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The urgent need for field detection of uranium in seawater is 2-fold: to provide prompt guidance for uranium extraction and to prevent human exposure to nuclear radiation. However, current methods for this purpose are largely hindered by bulky instrumentation, high costs of developed materials, and severe matrix interferences, which limit their further application in the field. Herein, we demonstrated a portable and label-free strategy for the field detection of uranyl in seawater based on the efficient photocleavage of DNA. Further experiments confirmed the generation of ultraviolet (UV) light-induced reactive oxygen species (ROS), such as O 2 and •OH, which fragmented oligomeric DNA in the presence of uranyl and UV light. Detailed studies showed that DNA significantly enhances uranyl absorption in the UV-visible region, leading to the generation of more ROS. A fluorescence system for the selective detection of uranyl in seawater was established by immobilizing two complementary oligonucleotides with the fluorescent dye SYBR Green I. The strategy of UV-induced photocleavage offers high selectivity, excellent interference immunity, and high sensitivity for uranyl, with a detection limit of 6.8 nM. Additionally, the fluorescence can be visually detected using a 3D-printed miniaturized device integrated with a smartphone. This method has been successfully applied to the on-site detection of uranyl in seawater in 18 Chinese coastal cities and along the coast of Hainan Island within 3 min for a single sample. The sample testing and field analysis results indicate that this strategy has promising potential for real-time monitoring of trace uranyl in China's coastal waters. It is expected to be utilized for the rapid assessment of nuclear contamination and nuclear engineering construction.•- and •OH, which fragmented oligomeric DNA in the presence of uranyl and UV light. Detailed studies showed that DNA significantly enhances uranyl absorption in the UV-visible region, leading to the generation of more ROS. A fluorescence system for the selective detection of uranyl in seawater was established by immobilizing two complementary oligonucleotides with the fluorescent dye SYBR Green I. The strategy of UV-induced photocleavage offers high selectivity, excellent interference immunity, and high sensitivity for uranyl, with a detection limit of 6.8 nM. Additionally, the fluorescence can be visually detected using a 3D-printed miniaturized device integrated with a smartphone. This method has been successfully applied to the on-site detection of uranyl in seawater in 18 Chinese coastal cities and along the coast of Hainan Island within 3 min for a single sample. The sample testing and field analysis results indicate that this strategy has promising potential for real-time monitoring of trace uranyl in China's coastal waters. It is expected to be utilized for the rapid assessment of nuclear contamination and nuclear engineering construction.

Invited for this issue's cover is the group of Huilei Yu at the East China University of Science and Technology. The image shows a sustainable biosynthesis route to nylon monomers from bio-based substrate α, ω-dicarboxylic acids. The Research Article itself is available at 10.1002/cssc.202301477., (© 2024 Wiley‐VCH GmbH.)

Four new indole- C -mannopyranoside alkaloids, neopetrosins A-D ( 1 - 4 ), together with one new diamine alkaloid, haliclorensin D ( 6 ), were isolated from the marine sponge Neopetrosia chaliniformis collected off Xisha Island in the South China Sea. Their structures and absolute configurations were determined by spectroscopic analysis, single-crystal X-ray diffraction, calculated electronic circular dichroism (ECD), and DP4+ probability analyses. Compounds 1 , 2 , and 4 exhibited in vivo hepatoprotective activity in a zebrafish model at a concentration of 20 μM.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus which was associated with severe diarrhea disease in pigs. SADS-CoV was first detected and identified as the causative agent of a devastating swine disease outbreak in southern China in 2017. Routine monitoring and early detection of the source of infection is therefore integral to the prevention and control of SADS-CoV infection. In this study, a SYBR green-based real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique was established for rapid detection and monitoring of this emerging virus. Specific primers were designed based on the conserved region within the M gene of the viral genome. The lowest detection limit of the RT-qPCR assay was 10 copies/μL. This assay was specific and had no cross-reaction with other 11 swine viruses. The positive rate of 84 clinical samples for the SYBR green-based RT-qPCR and the conventional RT-PCR was 73.81% (62/84) and 53.57% (45/84), respectively. These results demonstrated that the SYBR green-based RT-qPCR technique was an effectively diagnostic method with higher sensitivity than probe-based RT-qPCR and gel-based RT-PCR for detection and epidemiological investigations of SADS-CoV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Milk vetch dwarf virus (MDV) is a member of the genus Nanovirus, and its genome is composed of multiple circular 1-kb ssDNA components. In this study, we first determined that the diseased tobacco samples obtained in Zhucheng, Shandong Province were naturally infected with MDV using a polymerase chain reaction (PCR) assay. Subsequently, loop-mediated isothermal amplification (LAMP) was developed for the detection of MDV for the first time. The Mg 2+ and dNTP concentrations and the reaction temperature and time of the LAMP were optimized to 8 mM, 1.8 mM, 65 °C, and 60 min, respectively. The best ratio of the inner primers (FIP and BIP) to the outer primers (F3 and B3) was 2:1. The LAMP detection limit was 100 times greater than that of PCR. The nucleotide amplification could be clearly observed by adding SYBR Green I. The positive and negative reactions exhibit distinctly different colors in daylight; however, the positive reactions exhibit green fluorescence under a UV lamp. Therefore, the method is stable, sensitive and specific., (Copyright © 2018. Published by Elsevier B.V.)

Porcine circovirus 3 (PCV3) has been reported in cases of porcine dermatitis and nephropathy syndrome, reproductive failure, cardiac and multi-systemic inflammation. A SYBR green-based real-time quantitative PCR (qPCR) assay was established in this study to detect PCV3 in 203 clinical samples from suckling piglets affected by congenital tremors in China. The limit of detection (LOD) of PCV3 was 1.73×10 4 copies/μL for gel electrophoresis and 1.73×10 2 copies/μL for SYBR green-based real-time qPCR. The melt curve analysis showed a single melt peak at 82.5°C.The intra-assay coefficient of variation was up to 1.83% and the inter-assay coefficient of variation was up to 2.27%. The PCV3 positive detection rate of 203 clinical samples for the SYBR green-based real-time qPCR and the conventional PCR was 86.70% (176/203) and 26.60% (54/203), respectively. Each tissue detected in the SYBR green-based real-time qPCR showed a higher positive rate than that detected in the conventional PCR. These results indicated that the SYBR green-based real-time qPCR assay is a powerful method with high sensitivity, specificity and reproducibility for epidemiological investigations of PCV3., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25-153.00%, PCR efficiency of 99-100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50-7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32-28.90 ± 0.42) and melting curves (74.63-78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth.

In this work, a biosensor based on luminescence resonance energy transfer (LRET) from NaYF4:Yb,Tm upconversion nanoparticles (UCNPs) to SYBR Green I has been developed. The aptamers are covalently linked to UCNPs and hybridized with their complementary strands. The subsequent addition of SYBR Green allows SYBR Green I to insert into the formed double-stranded DNA (dsDNA) duplex and brings the energy donor and acceptor into close proximity, leading to the fluorescence of UCNPs transferred to SYBR Green I. When excited at 980 nm, the UCNPs emit luminescence at 477 nm, and this energy is transferred to SYBR Green I, which emits luminescence at 530 nm. In the presence of oxytetracycline (OTC), the aptamers prefer to bind to its corresponding analyte and dehybridize with the complementary DNA. This dehybridization leads to the liberation of SYBR Green I, which distances SYBR Green I from the UCNPs and recovers the UCNPs' luminescence. Under optimal conditions, a linear calibration is obtained between the ratio of I530 to I477 nm (I530/I477) and the OTC concentration, which ranges from 0.1 to 10 ng/ml with a limit of detection (LOD) of 0.054 ng/ml., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A real-time PCR for detection and quantification of M. ovipneumoniae was developed using 9 recently sequenced M. ovipneumoniae genomes and primers targeting a putative adhesin gene p113. The assay proved to be specific and sensitive (with a detection limit of 22 genomic DNA) and could quantify M. ovipneumoniae DNA over a wide linear range, from 2.2 × 10(2) to 2.2 × 10(7) genomes.

Avian encephalomyelitis virus (AEV) causes epidemic diseases in poultry worldwide. A SYBR Green real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay was developed for the rapid detection and quantitation of AEV in this study. A pair of specific primers was designed in the highly conserved VP1 gene of this virus. When comparing this assay with conventional RT-PCR, the rRT-PCR assay was 100 times more sensitive and could detect levels as low as 10 standard DNA copies of the AEV SX strain. The specificity of this technique was evaluated in five other avian pathogens. The AEV RNA was detected as early as three days post-infection in chicken embryos. All 18 clinical chicken brains collected from an AEV outbreak in Northwestern China were detected to be positive (100%) using the rRT-PCR assay. However, only 5 of the 18 samples were positive (28%) using the conventional RT-PCR. The results were confirmed by virus isolation in chicken embryos. This high sensitivity, specificity, and simplicity of the SYBR Green rRT-PCR approach can be a more effective method than the conventional one for AEV diagnosis and surveillance., (Copyright © 2014. Published by Elsevier B.V.)

Hantaan virus (HTNV), which belongs to the genus Hantavirus, causes hemorrhagic fever with renal syndrome (HFRS) mainly in China. The diagnosis of HFRS depends on clinical manifestations and serological tests. A SYBR Green I based one-step real-time PCR assay was established in this study to detect HTNV. The HTNV standard curves were generated by plotting mean cycle threshold (Ct) values versus 10-fold serial dilutions of a previous titrated HTNV stock over a wide range of concentrations (1×10(7) to 1PFU/ml). The minimum detection limit of the assay was 1PFU/ml, and it was 100-fold more sensitive than conventional RT-PCR. Melting curve analysis indicated that there were no primer-dimers and non-specific products in the assay. No cross-reaction was observed with Seoul virus (SEOV). The specificity of the asssay was also verified by nuleotide sequencing of the PCR products. Intra- and inter-assay variability data were analyzed to examine the reproducibility of the assay. HTNV viral loads in HFRS patients were also investigated with the assay. These results indicated that the one-step real-time PCR assay is useful for detecting HTNV and for monitoring the viral loads., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Duck Tembusu virus (DTMUV) is a single-stranded positive-sense RNA virus that causes disease to emerge in duck flocks and results in huge economic losses to the duck industry. However, no vaccines and control measures are available in China to date. Development of reliable and fast detection methods is necessary to prevent and control this disease. Therefore, a one-step SYBR Green real-time reverse transcription polymerase chain reaction (RT-PCR) method is established here for DTMUV detection. The results show that the method can specifically detect DTMUV without cross-reactions with selected avian pathogens. The sensitivity of the assay was 1000 times greater than that of a conventional RT-PCR and able to test as few as 20 copies from RNA standard samples. The coefficients of variations of inter- and intra-assay values ranged from 0.09% to 0.36% and 0.1% to 0.23%, respectively. Testing 168 field samples and 96 experimentally infected samples by conventional RT-PCR and the one-step SYBR Green real-time RT-PCR, the positive rates were 35.1% and 73.8% from field samples and 30.2% and 64.6% from infected samples. The one-step SYBR Green real-time RT-PCR developed in this study was shown to be a sensitive, specific, high-throughput, cost-effective, and simple diagnostic tool for the rapid detection and epidemiological surveillance of the emerging DTMUV infection.

Background: Co-infections of Apple chlorotic leaf spot virus (ACLSV) and Cherry green ring mottle virus (CGRMV) in peach is common in China and have resulted in significant yield reductions. A reliable, sensitive and quantitive method is needed to detect and distinguish between ACLSV and CGRMV in peach., Findings: We developed a sensitive and specific SYBR Green-I based RT-qPCR for the quantification of ACLSV and CGRMV in different peach tissues, and a duplex RT-qPCR system to detect ACLSV and CGRMV simultaneously. The RT-qPCR method was optimized using standard samples transcribed by the T7 Large Scale RNA Production System in vitro. The peach genes, RNA Polymerase subunit II (RPII) and Ubiquitin 10 (UBQ10), which were used as the internal controls for the quantification assay also showed good expression stability in this system. Single RT-qPCR assays showed that CGRMV in peach accumulates to a higher level than ACLSV. The detection limits of the duplex RT-qPCR assay were 10² and 10⁴ copies for ACLSV and CGRMV, respectively. The sensitivity of the duplex RT-qPCR was as high as RT-qPCR and higher than RT-PCR., Conclusions: The SYBR Green-I RT-qPCR assay provided a sensitive, specific and reliable method for the detection and quantification of ACLSV and CGRMV in different peach tissues. The duplex RT-qPCR system provided a sensitive and specific method to detect and differentiate between ACLSV and CGRMV in a single sample. This RT-qPCR assay could be a useful tool for the routine diagnosis of these two viruses and for disease epidemiology studies in peach orchards.

SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID(50) for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China.

Background: From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of the new virus related to Tembusu-related Flavivirus., Results: The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation., Conclusion: The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

This report describes a one-step real-time polymerase chain reaction assay based on SYBR Green I for detection of a broad range of duck circovirus (DuCV). Align with all DuCV complete genome sequences and other Genus Circovirus download from the GenBank (such as goose circovirus, pigeon circovirus), the primers targets to the replicate gene of DuCV were designed. The detection assay was linear in the range of 1.31 × 102-1.31 × 107 copies/μL. The reaction efficiency of the assay using the slope (the slope was -3.349) and the Y-intercept was 37.01 from the linear equation was estimated to be 0.99 and the correlation coefficient (R2) was 0.993. A series of experiments were carried out to assess the reproducibility, sensitivity, and specificity of the assay, following by the low intra-assay and inter-assay CVs for CT values obtained with the standard plasmids. The intra-assay CVs were equal or less than 1.89% and the inter-assay CVs were equal or less than 1.26%. There was no cross-reaction occurred with nucleic acids extracted from RA (Riemerella anatipestifer), E. coli (Escherichia coli), Duck Cholera (Pasteurella multocida), Avian influenza virus, avian paramyxovirus, Muscovy duck parvovirus, Duck reovirus, Duck hepatitis A virus as control templates. The nucleic acids extracted from samples of healthy ducks were used as negative controls. The assay was specific and reproducible. The established real time PCR was used to detect 45 DuCV-negative samples, which were tested using conventional PCR under the developed optimal conditions, each 15 for embryonated eggs, non-embryonated budgerigar eggs, newly hatched duck, the mixture of the lung, liver, spleen which were analysis for the presence of DuCV DNA, to conform that whether the DuCV can be transmitted vertically. Meanwhile, no positive result was shown by the real-time PCR method. The SYBR Green I-based quantitative PCR can therefore be practically used as an alternative diagnostic tool and a screening method for ducks infected with duck circovirus.

A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the rapid and specific detection of HCLV vaccine strain against classical swine fever. Four primers were designed for amplification of NS5B gene region with Bst DNA polymerase at a constant temperature of 65°C. The products showed ladder-like pattern on 2% agarose gel, and can be visualised after addition of SYBR Green I dye. The detection limit of the assay was 5 copies of the HCLV genome per reaction. No cross-reaction with other porcine viruses including different wild-type CSFV strains and the bovine viral diarrhoea virus was observed. The agreement between the LAMP and TaqMan real-time RT-PCR assays was 94.4% for the detection of 72 batches of HCLV vaccine. The assay provides a rapid tool for the control of vaccine quality and can be an accompanying assay of the LAMP for wild-type CSFV described previously for differential diagnosis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The objective of this study was to develop and evaluate a loop-mediated isothermal amplification (LAMP) method to detect infectious laryngotracheitis virus (ILTV) from commercial broiler and layer flocks in southern China. A set of six specific primers was designed to recognize six distinct genomic sequences of thymidine kinase (TK) from ILTV. The entire assay duration was recorded at 40 min under isothermal condition at 63.5 degrees C. The amplified products were analyzed by electrophoresis and visual judgment by the SYBR Green I dyeing. LAMP assay was 10-fold more sensitive than the routine PCR assay, with a detection limit of 46 copies per reaction. In detecting ILTV, the LAMP assay detected all 5 strains previously isolated, did not cross-react with other avian pathogens, and obtained a 100% sensitivity in 43 positive clinical samples with reference to virus isolation. Therefore, the LAMP assay may be a good alternative method for specific diagnosis of ILTV infection in primary care facilities, and in less well-equipped laboratories., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)