labeled reporters. We optimized the reaction concentrations
of these ssDNA-FQ reporters for a naked-eye read-out of assay results (no transducing
component required for visualization). In particular, we developed a convolutional
neural network algorithm to standardize and automate the analytical colorimetric
assessment of images and integrated this into the MagicEye mobile phone software. A
field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a
ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was
established. We deployed RAVI-CRISPR in a single tube toward an instrument-less
colorimetric POCT format that required only a portable rechargeable hand warmer for
incubation. The RAVI-CRISPR was successfully used for the high-sensitivity detection of
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever
virus (ASFV). Our study demonstrates this RAVI-CRISPR/MagicEye system to be suitable for
distinguishing different pathogenic nucleic acid targets with high specificity and
sensitivity as the simplest-to-date platform for rapid pen- or bed-side testing.
A series of 4-phenyl-acyl-substituted 3-(2,5-dimethylphenyl)-4-hydroxy-1-azaspiro[4.5]dec-3-ene-2,8-dione derivatives were designed and synthesized, and their structures were characterized using (1)H NMR (or (13)C NMR), mass spectrometry, and elemental analysis. The bioactivities of the new compounds were evaluated. These compounds exhibited good inhibition activities against bean aphids (Aphis fabae) and carmine spider mite (Tetranychus cinnabarinus), and 4-phenyl acyl esters showed stronger bioactivity than 4-arylesterases and alkyl esters. The results showed that compound 8-I-e, which contains a para-methoxy group on the phenyl acyl, and compound 8-I-m, which contains a para-trifluoromethyl group on the phenyl acyl, displayed potent insecticidal activity against A. fabae and T. cinnabarinus respectively. The insecticidal activity showed a clear structure-activity relationship, confirming the importance of the flexible bridge. The DFT/B3LYP/6-31(d) level method was used to calculate molecular geometries and electronic descriptors. These factors included total energy, charge distribution, and the linear orbital level of the title compounds. Quantitative structure-activity relationship studies were performed on these compounds using quantum-chemical and physicochemical parameters as independent variables and insecticidal activity as a dependent variable. Insecticidal activity was most closely correlated (r > 0.8) with quantum chemical and physicochemical parameters.
This paper deals with sparse signal reconstruction by designing a discrete-time projection neural network. Sparse signal reconstruction can be converted into an L₁-minimization problem, which can also be changed into the unconstrained basis pursuit denoising problem. To solve the L₁-minimization problem, an iterative algorithm is proposed based on the discrete-time projection neural network, and the global convergence of the algorithm is analyzed by using Lyapunov method. Experiments on sparse signal reconstruction and several popular face data sets are organized to illustrate the effectiveness and performance of the proposed algorithm. The experimental results show that the proposed algorithm is not only robust to different levels of sparsity and amplitude of signals and the noise pixels but also insensitive to the diverse values of scalar weight. Moreover, the value of the step size of the proposed algorithm is close to 1/2, thus a fast convergence rate is potentially possible. Furthermore, the proposed algorithm achieves better classification performance compared with some other algorithms for face recognition.
Porcine enteric coronaviruses have caused immense economic losses to the global pig industry, and pose a potential risk for cross-species transmission. The clinical symptoms of the porcine enteric coronaviruses (CoVs) are similar, making it difficult to distinguish between the specific pathogens by symptoms alone. Here, a multiplex nucleic acid detection platform based on CRISPR/Cas12a and multiplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of four diarrhea CoVs: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). With this strategy, we realized a visual colorimetric readout visible to the naked eye without specialized instrumentation by using a ROX-labeled single-stranded DNA-fluorescence-quenched (ssDNA-FQ) reporter. Our method achieved single-copy sensitivity with no cross-reactivity in the identification and detection of the target viruses. In addition, we successfully detected these four enteric CoVs from RNA of clinical samples. Thus, we established a rapid, sensitive, and on-site multiplex molecular differential diagnosis technology for porcine enteric CoVs.
A novel support vector machine (SVM) classification model was established for distinguishing potent and weak/inactive insecticides. Classification model-based rational design of novel tetronic acid derivatives was then performed to choose the preferable site of spirotetramat for chemical modification.Afterwards, eleven C5 0 -oxime ether-derived spirotetramat analogues, which are indicated as "potent class", were synthesized and validated by biological assays, revealing that theoretical estimates are significantly consistent with experimental activities of these compounds. To be of interest, the most promising compound 91b exhibited excellent insecticidal and acaricidal activities. Moreover, molecular docking was further implemented to propose the possible interaction mode of acetyl-CoA carboxylase (ACCase) and compounds 91b, 91j, and 91k, providing some important and useful guidelines for further development.Scheme 1 The establishment of SVM classification model and its application in rational design of novel C5 0 -substituted spirotetramat derivatives as potent insecticidal and acaricidal agents. 49196 | RSC Adv., 2015, 5, 49195-49203 This journal is
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