The synergistic effect of multiple mycotoxins in cereals increases their toxicity. Therefore, the simultaneous detection of multiple mycotoxins in cereals is of great importance. Exonuclease III (Exo III) assisted electrochemical aptasensor has been used for mycotoxin detection, but simultaneous detection of two mycotoxins has not been previously reported. By utilizing Exo III assisted strand displacement, this study developed an electrochemical aptasensor strategy for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA). This method demonstrates excellent detection of AFB1 and OTA within the range of 0.001-500 ng mL-1, with detection limits as low as 0.229 pg mL-1 for AFB1 and 0.564 pg mL-1 for OTA. The electrochemical aptasensor was successfully applied to analyze grain samples, and the sensor has good specificity, stability and repeatability. The adapter provides simultaneous detection of AFB1 and OTA in food in a fast, simple and highly sensitive platform.

Kanamycin (KAN) has the potential to cause significant harm to human health, so the KAN residue present in food should be subject to strict regulation and control. Therefore, we constructed an enzyme-free ratiometric electrochemical aptasensor based on entropy-driven strand displacement reaction (ESDR) for sensitive detection of KAN in food. In the presence of KAN, it binds specifically to the aptamer, causing KAN-sensitive aptamer-tDNA (A-T) complex dissociation and the release of the tDNA, which triggers ESDR and results in an IFc/IMB maximum. In the absence of KAN, the release of the tDNA is not possible, leading to minimal IFc/IMB value. The aptasensor has a LOD of 74 fg/mL, enabling trace detection of KAN. In addition, the aptasensor demonstrated high specificity for KAN and remarkable reproducibility (RSD: 2.455%), which can be utilised for evaluating KAN concentrations in real samples. Moreover, the recovery rate of KAN in food samples ranged from 93.4% to 105.4%, suggesting that the ratiometric electrochemical aptasensor holds great potential in food safety detection.

The food contamination with Ochratoxin A (OTA) has highlighted the need to create precise, sensitive, and convenient techniques. Herein, we proposed a label-free and immobilization-free ratiometric homogeneous electrochemical aptasensor based on dual catalytic hairpin self-assembly (CHA) for OTA detection. Methylene blue (MB) and ferrocene (Fc) in solution were utilized as label-free signaling molecules, generating a response signal (IMB) and a reference signal (IFc), respectively. The ratio of IMB/IFc was utilized as a measure to quantify OTA. Dual CHA was exploited to increase the ratiometric signal and enhance the amplification efficiency. This aptasensor achieved trace-level detection for OTA over a linear range of lower concentrations (1.0 × 10-3 ng/mL-1.0 × 103 ng/mL) with LOD of 92 fg/mL. The aptasensor was successfully applied to detect OTA in cereal and wine, with comparable results of HPLC-MS/MS. This strategy provided a viable platform for rapid, sensitive, and accurate detection of OTA in food.

Listeria monocytogenes (L. monocytogenes), as one of the most common foodborne pathogens, has caused many foodborne diseases, posing a major threat to food and public health safety. Therefore, a novel electrochemical biosensor was proposed based on the combination of saltatory rolling circle amplification (SRCA) and nicking enzyme mediated amplification (NEMA) for detecting Listeria monocytogenes. When L. monocytogenes was present, abundant double-stranded DNA (dsDNA) were produced by SRCA reaction. SRCA products could be specifically recognized and cleaved by Nb.BsmI. SRCA-NEMA was performed to generate abundant single-stranded DNA (ssDNA) with the action of Bst DNA polymerase and Nb.BsmI. The hydrosulfuryl-modified hairpin DNA (HShpDNA) was immobilized on the electrode surface and G-quadruplex was used as signaling molecule. The current signal increased with target DNA concentration and the ratio of the current could be calculated as the result. Subsequently, the detection linear range for L. monocytogenes genomic DNA was 5.4 x 100-5.4 x 107 fg/mu L, with the detection limit of 2.13 fg/mu L. In comparison with ISO 11290-1:2017 method, the relative sensitivity, relative specificity and relative accuracy of the developed method were 100 %, 97.8 % and 98 %, respectively. Overall, the developed method provides an outstanding platform for sensitive detection of L. monocytogenes in food.

Listeria monocytogenes (L. monocytogenes) is one of the most prevalent foodborne pathogens. It causes intestinal infections as well as severe meningitis and septicemia, representing a significant risk to food safety and human health. Consequently, the present study proposes a novel method based on the combination of saltatory rolling circle amplification (SRCA) technology and Safranine O for the rapid, sensitive and visual detection of L. monocytogenes in food. In the presence of L. monocytogenes, the SRCA amplification reaction yields copious quantities of double-stranded DNA. Following the addition of Safranine O and the oxidant, Safranine O is oxidized to form Safranine O oligomer. The electrostatic interactions between the positively charged Safranine O oligomers and the negatively charged SRCA amplification product result in the formation of dark red visible aggregates, the absorbance value also decreases with the formation of aggregates. The method has low detection limit, high specificity, straightforward and time efficient (DNA amplification for 40 min, aggregation for 10 min). The visual qualitative detection limit was 2.7 × 101 CFU/mL, and the limit of detection quantitative detection was 3.4 CFU/mL. In comparison with the SRCA electrophoresis detection method, the visual detection limit was enhanced by 10 times. Comparison with ISO method, the sensitivity, specificity and accuracy of the method for the detection of real samples were 100

A new property of Bst DNA polymerase was discovered by our team, which led to the establishment of a new isothermal amplification method for SRCA nucleic acid, which was applied to food safety detection, investigated the amplification mechanism, and the SRCA version 1.0 amplification mechanism was proposed. This study proposes a new SRCA amplification mechanism (version 2.0) by using the autosynthetic sequences CDG2040 and CDG2068, as well as the Listeria monocytogenes hlyA gene and Vibrio parahaemolyticus toxR gene as target sequences to further investigate the amplification mechanism. The initiation of the SRCA reaction depends primarily on the spatial structure of the non-closed circle formed by the discontinuous complementation of the bases on both sides of the target sequence. The DNA fragments that connect the target sequences in the SRCA reaction are the sum of the complementary sequences of adjacent partial sequences on both sides of the target sequence.

Aflatoxin B1 is a potent carcinogen widely found in agricultural products and feed. Therefore, investigating the development of convenient, sensitive, and precise techniques for detecting AFB1 is critical. This work proposes a ratiometric electrochemical aptasensor based on DNAzyme-driven tripedal DNA walker for enzyme-free AFB1 detection. The design of multiple legs reduces the moving time on the electrode surface and enhances the value of signal response. In this approach, the tagging of ferrocene and methylene blue as signal switch molecules diminishes the impact of interferences and significantly enhances the reproducibility (RSD: 2.825%). Meanwhile, the tripedal DNA walker and DNAzyme dual signal amplification enabled the sensor to detect AFB1 traces in cereals.We achieved trace detection of AFB1 with a detection limit of 61 fg/mL and a wide linear range from 0.0001 to 10 ng/mL. Furthermore, the aptasensor success rate was comparable to that of HPLC-PMP. The results demonstrate enormous potential for detection. Furthermore, the method can detect various targets by altering the target aptamer, displaying attractive scalability. This suggests its tremendous prospective in detecting mycotoxins in food in the future.

目的:通过综述神经特异性烯醇化酶(neuron-specific enolase,NSE)检测方法研究进展,为 NSE灵敏准确定量检测方法开发提供有力解决方案.方法:结合文献和数据库搜索,总结NSE的结构特征与基本性质,系统介绍近年来基于不同配体的NSE检测方法与技术的研究进展.结果:作为目前最可靠的小细胞肺癌诊断标志物,NSE已被应用于开发多种检测方法与技术,并被应用于临床中肺癌的诊断、预后及疗效评估.结论:目前尚无统一的金标准方法来检测NSE,但已经开发出了多种基于不同配体的检测方法,它们可以用于恶性肿瘤等重大疾病的早期诊断、病情监控和预后评估,为临床提供技术支持.

A highly purified and bioactive immunoglobulin G monoclonal antibody against receptor-binding domain of SARS-CoV-2 (RBD-IgG-MAb) has been accurately quantified by amino acid determination using isotope dilution liquid chromatography–mass spectrometry. Absolute quantification of RBD-IgG-MAb was achieved by averaging 4 amino acid certified reference materials, which allows the quantitative value (66.1 ± 5.8 μg/L) to be traced to SI unit (mol). Afterwards, the RBD-IgG-MAb was employed as control and calibration compound for the development of a point-of-care testing (POCT) system based on colloidal gold lateral flow immunoassay, which aimed to rapidly and accurately detect the level of protective RBD-IgG after vaccination. Under the detection parameters, a sigmoidal curve has been plotted between signal intensity and the logarithmic concentration for quantitative detection with the limit of detection of about 0.39 μg/mL. The relative standard deviations of intra-assay and inter-assay were lower than 2.3

Colorimetry has emerged as a promising option for pathogenic detection. Herein, a novel method based on saltatory rolling circle amplification (SRCA) combined with photosensitization colorimetric assay (SRCA-C) was developed for rapid and sensitive detection of Salmonella in food. The target identification and signal amplification are realized by SRCA to produce a large amount of double-stranded DNA. Subsequently, SYBR Green I as photosensitizer and 3,3',5,5"-tetramethylbenzidine (TMB) as colorimetric oxidase substrate are added to the colorimetric reaction system. The photosensitization reaction occurs under cyan LED irradiation, and TMB is oxidized to convert the double-stranded DNA signal into absorbance signal, thus realizing the detection of Salmonella. This colorimetric method benefits from convenience (label-free and no requirement of costly and complex equipment), short detection time (DNA amplification for 50 min and color development for 15 min) and high sensitivity (detected as low as 13 CFU/mL of Salmonella in pure culture). Five target strains and 10 nontarget strains were used to validate the specificity of the method. The recovery of Salmonella in the spiked milk samples ranged from 98.4% to 118.4%. Compared with the standard culture method, this method was 100% sensitivity, 98.2% specificity, and 98.3% accuracy for the detection of the real samples. Overall, this method highlights the importance for convenient, rapid, and sensitive detection of Salmonella in food.

Immunochromatographic assay (ICA) plays an important role in in vitro diagnostics because of its simpleness, convenience, fastness, sensitivity, accuracy, and low cost. The employment of magnetic nanoparticles (MNPs), possessing both excellent optical properties and magnetic separation functions, can effectively promote the performances of ICA. In this study, an ICA based on MNPs (MNP-ICA) has been successfully developed for the sensitive detection of carcinoembryonic antigen (CEA). The magnetic probes were prepared by covalently conjugating carboxylated MNPs with the specific monoclonal antibody against CEA, which were not only employed to enrich and extract CEA from serum samples under an external magnetic field but also used as a signal output with its inherent optical property. Under the optimal parameters, the limit of detection (LOD) for qualitative detection with naked eyes was 1.0 ng/mL, and the quantitative detection could be realized with the help of a portable optical reader, indicating that the ratio of optical signal intensity correlated well with CEA concentration ranging from 1.0 ng/mL to 64.0 ng/mL (R-2 = 0.9997). Additionally, method comparison demonstrated that the magnetic probes were beneficial for sensitivity improvement due to the matrix effect reduction after magnetic separation, and the MNP-ICA is eight times higher sensitive than ICA based on colloidal gold nanoparticles. The developed MNP-ICA will provide sensitive, convenient, and efficient technical support for biomarkers rapid screening in cancer diagnosis and prognosis.

It is urgently necessary to develop convenient, reliable, ultrasensitive and specific methods of ochratoxin A determination in food safety owing to its high toxicity. In the present study, an ultrasensitive and labeled-free fluorescent aptamer sensor combining real-time fluorescence with strand displacement amplification (SDA) was fabricated for the determination of OTA. In the presence of OTA, the OTA–aptamer combines with OTA, thus opening hairpins. Then, SDA primers specifically bind to the hairpin stem, which is used for subsequent amplification as a template. SDA amplification is initiated under the action of Bst DNA polymerase and nicking endonuclease. The amplified products (ssDNA) are dyed with SYBR Green II and detected with real-time fluorescence. The method has good linearity in the range of 0.01–50 ng mL−1, with the lowest limit of detection of 0.01 ng mL−1. Additionally, the fluorescent aptamer sensor shows outstanding specificity and reproducibility. Furthermore, the sensor shows excellent analytical performance in the artificial labeled detection of wheat and oat samples, with a recovery rate of 96.1~100%. The results suggest that the developed sensor has a promising potential application for the ultrasensitive detection of contaminants in food.

As a marine food-borne pathogen, Vibrio parahaemolyticus (V. parahaemolyticus) can cause human gastrointestinal disorders and pose a serious threat to food safety and public health worldwide. Using a portable scanner, herein, a real-time fluorescence saltatory rolling circle amplification (RF-SRCA) has been established for the rapid detection of V. parahaemolyticus in seafood targeting toxR gene. The RF-SRCA results could be effectively determined within 20-60 min via real-time fluorescence curve. Significant specificity of RF-SRCA was exhibited against 12 V. parahaemolyticus strains and 29 non-V. parahaemolyticus strains. The sensitivity and detection limit of V. parahaemolyticus in artificially contaminated raw oyster by RF-SRCA were 3.2 x 10(0) fg mu L-1 and 2.3 x 10(0) CFU g(-1), respectively. Compared with SRCA by electrophoresis, RF-SRCA has higher sensitivity, lower detection limit, and avoids complicated electrophoresis. Compared with visual SRCA, the RF-SRCA takes shorter time, provides more accurate results and can eliminate the risk of cross-contamination. Moreover, 236 seafood samples were investigated for V. parahaemolyticus contamination, and the results showed 100% sensitivity, 95.88% specificity and 98.31% accuracy compared with the ISO method. Therefore, RF-SRCA possesses great potential as an accurate, specific, sensitive, rapid and convenient molecular diagnostic method for V. parahaemolyticus detection from various seafoods.

The aim of this study was to establish a rapid and visual nucleic acid isothermal amplification method for the detection of viable Staphylococcus aureus (S. aureus) in pork and pork products. The method was based on saltatory rolling circle amplification (SRCA) combined with propidium monoazide (PMA). Eighteen S. aureus strains and 29 non-S. aureus strains were used for the specificity evaluation. The sensitivity and detection limit of PMA–SRCA assay through fluorescence visualization were 2.6 × 101 CFU/mL and 6.6 × 101 CFU/mL, respectively. Compared with PMA–PCR assay, the PMA–SRCA method by fluorescence visualization exhibited a 1000-fold higher sensitivity. A total of 123 pork samples were tested for S. aureus contamination, and the results indicated that the relative sensitivity, specificity and accuracy of PMA–SRCA method were 100, 99.12 and 99.19%, respectively, compared with ISO method. In conclusion, the PMA–SRCA assay can reduce the false-positive detection rate of S. aureus in pork and pork products.

本研究建立了一种叠氮溴化丙锭(PMA)与实时荧光跨越式滚环等温扩增(RF-SRCA)相结合的方法,可以高效灵敏检测乳中活的单增李斯特氏菌.以hlyA基因为靶点,设计并筛选出特异性引物,通过对PMA的工作浓度、暗孵育时间和光反应时间进行优化,确定了最佳的处理条件.此外,对该方法的特异性、灵敏度及检出限进行了分析.结果表明,该方法引物特异性良好,6株单增李斯特氏菌结果均为阳性,12株非单增李斯特氏菌结果均为阴性;当PMA工作浓度为30 μmol/L、暗孵育10 min、光反应15 min时,所建立的方法灵敏度为3.9 CFU/mL,人工污染乳制品的检出限为1.56×10 CFU/mL.综上所述,本研究所建立的PMA-RF-SRCA方法特异性强,灵敏度高,为检测食品中活的单增李斯特氏菌提供了新的思路.

Salmonella enterica serovar Typhimurium (S. enterica ser. Typhimurium) is one of the main causes of human bacterial gastroenteritis. Therefore, there is an urgent need to develop a highly specific and accurate method for S. enterica ser. Typhimurium detection in food. In the study, we constructed a ratiometric electrochemical biosensor based on saltatory rolling circle amplification (SRCA) and dual-signal electrochemical readout. The mercapto-modified β-cyclodextrin (SH-β-CD) was immobilized on the electrode surface by combining with gold nanoparticles (AuNPs) to form Au–S bond. After SRCA reaction with specially designed primers, a large number of amplified products with ferrocene (Fc) were obtained within 1 h. These products could bind to β-CD through host-guest interactions. In addition, the current had a significant change due to the insertion of methylene blue into the SRCA products. By optimizing the reaction conditions, the electrochemical biosensor displayed a wide detection range from 30 fg/μL to 30 ng/μL with a low detection limit of 15.8 fg/μL. Furthermore, by testing food samples, the ratiometric electrochemical biosensor showed consistency with the real-time fluorescence quantitative PCR (RT-qPCR). In conclusion, this electrochemical biosensor may be a promising alternative detection method for S. enterica ser. Typhimurium.

Meat adulteration is a form of economic fraud and a global issue diminishing consumer confidence. Consequently, a sensitive and reliable technique for meat species identification is needed to protect meat quality. The present work proposes a rapid, nucleic acid, isothermal amplification method, with SYBR Green I visualization called saltatory rolling circle amplification (SRCA) to identify horsemeat in beef products. Primers were designed to specifically amplify the mitochondrial cytochrome b gene regions of different lengths of horsemeat DNAs. Fresh muscle tissue samples from ten different species were used to confirm the specificity of SRCA. The sensitivity of SRCA was evaluated and found to be 6.3 × 101 fg/μL. The detection limit of SRCA was 0.01% when evaluated with artificially contaminated beef in horsemeat. Compared with conventional PCR approaches, SRCA assay achieved at least 100-fold higher sensitivity and tenfold lower detection limits. In addition, the sensitivity, specificity, and accuracy of the SRCA system were calculated to be 100.00, 98.41, and 98.48%, respectively. Consequently, the present study offers a highly sensitive and visual technique for determining meat adulteration by discriminating horsemeat.

目的 建立可视化-跨越式滚环等温扩增技术检测肉制品中鼠肉掺假的分析方法.方法 选取鼠的线粒体cyt b基因为靶基因,设计并筛选出一对引物,选择9个不同物种的新鲜肌肉组织样本为研究对象,验证跨越式滚环等温扩增技术(saltatory rolling circle isothermal amplification,SRCA)方法的特异性;测定该技术的灵敏度以及人工污染样品中鼠肉成分的检出限,验证SRCA方法的准确性.结果 SRCA荧光可视化法检测鼠肉DNA的灵敏度为7.3×100 fg/μL,与传统PCR方法相比,SRCA荧光可视化法的灵敏度提高了1000倍.在人工添加鼠肉的模拟掺假样品中,SRCA方法可检测到含量为0.01％的鼠肉.结论 SRCA技术可以灵敏、快速、准确地检测出肉制品中的鼠肉掺假成分,适用于基层单位对肉制品掺假的快速检测.

为保障转基因大豆的可追溯性以及消费者的知情权和选择权,开发快速、灵敏、准确的转基因大豆检测方法显得尤为重要.文章建立了一种用于检测转基因大豆及其制品的可视化跨越式滚环等温扩增(Saltatory rolling circle amplification,SRCA)技术.根据抗除草剂草甘膦CP4-EPSPS基因设计引物,进行特异性验证,并对该方法的灵敏度和检出限进行研究.通过检测68份实际样品,对该方法的相对敏感度、相对特异性和相对符合率进行评价.结果表明:可视化SRCA方法具有良好的特异性,其检测转基因大豆的灵敏度为8.8×100 fg/μL,是SRCA凝胶电泳法的10倍,是PCR方法的1000倍.在人工加标样品中,可视化SRCA方法的检出限为0.01％(w/w),均显著低于SRCA凝胶电泳法和PCR方法.与行业检测标准(SN/T 1204-2016)相比,可视化SRCA方法的相对敏感性、相对特异性和相对符合率分别为100.00％、98.41％、98.52％.可视化SRCA方法具有操作简便、灵敏度高、特异性强、检出限低等优点,可以灵敏、高效地检测转基因成分,有利于在检测机构的推广应用.

建立一种新型的单核细胞增生李斯特氏菌检测方法,即通过可视化跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)技术检测单核细胞增生李斯特氏菌.根据单核细胞增生李斯特氏菌hlyA基因序列设计引物,进行特异性验证,对灵敏度和人工污染样品的检出限进行测定.通过检测70种实际食品样品对SRCA方法的敏感性、特异性和符合率进行评价.结果 表明:所有单核细胞增生李斯特氏菌呈阳性结果,非单核细胞增生李斯特氏菌呈阴性结果,说明该方法特异性良好.SRCA方法的灵敏度为8.9×100 fg/μL,是传统聚合酶链式反应(polymerase chain reaction,PCR)方法的l 000倍,检出限为2.8×100 CFU/g,是传统PCR方法的1/1000.在实际样品的检测中,SRCA方法与GB 4789.30-2016《食品微生物学检验单核细胞增生李斯特氏菌》方法进行比较,其敏感性、特异性和符合率分别为100％、97.01％和97.14％.可视化SRCA技术具有操作简便、设备简单、特异性强、灵敏度高、检出限低、检测成本低等优点,适合在基层单位和中小型食品企业中推广应用.