Development and optimization of a loop-mediated isothermal amplification (LAMP) assay for the species-specific detection of Penicillium expansum

Frisch, Lisa M.; Mann, Magdalena A.; Marek, David N.; Niessen, Ludwig

doi:10.1016/j.fm.2020.103681

Cited by 14 publications

(11 citation statements)

References 25 publications

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“…The designed primer set LAMP could only amplify A. fabacearum , indicating its good specificity. By detecting the sensitivity of LAMP, we concluded that the LAMP primer set had a high sensitivity, and the lowest detection limit reached 5 × 10 −7 ng/μL This detection limit is lower than previously reported for the detection of potato late blight ( P. infestans ), blue mold decay ( Penicillium expansum ), maize ear and stalk rot diseases ( Fusarium temperatum ) and dried chickpea root rot ( Rhizoctonia bataticola ) [ 29 , 35 , 50 , 51 ]. The LAMP method established in this study is more precise and more sensitive than conventional PCR.…”

Section: Discussionmentioning

confidence: 82%

First Report of Crown Gall of Kiwifruit (Actinidia deliciosa) Caused by Agrobacterium fabacearum in China and the Establishment of Loop-Mediated Isothermal Amplification Technique

Shi

Zhang

et al. 2021

IJMS

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Kiwifruit is moderately sweet and sour and quite popular among consumers; it has been widely planted in some areas of the world. In 2019, the crown gall disease of kiwifruit was discovered in the main kiwifruit-producing area of Guizhou Province, China. This disease can weaken and eventually cause the death of the tree. The phylogeny, morphological and biological characteristics of the bacteria were described, and were related to diseases. The pathogenicity of this species follows the Koch hypothesis, confirming that A. fabacearum is the pathogen of crown gall disease of kiwifruit in China. In this study, Loop-mediated isothermal amplification (LAMP) analysis for genome-specific gene sequences was developed for the specific detection of A. fabacearum. The detection limit of the LAMP method is 5 × 10−7 ng/μL, which has high sensitivity. At the same time, the amplified product is stained with SYBR Green I after the reaction is completed, so that the amplification can be detected with the naked eye. LAMP analysis detected the presence of A. fabacearum in the roots and soil samples of the infected kiwifruit plant. The proposed LAMP detection technology in this study offers the advantages of ease of operation, visibility of results, rapidity, accuracy and high sensitivity, making it suitable for the early diagnosis of crown gall disease of kiwifruit.

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Section: Discussionmentioning

confidence: 82%

First Report of Crown Gall of Kiwifruit (Actinidia deliciosa) Caused by Agrobacterium fabacearum in China and the Establishment of Loop-Mediated Isothermal Amplification Technique

Shi

Zhang

et al. 2021

IJMS

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“…While violet changed to blue for HNB [ 55 , 64 ], dark blue changed to light blue for malachite green [ 69 ], and dark yellow changed to yellow for calcein [ 63 ], which were less discernable color changes based on their effects, disadvantage for these dyes [ 70 ]. Another advantage of the NR dye is its ability to easily visualize the color change of the reaction by naked eye without the risk of post-contamination from re-opening the tube [ 67 , 71 , 72 ]. This advantage is due to the release of hydrogen ions in isothermal amplification, causing NR to be protonated and changed to cationic form, affecting its contrast color shifts from yellow to pink [ 73 ].…”

Section: Discussionmentioning

confidence: 99%

“…This advantage is due to the release of hydrogen ions in isothermal amplification, causing NR to be protonated and changed to cationic form, affecting its contrast color shifts from yellow to pink [ 73 ]. Thus, it is most reasonable to utilize NR as an appropriate pH-sensitive dye for distinct positive and negative differentiation in LAMP assay [ 66 ] for detecting patulin-producing Penicillium species [ 67 ], species-specific of P. expansum [ 71 ], Photobacterium spp. [ 74 ], and African swine fever virus (ASFV) [ 72 ].…”

Section: Discussionmentioning

confidence: 99%

Development of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detecting Feline Coronavirus

Rapichai

Saejung

Khumtong

et al. 2022

Animals

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Feline infectious peritonitis (FIP) is a worldwide fatal disease caused by a mutant feline coronavirus (FCoV). Simple and efficient molecular detection methods are needed. Here, sensitive, specific, rapid, and reliable colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect the ORF1a/1b gene of FCoV from cats with suspected FIP using neutral red as an indicator. Novel LAMP primers were specifically designed based on the gene of interest. The isothermal assay could visually detect FCoV at 58 °C for 50 min. The RT-LAMP assay was highly specific and had no cross-reactivity with other related feline viruses. The detection limit of FCoV detection by RT-LAMP was 20 fg/µL. A blind clinical test (n = 81) of the developed RT-LAMP procedure was in good agreement with the conventional PCR method. In the light of its performance specificity, sensitivity, and easy visualization, this neutral-red-based RT-LAMP approach would be a fruitful alternative molecular diagnostic tool for veterinary inspection of FCoV when combined with nucleotide sequencing or specific PCR to affirm the highly virulent FIP-associated FCoV.

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“…A developing yellow colour in a positive LAMP reaction is easily distinguishable from a pink negative sample [157][158][159][160]. The opposite colorization is also available [161][162][163]. There are commercial premixes containing pH indicators that can provide detection without opening the tube after the LAMP mix to prevent possible contamination.…”

Section: Colorimetric Detection Of Lamp Ampliconsmentioning

confidence: 99%

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Иванов

Safenkova

Zherdev

et al. 2021

Plants

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Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

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Development and optimization of a loop-mediated isothermal amplification (LAMP) assay for the species-specific detection of Penicillium expansum

Cited by 14 publications

References 25 publications

First Report of Crown Gall of Kiwifruit (Actinidia deliciosa) Caused by Agrobacterium fabacearum in China and the Establishment of Loop-Mediated Isothermal Amplification Technique

First Report of Crown Gall of Kiwifruit (Actinidia deliciosa) Caused by Agrobacterium fabacearum in China and the Establishment of Loop-Mediated Isothermal Amplification Technique

Development of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detecting Feline Coronavirus

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

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