Integrating CRISPR-Cas12a and rolling circle-amplified G-quadruplex for naked-eye fluorescent "off-on" detection of citrus Alternaria.

Alternaria is a plant pathogen that spreads globally and is prone to causing citrus brown spot disease and metabolizing mycotoxins, thus seriously hindering the development of this economic crop industry. Herein, a "label-free" and "turn on" visual fluorescent assay for citrus Alternaria based on CRISPR-Cas12a and rolling circle amplification (RCA) was described. Using ssDNA complementary to RCA primer as a trans-cleavage substrate for CRISPR-Cas12a, the two systems of CRISPR-Cas12a and RCA-amplified G-quadruplex were skillfully integrated. By using a portable light source for excitation, the positive sample produced obvious red fluorescence, while the negative sample remained almost colorless, making them easy to differentiate with the naked eye. In addition, the specificity was demonstrated by distinguishing Alternaria from other citrus disease related pathogens. Moreover, the practicality was verified by analyzing cultured Alternaria and Alternaria in actual citrus leaf and fruit samples. Therefore, this method may contribute to the on-site diagnosis of Alternaria.

Determination, temporal variation and potential health risk assessment of pesticide residues in grapes from South and Southwest China.

Pesticide residues in grapes from South and Southwest China were determined using the QuEChERS procedure and UHPLC-MS/MS and GC-MS/MS methods. The 4-year monitoring and survey showed 94.6% of the 1341 samples of grapes collected from eight main production areas contained one or multiple pesticide residues (above the respective LOQs). Overall, 40 pesticides were detected, including 24 fungicides, 12 insecticides, 2 acaricides and 2 plant growth modulators, of which one pesticide was unauthorised for use in treating grapes. Two or more pesticide residues were discovered in 87.4% of the samples (above the respective LOQs), and pesticide residues in 5.7% of the samples exceeded the MRLs, such as difenoconazole, cyhalothrin, propiconazole, etc. The main risk factors affecting the safety of grape before 2019 were difenoconazole, cyhalothrin and cyazofamid. After 2019, however, the frequency of occurrence of the above pesticides significantly declined, and the banned or restricted pesticides including omethoate were not found, which was credited to the stricter supervision and management policies by local governments. Despite the high detection rates and multi-residue occurrence of pesticides in grapes, about 84% of the samples were compliant with regulatory standards. Moreover, the accumulative chronic diet risk determined from ADI is very low. This study and timely monitoring can ensure that grape growers comply with GAP and minimise the occurrence of residues.

Determination, Quality, and Health Assessment of Pesticide Residues in Kumquat in China.

Pesticide residues in kumquat fruits from China, and the quality and chronic/acute intake risks in Chinese consumers, were assessed using the QuEChERS procedure and UHPLC-MS/MS and GC-MS/MS methods. Our 5-year monitoring and survey showed 90% of the 573 samples of kumquat fruits collected from two main production areas contained one or multiple residual pesticides. Overall, 30 pesticides were detected, including 16 insecticides, 7 fungicides, 5 acaricides, and 2 plant growth modulators, of which 2 pesticides were already banned. Two or more residual pesticides were discovered in 81% of the samples, and pesticide residues in 9.4% of the samples surpassed the MRLs, such as profenofos, bifenthrin, triazophos, avermectin, spirodiclofen, difenoconazole, and methidathion. The major risk factors on the safety of kumquat fruits before 2019 were profenofos, bifenthrin, and triazophos, but their over-standard frequencies significantly declined after 2019, which was credited to the stricter supervision and management policies by local governments. Despite the high detection rates and multi-residue occurrence of pesticides in kumquat fruits, about 81% of the samples were assessed as qualified. Moreover, the accumulative chronic diet risk determined from ADI is very low. To better protect the health of customers, we shall formulate stricter organic phosphorus pesticide control measures and stricter use guidelines, especially for methidathion, triazophos, chlorpyrifos, and profenofos. This study provides potential data for the design of kumquat fruit quality and safety control guidelines and for the reduction in health risks to humans.

Simultaneous determination of the nematicide fluensulfone and its two major metabolites in soils by ultra-high performance liquid chromatography-tandem mass spectrometry.

A fast and sensitive method for simultaneously detecting nonfumigant nematicide fluensulfone (FSF) and its two major metabolites [3,4,4-trifluorobut-3-ene-1-sulfonic acid (BSA) and 5­chloro-1,3-thiazole-2-sulfonic acid (TSA)] in different types of agricultural soils (black soil, krasnozem, sierozem) was established and validated through ultra-high performance liquid chromatography-tandem mass spectrometry. The samples were prepared by a modified quick, easy, cheap, effective, rugged, and safe method. The soil samples were firstly extracted with acetonitrile/water (4/1) and then purified with multi-walled carbon nanotubes (MWCNTs). Parameters influencing purification efficiency and recoveries, such as the type and the amount of sorbent were evaluated and compared. The overall average recoveries of three target analytes in soils were in the range of 73.1%-113.9% and the relative standard deviations (including intra-day and inter-day precision) were less than 12.7%. The limit of quantification was 5 µg/kg for all three compounds. The established method was successfully applied to examine the degradation of FSF and the formation of its two major metabolites in three different types of soil, indicating its efficacy in investigating the environmental behavior of FSF in agricultural soil system.

Construction of a portable immunosensor for the sensitive detection of carbendazim in agricultural products using a personal glucose meter.

Portable and sensitive detection of carbendazim (CBD) is highly desirable for food safety and environmental protection. Herein, a portable immunosensor for the sensitive detection of CBD is proposed based on alkaline phosphatase (ALP)-labeled and secondary antibody-modified gold nanoparticles (AuNPs). The quantification is based on ALP catalyzing the dephosphorylation of glucose-1-phosphate disodium salt to generate glucose, thus converting the concentration of CBD into glucose, thereby realizing the portable detection of CBD by personal glucose meter. Benefiting from signal amplification strategy that integrates the large specific surface area of AuNPs, the enzymatic reactions of terminal deoxynucleotidyl transferase and ALP, a low detection limit of 0.37 ng/mL for CBD is achieved. When this portable method is used to analyze citrus fruit, canned citrus, and cabbage, good-consistency results are obtained with the UPLC-MS/MS method. The good performance demonstrates the great potential of this portable method for CBD monitoring in resource-poor settings.

Simultaneous determination of pyflubumide and its metabolite in vegetables and fruits by ultrahigh performance liquid chromatography-tandem mass spectrometry.

A rapid and cost-effective analytical method based on ultrahigh-performance liquid chromatography-tandem mass spectrometry was designed and verified for simultaneously monitoring the novel acaricide pyflubumide and its metabolite (pyflubumide-des(2-methyl-1oxopropyl)) in vegetables and fruits. After the extraction with acetonitrile, the samples were purified by dispersive solid-phase extraction with multi-walled carbon nanotubes. Detection of the two target analytes was achieved within 3.0 min using a positive electrospray ionization mode. The average recovery, intra-day precision, and inter-day precision of the two analytes at three spiked levels (2, 20, and 100 µg/kg) were 75.0-101.0, 0.4-4.4, and 0.6-5.3%, respectively. The limit of quantification of two compounds was 2 µg/kg, which was far below the maximum residue limits of pyflubumide in foods established by Japan and South Korea. Finally, the concentrations of pyflubumide and its metabolite in the samples were 16.6 and 7.8 µg/kg respectively, which verified the practicability and reliability of the method. The method was used to efficiently detect pyflubumide and its metabolite in real samples and was confirmed to be robust and effective for routinely analyzing both pyflubumide and its metabolite in vegetable and fruit samples.

CRISPR-Cas12a based fluorescence assay for organophosphorus pesticides in agricultural products.

Herein, we propose a sensitive fluorescent assay for organophosphorus pesticides (OPs) detection based on a novel strategy of activating the CRISPR-Cas12a system. Specifically, acetylcholinesterase (AChE) hydrolyzes acetylthiocholine into thiocholine (TCh). Subsequently, TCh induces the degradation of MnO2 nanosheets and generates sufficient Mn2+ ions to activate the Mn2+-dependent DNAzyme. Then, as the catalytic product of activated DNAzyme, the short DNA strand activates the CRISPR-Cas12a system to cleave the fluorophore-quencher-labeled DNA reporter (FQ) probe effectively; thus, increasing the fluorescence intensity (FI) in the solution. However, in the presence of OPs, the activity of AChE is suppressed, resulting in a decrease in FI. Under optimized conditions, the limits of detection for paraoxon, dichlorvos, and demeton were 270, 406, and 218 pg/mL, respectively. Benefiting from the outstanding MnO2 nanosheets properties and three rounds of enzymatic signal amplification, the proposed fluorescence assay holds great potential for the detection of OPs in agricultural products.

Determination of five pesticides in kumquat: Dissipation behaviors, residues and their health risk assessment under field conditions.

The present study was carried out to profile the dissipation patterns and residues of five pesticides (triazophos, profenofos, chlorpyrifos, etoxazole and bifenthrin) on kumquat using QuEChERS method coupled with HPLC-MS/MS. The corresponding dietary health risks were also estimated. In the method validation, satisfactory results of good linearity (r2 ≥ 0.9956), sensitivity (limits of quantification ≤0.01 mg/kg), recoveries (71.0-95.7%) with relative standard deviations (0.70-9.4%) were obtained. The half-lives of the five pesticides in kumquat were 13.6-38.5 d under field conditions according to first-order kinetics. Based on the final residue experiment, dietary exposure risks of profenofos, chlorpyrifos, etoxazole and bifenthrin were all acceptably low, with RQc and RQa values of 0.00199-0.122 and 0.00145-0.200, respectively. However, exposure intake of triazophos posed unacceptable acute and chronic health risks for Chinese residents, especially for children with RQa and RQc up to 4.25 and 2.19. Forbidden use suggestion of triazophos and recommended MRLs of profenofos and bifenthrin were put forward in kumquat for safe production and consumption. This work was significant in providing guidance on appropriate application and MRL establishment of pesticides in kumquat.

A multicolor enzyme-linked immunoassay method for visual readout of carbendazim.

Enzyme-linked immunosorbent assay (ELISA) with high specificity and sensitivity is one of the most popular techniques for detecting carbendazim (CBD), a commonly used benzimidazole fungicide in agriculture. However, the traditional ELISA based on the horseradish peroxidase (HRP)-3,3',5,5'-tetramethylbenzidine (TMB) system for CBD only displays the yellow color of TMB2+ from deep to light, making it difficult for the naked eye to judge whether CBD in fruits and vegetables exceeds the maximum residue limit. In this article, we intend to improve the traditional ELISA method to establish a multicolor signal output ELISA to achieve visual semiquantitative detection of CBD. This method is based on the optical properties of gold nanorods (AuNRs). After introducing AuNRs into TMB2+ solution, which was produced by the HRP-TMB system of traditional ELISA, AuNRs were quickly etched by TMB2+. Consequently, the longitudinal localized surface plasmon resonance peak of AuNRs shows a clear blue shift and a vivid color change. Different concentrations of CBD generate different amounts of TMB2+, which in turn leads to different etching degrees of AuNRs, and ultimately results in a rainbow-like color change. As a result, CBD from 0.08 to 100 ng mL-1 can be easily distinguished by the naked eye, which does not require any large instruments. Moreover, the colors displayed by 0.49 ng mL-1 (purple) and 0 ng mL-1 (pink) are significantly different from each other. It should be noted that 0.49 ng mL-1 is far below the most stringent maximum residue limit of CBD in the world. Additionally, the quantitative determination of CBD spiked in canned citrus, citrus fruits, chives, and cabbage samples showed satisfactory recoveries. The good performance of the AuNR-based ELISA makes it have a wide range of application prospects in food safety and international trade.

A sensitive and practical ELISA for analyzing naringenin in pummelo and herb samples.

Naringenin, a flavonoid compound found in pummelo, is a key biological active compound in some traditional Chinese medicines, including Citri reticulatae pericarpium, Citri reticulatae pericarpium viride, Aurantii fructus immaturus, and Aurantii fructus. These Chinese medicinal preparations are the peels or immature fruits of certain citrus species. Aiming at detecting naringenin in complex matrices such as pummelo and traditional Chinese medicines, we put forward a sensitive and practical indirect competitive enzyme-linked immunosorbent assay (icELISA) based on anti-naringenin monoclonal antibodies (anti-Nar-mAbs). The median inhibitory concentration (IC50) was 4.43 ng/mL, and the working range was 1.15-15.81 ng/mL. The findings of the icELISA for the analysis of naringenin in pummelo and herb samples had a good correlation with the ultra performance liquid chromatography (UPLC) methodology and showed good accuracy and reproducibility. These data demonstrated that the developed icELISA is reliable, accurate, and suitable for detecting naringenin in pummelo and traditional Chinese medicines.

Portable and quantitative detection of carbendazim based on the readout of a thermometer.

The detection of carbendazim (CBZ) is important for food safety and human health. However, most current analytical methods require large instruments and highly trained operators. In order to solve this problem, herein, an innovative portable and quantitative photothermal assay platform relying on a thermometer readout for the detection of CBZ has been developed. Gold nanoparticles (AuNPs), which exhibit a strong distance-dependent photothermal effect under specific laser irradiation, were utilized as indicators. The CBZ aptamer was introduced to protect AuNPs from salt-mediated aggregation. When CBZ is present, the binding event between CBZ and aptamer leads to the loss of the aptamer protective effect on AuNPs, and AuNP aggregation occurs. Under 650-nm laser irradiation, the increase in temperature associated with an AuNP-dependent photothermal effect is highly related to the CBZ concentration. Having the advantages of user-friendliness, low cost, quick response, and portability, this method has great potential for on-site applications.

Occurrence, temporal variation, quality and safety assessment of pesticide residues on citrus fruits in China.

Citrus is highly consumed in China and considered a major source of pesticide carrier in human diet. In this paper, pesticide residues in mandarins, tangerines and oranges from China were evaluated, as well as the quality and long-term dietary risks for the Chinese consumers. Temporal variations for eight MRL non-compliant pesticides were also investigated. 106 pesticides were analyzed using QuEChERS procedure and UPLC-MS/MS, GC-MS and GC methods. Forty different pesticides were detected in 86% of the 2922 samples from 2013 to 2018. Chlorpyrifos was the most frequently detected pesticide with a detection rate of 40%. Residues of eight pesticides in 3.8% of the total samples exceeded their MRLs. The most severely excessive pesticide was bifenthrin with 283% MRL. The occurrences and exceeding rates of eight pesticides presented clear temporal variations during the six harvesting years. Isocarbophos, carbofuran and triazophos were the main risk factors threatening the citrus safety pre-2015, whereas profenofos and bifenthrin gradually became dominant after 2016, coinciding with government control measures. The index of quality for residues (IqR) levels of 88% of the samples were below 1.0, which indicated a satisfactory quality of citrus fruits in China, although 70% of the samples contained two or more residues. The cumulative chronic dietary risks were acceptable for both the Chinese general population and children and would not pose health risks. However, more strictly enforced control measures for methidathion and isocarbophos, and reduced usage of triazophos, quinalphos, profenofos and bifenthrin should be pursued to further improve the safety of citrus fruits.

Facile and sensitive fluorescence sensing of alkaline phosphatase activity using NMM/G-quadruplex.

Herein, we are presenting an approach that utilizes the λ exonuclease (λ exo) cleavage reaction in combination with the formation of G-quadruplex, thereby providing a label-free fluorometric tool for simply and accurately determining alkaline phosphatase (ALP) activity and inhibition. A hairpin probe (HP) with 5'-phosphoryl termini and 3'-end containing a G-rich region, is designed. Taking advantage of the efficient enzyme reactions, namely the λ exo cleavage reaction, the G-rich DNA fragment is released from HP and folds into a stable G-quadruplex in the presence of potassium ions, thus greatly enhance the fluorescence of N-methyl mesoporphyrin IX (NMM) (a specific G-quadruplex binder). However, in the presence of ALP, the 5'-phosphoryl of the HP is dephosphorylated. The yielding 5'-hydroxyl end product hampers the λ exo cleavage reaction. HP maintains its stem-loop structure. Thus the formation of the G-quadruplex is prohibited, and this results in weak fluorescence of NMM. The fluorescence intensity exhibits a linear correlation to ALP concentration in the range of 1-50 U/L with a detection limit of 0.75 U/L. Additionally, inhibition effect of sodium orthovanadate has also been investigated. This study offers a simple yet sensitive method for ALP activity assay.

Dissipation and residue of forchlorfenuron in citrus fruits.

Field trials were carried out in three provinces of China to study the dissipation and residue of forchlorfenuron in citrus fruits. The results had shown that the degradation rate of forchlorfenuron in citrus fruits followed the first-order kinetics equation C = A∙eBt. The half-lives of forchlorfenuron were 15.8-23.0 days, the final residues of forchlorfenuron in pulp were all ≤0.002 mg/kg, and most of the residues were concentrated in the peel. The risk assessment revealed that no significant potential health risk would be induced by forchlorfenuron in citrus fruits. Therefore, it could be safe to apply forchlorfenuron in citrus fruits, and the results of this study could also be regarded as a reference to the setting of maximum residue limit for forchlorfenuron in citrus fruits in China.