Rapid detection of the pathogenic fungi causing blackleg of Brassica napus using a portable real-time fluorescence detector

Lei, Rong; Kong, Jun; Qiu, Yu; Chen, Naizhong; Zhu, Shuifang; Wang, Xinyi; Wu, Pinshan

doi:10.1016/j.foodchem.2019.02.089

Cited by 30 publications

(27 citation statements)

References 34 publications

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“…The exponential amplification can be detected within 30 min in a temperature range of 37 to 42 °C [ 19 ]. Recently, several applications of RPA in DNA and RNA target detection have been proved [ 20 , 21 ], and the integration of RPA in different platforms has been reported [ 22 , 23 , 24 ]. Lateral flow dipsticks (LFD) has great application prospects in the detection field due to easy-to-use, high sensitivity, less time consuming and low-cost [ 25 , 26 , 27 ].…”

Section: Introductionmentioning

confidence: 99%

Recombinase Polymerase Amplification Based Multiplex Lateral Flow Dipstick for Fast Identification of Duck Ingredient in Adulterated Beef

Zhang

Zhou

et al. 2020

Animals

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Meat adulteration has become a global social problem. In order to protect consumers from meat adulteration, several methods have been developed to identify meat species. However, the conventional methods are labor-intensive, time-consuming and require instruments. In the present study, a rapid and visual method based on recombinase polymerase amplification (RPA) and multiplex lateral flow dipstick (MLFD) was developed to detect duck ingredient in adulterated beef. Using recombinase and strand displacement polymerase enable RPA to amplify different double-labeled DNA amplicons at room temperature, which can be further detected by MLFD. The whole reaction process can be finished within 35 min, and the results can be determined by naked eyes. As low as 5% of duck ingredient in adulterated beef can be easily measured. Moreover, we confirmed that our new method held good potential in the detection of commercially processed meat samples. In conclusion, this study reported a useful animal derived meat adulteration detection method, which have potential application in future.

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Section: Introductionmentioning

confidence: 99%

Recombinase Polymerase Amplification Based Multiplex Lateral Flow Dipstick for Fast Identification of Duck Ingredient in Adulterated Beef

Zhang

Zhou

et al. 2020

Animals

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“…Repeatability of RPA assay. During RPA amplification, fluorescence intensity can easily be affected by the concentration of the probe and DNA template, even by the pipette due to the small volume of DNA template 40 . The repeatability of the RPA assay was therefore evaluated by measuring 0.02 ng (4.1 × 10 6 DNA copies) and 20 fg (4.1 × 10 3 copies) of pUC57-bc48 plasmid DNA four times (Fig.…”

Section: Resultsmentioning

confidence: 99%

Rapid isothermal duplex real-time recombinase polymerase amplification (RPA) assay for the diagnosis of equine piroplasmosis

Lei

Wang

Zhang

et al. 2020

Sci Rep

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movement of horses 6,7. Importation of carrier animals with no obvious signs of disease is a major risk factor for the introduction of EP into non-enzootic areas 1. Therefore, developing sensitive and specific diagnostic methods is essential for identifying asymptomatic equines carrying these parasites. Current diagnostic methods include aetiological diagnostics 8,9 , immunological diagnostics 10-14 and molecular diagnostics 9,15-33. Of these, molecular diagnostics is widely recognized due to its accuracy and sensitivity 9,34. Cortes et al. developed a multinested PCR assay for simultaneous detection of the equine piroplasmids T. equi and B. caballi by amplification of five genetic markers (18S rRNA, β-tubulin, cytB, ema-1 and rap-1) 23. To identify the species of piroplasmid in an infected horse, nested PCR assay is commonly used to amplify long fragments of 18S rRNA, followed by sequence analysis 22. However, PCR-based diagnosis requires a thermocycler, skilled personnel and a long detection time, which made it inappropriate for field diagnostic applications. As an alternative, recombinase polymerase amplification (RPA) has emerged as a novel isothermal technique for molecular diagnosis of various infectious diseases 35 , including the protozoan parasites 36 Theileria annulata 37 and Babesia gibsoni 38. Compared to PCR-based assay and loop-mediated isothermal amplification (LAMP), RPA is more rapid (<20 min), simpler to perform as it requires a lower temperature (37-42 °C) and has an acceptable sensitivity 39,40. RPA-lateral flow assay has been used for rapid detection of Trichinella 41 , Perkinsus beihaiensis 42 , Plasmodium knowlesi 43 , Babesia gibsoni 38 , Protozoan parasites 36 , Theileria annulata 37 , Fasciola hepatica 44 , Schistosoma japonicum 45 , Schistosoma haematobium 46 , Leishmania donovani 47 , Intestinal Protozoa 48 , Giardia 49 , Plasmodium falciparum 50. To avoid the "ghost band" and the false-positive results, the primers, probes and the detection procedure have to be carefully designed 39. To speed up the clearance of an imported horse at a port, or detect a piroplasmosis infection in the field or in low-resource underserved rural communities, we also adapted RPA to develop a duplex detection of both T. equi and B. caballi as an alternative to duplex qPCR 26,30. Various genomic sites have been used in species identification, phylogenetic and genotype studies of both T. equi and B. caballi with PCR-based molecular techniques, including the small subunit ribosomal RNA gene (18S rRNA) 11,22,51-54 ; and genomic sites targeted by qPCR assay have included the ema-1 gene of T. equi and the 48 kDa merozoite rhoptry protein (bc48) gene of B. caballi 30. These sites have been used to create a duplex real-time PCR for simultaneous detection of both parasites using the ema-1 gene of T. equi and the bc48 gene of B. caballi 30 ; or the 18S rRNA gene of B. caballi and the ema-1 gene of T. equi 26. In this study, we designed primers and fluorescent probes to target the 18S rRNA gene of both T. equi and B. ...

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“…Several examples of portable devices exploiting fluorescence detectors for POC plant pathogen analysis can be found. Such devices combine fluorescence detectors with several amplification methods, such as qPCR (Koo et al, 2013;DeShields et al, 2018;Fotiou et al, 2019), LAMP (Larrea-Sarmiento et al, 2018;Tahzima et al, 2019;Xia et al, 2019), RCA (Zhang et al, 2019), and RPA (Lei et al, 2019). A possible alternative to the classical fluorescence-based detection methods is the so-called surface-enhanced Raman scattering (SERS), a technique that exploits the signal enhancement given by metal nanoparticles surfaces upon laser excitation (Kneipp et al, 1997).…”

Section: Detection Methodsmentioning

confidence: 99%

“…Nonetheless, several commercial kits have been tested, and some of them showed characteristics that could be suitable for POC analysis, especially those based on the use of magnetic beads for nucleic acid purification (DeShields et al, 2018). Several works can be found in literature exploiting this extraction principle on various plant species and pathogens, such as detection of the fungus Leptosphaeria maculans, causing Phoma stem canker (black leg) of Brassica napus (Lei et al, 2019). Other examples include detection of soil-borne pathogens of potatoes (DeShields et al, 2018); Alternaria panax Whetz infecting ginseng (Wei et al, 2018); Puccinia striiformis f.sp.…”

Section: Nucleic Acid Extractionmentioning

confidence: 99%

“…All these characteristics make RPA a very easy-to-use approach, especially in developing countries when fast analysis in low resource environments is needed (Wambua et al, 2017;Ghosh et al, 2018;Silva et al, 2018). The possibility to multiplex the reaction can further increase the speed and reduce the costs of RPA (Lau et al, 2016;Lei et al, 2019). Several RPA-based portable devices are already available for POC detection of human pathogens (Ereku et al, 2018;Tsaloglou et al, 2018), and assays for plant pathogen detection are rapidly developing (Zhang et al, 2014;Hammond and Zhang, 2016;Gaige et al, 2018).…”

Section: Isothermal Nucleic Acid Amplificationmentioning

confidence: 99%

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Molecular Approaches for Low-Cost Point-of-Care Pathogen Detection in Agriculture and Forestry

Baldi

Porta

2020

Front. Plant Sci.

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Early detection of plant diseases is a crucial factor to prevent or limit the spread of a rising infection that could cause significant economic loss. Detection test on plant diseases in the laboratory can be laborious, time consuming, expensive, and normally requires specific technical expertise. Moreover, in the developing countries, it is often difficult to find laboratories equipped for this kind of analysis. Therefore, in the past years, a high effort has been made for the development of fast, specific, sensitive, and cost-effective tests that can be successfully used in plant pathology directly in the field by low-specialized personnel using minimal equipment. Nucleic acid-based methods have proven to be a good choice for the development of detection tools in several fields, such as human/animal health, food safety, and water analysis, and their application in plant pathogen detection is becoming more and more common. In the present review, the more recent nucleic acid-based protocols for point-of-care (POC) plant pathogen detection and identification are described and analyzed. All these methods have a high potential for early detection of destructive diseases in agriculture and forestry, they should help make molecular detection for plant pathogens accessible to anyone, anywhere, and at any time. We do not suggest that on-site methods should replace lab testing completely, which remains crucial for more complex researches, such as identification and classification of new pathogens or the study of plant defense mechanisms. Instead, POC analysis can provide a useful, fast, and efficient preliminary on-site screening that is crucial in the struggle against plant pathogens.

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Rapid detection of the pathogenic fungi causing blackleg of Brassica napus using a portable real-time fluorescence detector

Cited by 30 publications

References 34 publications

Recombinase Polymerase Amplification Based Multiplex Lateral Flow Dipstick for Fast Identification of Duck Ingredient in Adulterated Beef

Recombinase Polymerase Amplification Based Multiplex Lateral Flow Dipstick for Fast Identification of Duck Ingredient in Adulterated Beef

Rapid isothermal duplex real-time recombinase polymerase amplification (RPA) assay for the diagnosis of equine piroplasmosis

Molecular Approaches for Low-Cost Point-of-Care Pathogen Detection in Agriculture and Forestry

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