A novel high sensitive, specificity duplex enzyme-activated differentiating probes PCR method for the SNP detection and differentiation of MS-H vaccine strains from wild-type Mycoplasma synoviae strains.

Mycoplasma synoviae (MS) is a contagious pathogen that poses a significant threat to the poultry industry. Detection plays an important role in the prevention and control of MS, particularly in differentiating between wild-type MS and live attenuated vaccine strains for vaccination selection and culling of animals with wild-type only. The live attenuated ts+ vaccine strain MS-H is recognized as the most effective and widely used vaccine. In this study, we have developed a method called double enzyme-activated differentiation probes PCR (DEA-probes PCR) for the differentiation of MS-H vaccine strain from wild-type strain by targeting the single nucleotide polymorphism (SNP) of the 367th nucleotide in the Obg gene sequence. We developed 2 modified probes with the ribonucleotide insert. When the probe perfectly complements with the target, the ribonuclease H2 (RNase H2) will cleave the ribonucleotide, resulting in the generation of fluorescent signal. With a detection limit of 5.8 copies/µL, the DEA-probes PCR method demonstrates 100% specificity in distinguishing wild-type MS from MS-H strains in 1 h. The method demonstrated great performance in real application of 100 superior palate cleft swab samples from chickens in poultry farms. Twenty-eight samples were detected as MS positive, consistent with the results of the Chinese industry standard method. Additionally, our method was able to distinguish 19 wild-type MS strains from 9 MS-H vaccine strains. The DEA-probes PCR method is rapid, specific and sensitive for SNP detection, overcoming the misidentification in MS detection and differentiation. It can be also applied to the differentiation of infected from vaccinated animals (DIVA) for other pathogens.

General room-temperature Suzuki-Miyaura polymerization for organic electronics.

π-Conjugated polymers (CPs) have broad applications in high-performance optoelectronics, energy storage, sensors and biomedicine. However, developing green and efficient methods to precisely synthesize alternating CP structures on a large scale remains challenging and critical for their industrialization. Here a room-temperature, scalable and homogeneous Suzuki-Miyaura-type polymerization reaction is developed with broad generality validated for 24 CPs including donor-donor, donor-acceptor and acceptor-acceptor connectivities, yielding device-quality polymers with high molecular masses. Furthermore, the polymerization protocol significantly reduces homocoupling structural defects, yielding more structurally regular and higher-performance electronic materials and optoelectronic devices than conventional thermally activated polymerizations. Experimental and theoretical studies reveal that a borate transmetalation process plays a key role in suppressing protodeboronation, which is critical for large-scale structural regularity. Thus, these results provide a general polymerization tool for the scalable production of device-quality CPs with alternating structural regularity.

High Levels of Antibiotic Resistance in MDR-Strong Biofilm-Forming Salmonella Typhimurium ST34 in Southern China.

Salmonella enterica subsp. enterica serovar Typhimurium (S. typhimurium) is an important zoonotic pathogen with important public health significance. To understand S. typhimurium's epidemiological characteristics in China, multi-locus sequence typing, biofilm-forming ability, antimicrobial susceptibility testing, and resistant genes of isolates from different regions and sources (human, food) were investigated. Among them, ST34 accounted for 82.4% (243/295), with ST19 ranking second (15.9%; 47/295). ST34 exhibited higher resistance levels than ST19 (p < 0.05). All colistin, carbapenem, and ciprofloxacin-resistant strains were ST34, as were most cephalosporin-resistant strains (88.9%; 32/36). Overall, 91.4% (222/243) ST34 isolates were shown to have multidrug resistance (MDR), while 53.2% (25/47) ST19 isolates were (p < 0.05). Notably, 97.8% (45/46) of the MDR-ACSSuT (resistance to Ampicillin, Chloramphenicol, Streptomycin, Sulfamethoxazole, and Tetracycline) isolates were ST34, among which 69.6% (32/46) of ST34 isolates were of human origin, while 30.4% (14/46) were derived from food (p < 0.05). Moreover, 88.48% (215/243) ST34 showed moderate to strong biofilm-forming ability compared with 10.9% (5/46) ST19 isolates (p < 0.01). This study revealed the emergence of high-level antibiotic resistance S. typhimurium ST34 with strong biofilm-forming ability, posing concerns for public health safety.

An Efficient Direct Arylation Polycondensation via C-S Bond Cleavage.

The direct arylation polycondensation (DArP) has become one of the most important methods to construct conjugated polymers (CPs). However, the homocoupling side-reactions of aryl halides and the low regioseletive reactivities of unfunctionalized aryls hinder the development of DArP. Here, an efficient Pd and Cu co-catalyzed DArP was developed via inert C-S bond cleavage of aryl thioethers, of which robustness was exemplified by over twenty conjugated polymers (CPs), including copolymers, homopolymers, and random polymers. The capture of oxidative addition intermediate together with experimental and theoretic results suggested the important role of palladium (Pd) and copper (Cu) co-catalysis with a bicyclic mechanism. The studies of NMR, molecular weights, trap densities, two-dimensional grazing-incidence wide-angle X-ray scattering (2D-GIWAXS), and the charge transport mobilities revealed that the homocoupling reactions were significantly suppressed with high regioselectivity of unfunctionalized aryls, suggesting this method is an excellent choice for synthesizing high performance CPs.

Organic Optoelectronic Synapses for Sound Perception.

The neuromorphic systems for sound perception is under highly demanding for the future bioinspired electronics and humanoid robots. However, the sound perception based on volume, tone and timbre remains unknown. Herein, organic optoelectronic synapses (OOSs) are constructed for unprecedented sound recognition. The volume, tone and timbre of sound can be regulated appropriately by the input signal of voltages, frequencies and light intensities of OOSs, according to the amplitude, frequency, and waveform of the sound. The quantitative relation between recognition factor (ζ) and postsynaptic current (I = Ilight - Idark) is established to achieve sound perception. Interestingly, the bell sound for University of Chinese Academy of Sciences is recognized with an accuracy of 99.8%. The mechanism studies reveal that the impedance of the interfacial layers play a critical role in the synaptic performances. This contribution presents unprecedented artificial synapses for sound perception at hardware levels.

A novel linear displacement isothermal amplification with strand displacement probes (LDIA-SD) in a pocket-size device for point-of-care testing of infectious diseases.

Nucleic acid amplification is crucial for disease diagnosis, especially lethal infectious diseases such as COVID-19. Compared with PCR, isothermal amplification methods are advantageous for point-of-care testing (POCT). However, complicated primer design limits their application in detecting some short targets or sequences with abnormal GC content. Herein, we developed a novel linear displacement isothermal amplification (LDIA) method using two pairs of conventional primers and Bacillus stearothermophilus (Bst) DNA polymerase, and reactions could be accelerated by adding an extra primer. Pseudorabies virus gE (high GC content) and Salmonella fimW (low GC content) genes were used to evaluate the LDIA assay. Using strand displacement (SD) probes, a LDIA-SD method was developed to realize probe-based specific detection. Additionally, we incorporated a nucleic acid-free extraction step and a pocket-sized device to realize POCT applications of the LDIA-SD method. The LDIA-SD method has advantages including facile primer design, high sensitivity and specificity, and applicability for POCT, especially for amplification of complex sequences and detection of infectious diseases.

Retina-Inspired Organic Photonic Synapses for Selective Detection of SWIR Light.

Photonic synapses with the dual function of optical signal detection and information processing can simulate human visual system. However, photonic synapses with selective detection of short-wavelength infrared (SWIR) light have never been reported, which can not only broaden the human vision region but also integrate neuromorphic computation and infrared optical communication. Here, organic photonic synapses based on a new donor-acceptor copolymer P1 are fabricated, which exhibit excellent synaptic characteristics with selective detection for SWIR and extremely low energy consumption (2.85 fJ). The working mechanism is rooted in energy level barriers and unbalanced charge transportation. Moreover, these photonic synapses demonstrate excellent performance in multi-signal logic editing, letter imaging and memory with noise reduction function. This contribution provides ideas of constructing selective-response synapses for artificial visual system and neuromorphic computing.

A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) causes watery diarrhea, vomiting, and 30-40% mortality in newborn piglets. A simple, rapid, and sensitive method for PDCoV detection is valuable in its surveillance and control. Here, we developed a novel, cleaved probe-based reverse transcription loop-mediated isothermal amplification (CP-RT-LAMP) method for PDCoV detection. A cleaved probe with a ribonucleotide insertion that targeted the N gene of PDCoV was designed. During the reaction, the enzyme ribonuclease H2 is activated only when the cleaved probe is perfectly complementary to the template, leading to the hydrolytic release of a quencher moiety and signal output. This method can be easily used on a real-time fluorescence quantitative equipment or an on-site isothermal instrument combined with a smartphone. The specificity assay showed no cross-reactivity with other porcine enteric pathogens. This method had a detection limit of 25 copies/µL, suggesting comparable sensitivity with reverse transcription quantitative PCR (RT-qPCR). In detecting 100 clinical samples (48 fecal swab specimens and 52 intestinal specimens), the detection rate of the CP-RT-LAMP method (26%) was higher than that of RT-qPCR (17%). Thus, it is a highly specific and sensitive diagnostic method for PDCoV, with a great application potential for monitoring PDCoV in the laboratory or point-of-care testing in the field.

Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus.

African Swine Fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and lethal viral disease of pigs. However, commercial vaccines are not yet available, and neither are drugs to prevent or control ASF. Therefore, rapid, accurate on-site diagnosis is urgently needed for detection during the early stages of ASFV infection. Herein, a cleaved probe-based loop-mediated isothermal amplification (CP-LAMP) detection method was established. Based on the original primer sets, we targeted the ASFV 9GL gene sequence to design a probe harboring a ribonucleotide insertion. Ribonuclease H2 (RNase H2) enzyme activity can only be activated when the probe is perfectly complementary, resulting in hydrolytic release of a quencher moiety, and consequent signal amplification. The method displayed robust sensitivity, with copy number detection as low as 13 copies/µL within 40 min at constant temperature (62°C). Visualization of the fluorescence product was employed using a self-designed 3D-printed visualization function cassette, and the CP-LAMP method achieved specific identification and visual detection of ASFV. Moreover, coupling the dual function cassette and smartphone quantitation makes the CP-LAMP assay first user-friendly, cost-effective, portable, rapid, and accurate point-of-care testing (POCT) platform for ASFV.

The Aryl Sulfide Synthesis via Sulfide Transfer.

Aryl sulfides are in great demands in drugs and materials sciences. To avoid using nucleophilic and noxious thiols, many efforts have been focused on exploring novel sulfide resources. Herein, a reductive Pd-catalyzed, Ni-mediated method to synthesize aryl sulfides via a sulfide transfer reaction is developed. The utility and scope of this reaction is exemplified by various aryl electrophiles and aryl sulfides. Mechanistic studies reveal two competing catalytic cycles of sulfide transfer and aryl transfer in this reaction, where the former one is favored over the later one because of the large energy barrier difference during the transmetalation. Moreover, two important chemicals are late-stage functionalized by this method, exhibiting the potential applications in drugs and materials science.

Uncertainty investigation of plume-chasing method for measuring on-road NOx emission factors of heavy-duty diesel vehicles.

The plume-chasing method has shown great advantages in measuring on-road emission factors (EFs) compared with regulatory methods like dynamometer and portable emission measurement systems (PEMS). In this study, a new on-board measurement system incorporating ultrasonic anemometers and solid-state Lidar was developed to investigate the uncertainties of on-road emission factors measured by plume-chasing method due to variables such as on-road wind velocity, chasing speed, chasing distance, and turbulent kinetic energy (TKE). A series of PEMS-chasing experiments for heavy-duty diesel vehicles (HDDVs) were conducted on both highways and local roadways in Beijing, China. Our analysis demonstrated that the differences in EF estimations between concurrent plume-chasing and PEMS measurement decreased with increasing chasing speed as a result of greater vehicle-induced TKE in the wake between HDDV and the mobile platform, whereas the effect of chasing distance on EF estimations appeared insignificant within the tested distance range (12-22 m). In the case of strong crosswinds, overprediction of chasing-based EFs was observed due to convective plume mixing from surrounding vehicular sources. The findings of this study contribute greatly to interpret emission factors measured by the plume-chasing method, and also calls for a future study to develop real-time EF correction algorithms for large-scale mobile chasing measurements.

Highly Prevalent Multidrug-Resistant Campylobacter spp. Isolated From a Yellow-Feathered Broiler Slaughterhouse in South China.

The purpose of this study was to investigate the prevalence, antimicrobial resistance, virulence genes, and genetic diversity of Campylobacter spp. along the yellow-feathered broiler slaughtering line in Southern China from December 2018 to June 2019. A total of 157 Campylobacter spp. isolates were identified from 1,102 samples (including 53.6% (75/140) of live chicken anal swab samples, 27.5% (44/160) of defeathering samples, 18.1% (29/160) of evisceration samples, 2.1% (3/140) of washing samples, 1.4% (2/140) of chilling samples, and 1.1% (4/362) of environmental samples). The prevalence of Campylobacter spp. was 14.2%, including 43.9% Campylobacter jejuni, 53.5% Campylobacter coli, and 2.5% other Campylobacter species. The highest antimicrobial resistance rate was found to be against sulfamethoxazole (138/157, 87.9%), and 90.4% (142/157) of the isolates were multidrug resistant (MDR). Examination of resistance-related genes revealed the double base mutated Thr-86-Ile, which informed ACA-TTA, with an Arg-79-Lys substitution in gyrA. Eleven virulence-associated genes (cadF, cdtA, cdtB, ciaB, flaA, imaA, dnaJ, plaA, virB11, racR, and cdtC) were also detected by a polymerase chain reaction (PCR) analysis, and cadF (81.5%) was the most prevalent. Based on an analysis of pulsed-field gel electrophoresis (PFGE) results, we found that Campylobacter spp. could be cross-contaminated throughout the entire slaughtering line. These results show that it is imperative to study the Campylobacter spp. from the yellow-feathered broiler along the slaughtering line in China to develop preventative and treatment measures for the poultry industry, as well as food safety and public health.

Characterization of the emerging multidrug-resistant Salmonella enterica serotype Kentucky ST314 in China.

Salmonella enterica serotype Kentucky (S. Kentucky) is an important Salmonella serotype with multiple sequence types (ST) with a worldwide incidence. We identified 8 STs from 180 strains of S. Kentucky, and ST314 emerged as the most commonly encountered ST. Drug susceptibility testing revealed that ST314 had multiple resistance properties, and 75.5% of the strains were resistant to three or more classes of antimicrobials. The rate of resistance to chloramphenicol, florfenicol, sulfafurazole and tetracycline were greater than 60%. The rates of ST314 resistance to quinolones were as follows: ciprofloxacin, 32.1%; nalidixic acid, 16%; and ofloxacin, 7.5%. Investigating the mechanism of quinolone resistance of ST314 revealed that mutations in the quinolone resistance-determining regions were rare, and resistance mainly occurred due to the resistance genes carried by plasmids. Only 1.9% (2/106) of ST314 strains had mutations in the quinolone resistance-determining regions (QRDR). The drug resistance genes of ST314 were primarily of plasmid-mediated quinolone resistance (PMQR). The detection rate of Salmonella genomic island 1 (SGI1) in ST314 was 12.3%. XbaI-pulsed-field gel electrophoresis revealed that S. enterica Kentucky ST314 was capable of cross-regional and cross-host transmission in China. We found ST314 to be the dominant S. Kentucky ST in China, and it carried multidrug resistance. This is the first report about the emergence of quinolone-resistant S. enterica Kentucky ST314 in China, which is different from previous reports, and the findings of the present study suggest that the mechanism of quinolone resistance in these strains are plasmid-mediated. Notably, plasmids carrying resistance genes may promote the rapid spread of ciprofloxacin resistance.

Competitive activation cross amplification combined with smartphone-based quantification for point-of-care detection of single nucleotide polymorphism.

In this study, we firstly propose a novel smartphone-assisted visualization SNP genotyping method termed competitive activation cross amplification (CACA). The mutation detection strategy depends on the ingenious design of both a start primer and a verification probe with ribonucleotide insertion through competitive combination and perfect matching with the target DNA, Meanwhile, the RNase H2 enzyme was utilized to specifically cleave ribonucleotide insertion and achieve extremely specific dual verification. Simultaneously, the results allow both colorimetric and fluorescence product dual-mode visualization by using self-designed 3D-printed dual function cassette. We validated this novel CACA by analyzing the Salmonella Pullorum rfbS gene at the 237th site, successfully solve the current bottleneck of specific identification and visual detection of this pathogen. The concentration detection limits of the plasmid and genomic DNA were 1500 copies/µL and 3.98 pg/µL, respectively, and as low as the presence of 0.1% mutant-type can be distinguished from 99.9% wild-type. Combined with a powerful hand-warmer, which can provide heating more than 60 °C for 20 h to realize power-free, dual function cassette and smartphone quantitation, our novel CACA platform firstly realizes user-friendly, cost-effective, portable, rapid, and accurate POC detection of SNP.

A one-step closed-tube enzyme-activated blocked probe assay based on SNP for rapid detection of Salmonella Pullorum.

Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) is an infectious bacterial pathogen in the poultry industry that causes systemic pullorum disease. This disease causes great losses in terms of the clinical production and quality of chicken products in breeding farms. However, an acknowledged usable rapid detection method for its specific identification has not been reported, and it is generally difficult to distinguish from fowl typhoid caused by Salmonella enterica serovar Gallinarum biovars Gallinarum. The development of a specific and rapid detection method for this pathogen is therefore needed. In the present study, we targeted the single-nucleotide mutation position 237 of the S. Pullorum rfbS gene to develop an enzyme-activated blocked probe for its clinical rapid detection. The method displayed robust specificity and reproducibility, and it achieved minimal detection limits of 21 copies/µL of copy number and 4.53 pg/µL of genomic DNA. Compared with traditional identification and PCR methods, this method performed better for the detection of 100 clinical actual samples and without false negative results. The entire process can be accomplished in a 1-step closed-tube operation, overcomes the difficulties currently associated with S. Pullorum detection, and provides a specific and rapid method with broad application potential for SNP detection.

Highly prevalent multidrug resistance and QRDR mutations in Salmonella isolated from chicken, pork and duck meat in Southern China, 2018-2019.

This study was undertaken to investigate the prevalence, serotype distribution and antimicrobial resistance in Salmonella isolated from retail meat in Southern China, and to characterize the major mechanisms that mediate the ciprofloxacin resistance of isolates. High levels of Salmonella contamination were detected in pork (67.0%), duck (50.5%) and chicken (46.2%). Thirty different serotypes were identified among 500 detected Salmonella isolates, as well as significant differences in serotypes between different retail meat samples. Notably, 405 (80.1%) isolates exhibited multidrug resistance (MDR). Meanwhile, we also found that 74 (14.8%) Salmonella isolates were resistant to ciprofloxacin and the major mechanisms underlying this resistance were investigated. The commonest mutations in gyrA S83F (40.5%) and D87N (35.1%), and in parC was T57S (71.6%) and S80I (35.1%). Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) analysis revealed that the S. Kentucky isolates that were resistant to ciprofloxacin mostly belonged to ST198 (21/23, 91.3%) and PFGE revealed the presence of various genotypes. This study identified a diversity of Salmonella serotypes and a high prevalence of multidrug resistance (MDR) among Salmonella isolated from retail meat in Southern China, which indicates that foodborne Salmonella potentially constitutes a potential food safety risk.

A Sensitive, Highly Specific Novel Isothermal Amplification Method Based on Single-Nucleotide Polymorphism for the Rapid Detection of Salmonella Pullorum.

S. Pullorum (Salmonella enterica serovar Gallinarum biovars Pullorum) is an infectious pathogen that causes the acute systemic disease called Pullorum disease in poultry. This disease causes huge losses to the poultry industry and seriously affects the yield and quality of the chicken product. It is not easily distinguishable with fowl typhoid caused by S. Gallinarum (Salmonella enterica serovar Gallinarum biovars Gallinarum), hence the development of a specific and rapid detection method for this pathogen is highly desired. In this study, we propose a novel single-nucleotide polymorphism (SNP) detection strategy termed loop primer probe-introduced loop-mediated isothermal amplification (LP-LAMP) for S. Pullorum detection. Based on the original primer sets, we targeted the nucleotide position 237 of the rfbS gene sequence to design a new modified loop-primer probe with a ribonucleotide insertion, where activity of the enzyme ribonuclease H2 (RNase H2) is only activated when the probe is perfectly complementary, leading to the hydrolytic release of a quencher moiety and thus an amplified signal. The method exhibits robust specificity and a low detection limit as the copy number and genomic DNA is 21 copies/µL and 4.92 pg/µL, respectively. This method showed great performance in real sample testing of 130 samples of embryos, livers, and anal swabs from chickens in poultry farms. The experimental results are mainly consistent with traditional identification methods and a PCR method reported in the past. However, the other two methods still contain some false negative results, while our method is without miss detection. The entire closed-tube reaction process can be accomplished within 40 min at a constant temperature (61°C) without the need for expensive instruments or a complicated operation. The LP-LAMP strategy established in this study not only overcomes the existing difficulties of S. Pullorum rapid detection, it also provides a novel, sensitive, and highly specific detection platform for SNPs that is suitable for clinical use.

A rapid novel visualized loop-mediated isothermal amplification method for Salmonella detection targeting at fimW gene.

Salmonella infection causes huge losses in the poultry industry worldwide. With the aim to prevent infectious diseases caused by Salmonella and to achieve rapid visualized Salmonella detection in poultry production, we used cresol red as an indicator to develop a novel visualized loop-mediated isothermal amplification method that targets the Salmonella fimW gene firstly in related field. The detection limit was 7.3 × 101 CFU/mL, and the method was highly specific and showed a high clinical detection rate. The entire reaction can be completed in about 40 min and only requires a water bath at 62°C, which makes the method extremely suitable for application to poultry production.