Development and Evaluation of Isothermal Amplification Methods for Rapid Detection of Lethal Amanita Species

He, Zhengmi; Su, Yu-Ting; Li, Sainan; Long, Pan; Zhang, Ping; Chen, Zuohong

doi:10.3389/fmicb.2019.01523

Cited by 28 publications

(23 citation statements)

References 41 publications

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“…With no need for instruments and skilled operators, isothermal amplification has become a promising onsite detection method. But like other amplification technologies, its resolution depends on the binding specificity between the primers and templates, which can limit their accuracy (Boonham et al, 2014;He et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever

et al. 2019

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The mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and wild boars is caused by African swine fever virus (ASFV), can reach 100%. Since the first confirmed ASF outbreak in China on 3 August 2018, 156 ASF outbreaks were detected in 32 provinces. About 1,170,000 pigs were culled in order to halt further spread. There is no effective treatment or vaccine for it and the present molecular diagnosis technologies have trade-offs in sensitivity, specificity, cost and speed, and none of them cater perfectly to ASF control. Thus, a technology that overcomes the need for laboratory facilities, is relatively low cost, and rapidly and sensitively detects ASFV would be highly valuable. Here, we describe an RAA-Cas12a-based system that combines recombinase aided amplification (RAA) and CRISPR/Cas12a for ASFV detection. The fluorescence intensity readout of this system detected ASFV p72 gene levels as low as 10 aM. For on-site ASFV detection, lateral-flow strip readout was introduced for the first time in the RAA-Cas12a based system (named CORDS, Cas12abased On-site and Rapid Detection System). We used CORDS to detect target DNA highly specifically using the lateral-flow strip readout and the assay displayed no crossreactivity to other 13 swine viruses including classical swine fever (CSF). CORDS could identify the ASFV DNA target at femtomolar sensitivity in an hour at 37 • C, and only requires an incubator. For ease of use, the reagents of CORDS were lyophilized to three tubes and remained the same sensitivity when stored at 4 • C for at least 7 days. Thus, CORDS provide a rapid, sensitive and easily operable method for ASFV on-site detection. Lyophilized CORDS can withstand long-term transportation and storage, and is ready for field-based applications.

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Section: Introductionmentioning

confidence: 99%

Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever

et al. 2019

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“…This conclusion was confirmed by multiple independent repeated experiments ( Figure 4 ). In related studies, the LAMP detection method established by Chen Zuohong’s research team from Hunan Normal University for lethal Amanita species had a detection limit of 10 pg ( He et al, 2019 ). The sensitivity of the LAMP assay might be affected by many factors.…”

Section: Discussionmentioning

confidence: 99%

“…Isothermal amplification methods have been widely used for species identification of pathogenic fungi in recent years ( He et al, 2019 ; Qiu et al, 2019 ; Bodulev and Sakharov, 2020 ). Among them, loop-mediated isothermal amplification (LAMP) is the most widely used one, which has received increasing attention ( Lee, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

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Rapid and Visual Identification of Chlorophyllum molybdites With Loop-Mediated Isothermal Amplification Method

et al. 2021

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Chlorophyllum molybdites is a kind of common poisonous mushroom in China that is widely distributed in different areas. Food poisoning caused by accidentally eating C. molybdites has become more frequent in recent years. In 2019, there were 55 food poisoning incidents caused by eating this mushroom in China. Mushroom poisoning continues to be a common health issue of global concern. When mushroom poisoning occurs, an effective, simple, and rapid detection method is required for accurate clinical treatment or forensic analysis. For the first time, we established a loop-mediated isothermal amplification (LAMP) assay for the visual detection of C. molybdites. A set of specific LAMP primers was designed, and the specificity was confirmed against 43 different mushroom species. The LAMP method could detect as low as 1 pg of genomic DNA. Boiled mushrooms and artificial gastric-digested mushroom samples were prepared to test the applicability of the method, and the results showed that as low as 1% C. molybdites in boiled and digested samples could be successfully detected. The LAMP method can also be completed within 45 min, and the reaction results could be directly observed based on a color change under daylight by the naked eye. Therefore, the LAMP assay established in this study provides an accurate, sensitive, rapid, and low-cost method for the detection of C. molybdites.

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“…HRCA is an isothermal ampli cation approach and thus provides a rapid, simple and low-cost detection of speci c nucleic acid sequences (Nilsson et al 1994;Lizardi et al 1998). This approach has been widely used for clinic detection of human-pathogenic microfungi (Zhou et al 2008;Trilles et al 2014;Rodrigues et al 2015), and recently, was also reported for rapid detection of poisonous macrofungi (He et al 2019a(He et al , 2019b. Regarding lethal Amanita species, a more than two-nucleotide-long difference was evidenced to be valid for identi cation of α-amanitin gene (He et al 2019a).…”

Section: Discussionmentioning

confidence: 99%

ITS barcoding-based species identification for Sanghuangporus (Basidiomycota), a genus of medicinal mushrooms

Liu

Shen

Jiang

et al. 2020

Preprint

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“Sanghuang” is a kind of important medicinal mushrooms and taxonomically represented by members in the fungal genus Sanghuangporus. Species of Sanghuangporus referred to medicinal studies and industry are discriminated mainly by BLAST search of GenBank with ITS barcoding region as a query. However, the inappropriately labeled ITS sequences related to “Sanghuang” in GenBank restrict accurate species identification and, to some extent, the utilization of these medicinal resources. Here, we examined all available 271 ITS sequences related to “Sanghuang” from GenBank including 31 newly submitted sequences for this study. Of these sequences, more than half were mislabeled and the corresponding species names are corrected. The mislabeled sequences mainly came from strains by non-taxonomists. Based on the analyses of ITS sequences submitted by taxonomists, we treat Sanghuangporus toxicodendri as a later synonym of S. quercicola, and the intraspecific and interspecific differences are below 1.50% (but S. weirianus) and above 1.50%, respectively. Moreover, ten potential diagnostic sequences are provided for hyperbranched rolling circle amplification to rapidly detect three common commercial species, viz. S. baumii, S. sanghuang and S. vaninii. Generally, the current results provide a practical method for ITS barcoding-based species identification of Sanghuangporus, and will promote medicinal studies and industrial development from the taxonomic perspective.

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Development and Evaluation of Isothermal Amplification Methods for Rapid Detection of Lethal Amanita Species

Cited by 28 publications

References 41 publications

Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever

Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever

Rapid and Visual Identification of Chlorophyllum molybdites With Loop-Mediated Isothermal Amplification Method

ITS barcoding-based species identification for Sanghuangporus (Basidiomycota), a genus of medicinal mushrooms

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