Evaluating two approaches for using positive control in standardizing the avian influenza H5 reverse transcription recombinase polymerase amplification assay

Ahmed, Basem M.; Amer, Haitham A.; Kissenkoetter, Jonas; Wahed, Ahmed Abd El; Bayoumi, Mahmoud; Böhlken-Fascher, Susane.; El‐Gamal, Mahmoud A.; Yehia, Nahed; Yousif, Ausama; Shalaby, Mohamed A.

doi:10.1016/j.mcp.2020.101511

Cited by 6 publications

(6 citation statements)

References 25 publications

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“…Considering IAVs are negative-sense single-stranded RNA viruses 36 , we sought to investigate the level of conservation of DRACH motifs in the genomic sequence as well. Based on the previous H1N1 meta-data 4 , a total of nine putative m 6 A sites were identified across the entire HA vRNA which were not described before.…”

Section: Resultsmentioning

confidence: 99%

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Evolutionary conservation of the DRACH signatures of potential N6-methyladenosine (m6A) sites among influenza A viruses

Bayoumi

Munir

2021

Sci Rep

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The addition of a methyl group to the N6-position of adenosine (m6A) is considered one of the most prevalent internal post-transcriptional modifications and is attributed to virus replication and cell biology. Viral epitranscriptome sequencing analysis has revealed that hemagglutinin (HA) mRNA of H1N1 carry eight m6A sites which are primarily enriched in 5′-DRACH-3′ sequence motif. Herein, a large-scale comparative m6A analysis was conducted to investigate the conservation patterns of the DRACH motifs that corresponding to the reference m6A sites among influenza A viruses. A total of 70,030 complete HA sequences that comprise all known HA subtypes (H1–18) collected over several years, countries, and affected host species were analysed on both mRNA and vRNA strands. The bioinformatic analysis revealed the highest degree of DRACHs conservation among all H1 sequences that clustered largely in the middle and in the vicinity to 3′ end with at least four DRACH motifs were conserved in all mRNA sequences. The major HA-containing subtypes displayed a modest DRACH motif conservation located either in the middle region of HA transcript (H3) or at the 3′ end (H5) or were distributed across the length of HA sequence (H9). The lowest conservation was demonstrated in HA subtypes that infect mostly the wild type avian species and bats. Interestingly, the total number and the conserved DRACH motifs in the vRNA were found to be much lower than those observed in the mRNA. Collectively, the identification of putative m6A topology provides a foundation for the future intervention of influenza infection, replication, and pathobiology in susceptible hosts.

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Section: Resultsmentioning

confidence: 99%

“…The high mutation rate along with the reassortment of IAVs segmented nature can lead to the emergence of pandemics. Additionally, IAVs can infect a wide range of host species including humans, animal, and avian species suggesting the likely roles to cross species barriers and causing zoonotic fatal infections 36 – 38 .…”

Section: Discussionmentioning

confidence: 99%

Evolutionary conservation of the DRACH signatures of potential N6-methyladenosine (m6A) sites among influenza A viruses

Bayoumi

Munir

2021

Sci Rep

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“…26,40,41 The introduction of a target-specific endogenous probe and the CRISPR/Cas12a system allows the reaction system to produce green fluorescence and further prevents the RPA assay from false amplicons, ensuring the accuracy and ease of field visualization applications. 27,42 Here, we reported a visualization method for khapra beetle based on RPA and the CRISPR/Cas12a system for the first time. This also is the first application of RPA-CRISPR/Cas12a detection in a Coleopteran species.…”

Section: Discussionmentioning

confidence: 99%

New and rapid visual detection assay for Trogoderma granarium everts based on recombinase polymerase amplification and CRISPR/Cas12a

Zeng,

Zheng,

Stejskal

et al. 2023

Pest Management Science

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BACKGROUNDKhapra beetle (Trogoderma granarium Everts), one of the most important quarantine pests globally, is capable of causing severe infestation and huge economic loss to stored grain, and its interception rate has increased in major global trade countries over the past few years. However, difficulties remain in distinguishing this species with similar ones. In order to assist border ports and warehouses in khapra beetle's effective rapid identification as well as pest control at the early stages of monitoring or interception, we herein developed a new and rapid visual detection assay for T. granarium based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system.RESULTSWe designed and selected the first khapra beetle‐specific RPA primers and crRNA, and optimized the visualization reaction system (Cas12a/CrRNA = 100 nM/500 nM). With only a 37 °C‐heat‐source and a blue light torch, RPA and CRISPR/CAS12a‐based visualization assays can be completed within 40 min to differentiate between khapra beetle and nine similar Dermestidae species. After DNA extraction using a kit (4–5 h) or a simple method (5 min), the specific amplicons were obtained after a 15 min RPA reaction at 37 °C, followed by a 15 min color reaction under 37 °C in dark conditions using a CRISPR/CAS12a system and a fluorescent probe (5′‐FAM/3′‐BHQ1 labeled). This method is ingenious to low levels of DNA (10−1 ng μL−1) and meets the sensitivity requirements for detecting a single khapra beetle's egg (≈0.7 mm).CONCLUSIONOur specificity and sensitivity analysis inferred that the present visualization system is effective to quickly and uniquely detect khapra beetle at room temperature (37 °C), thereby preventing this species before they spread widely. Our study is suitable for being pushed forward in storage pest management, and provides value as a reference for monitoring and identification of other pests. © 2023 Society of Chemical Industry.

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“…Furthermore, amplification products of similar size from multiplexed targets are difficult to separate by electrophoresis in agarose gels. This drawback may be overcome by real‐time monitoring with exo probes (Ahmed et al ., 2020), or by visualization with lateral flow dipstick and nfo probes (Rames and Macdonald, 2019), but the need for probes restricts this method due to the complexity of probe design (TwistDx, 2018). Our method involves a second step of specific LbCas12a/crRNA recognition of amplicons that contain the target sequence.…”

Section: Discussionmentioning

confidence: 99%

Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay

Jiao

Kong

Han

et al. 2020

Plant Biotechnology Journal

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Summary Co‐infection of apple trees with several viruses/viroids is common and decreases fruit yield and quality. Accurate and rapid detection of these viral pathogens helps to reduce losses and prevent virus spread. Current molecular detection assays used for apple viruses require specialized and expensive equipment. Here, we optimized a CRISPR/Cas12a‐based nucleic acid detection platform for the diagnosis of the most prevalent RNA viruses/viroid in apple, namely Apple necrotic mosaic virus (ApNMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd). We detected each RNA virus/viroid directly from crude leaf extracts after simultaneous multiplex reverse transcription‐recombinase polymerase amplification (RT‐RPA) with high specificity. Positive results can be distinguished by the naked eye via oligonucleotide‐conjugated gold nanoparticles. The CRISPR/Cas12a‐RT‐RPA platform exhibited comparable sensitivity to RT‐qPCR, with limits of detection reaching 250 viral copies per reaction for ASPV and ASGV and 2500 copies for the others. However, this protocol was faster and simpler, requiring an hour or less from leaf harvest. Field tests showed 100% agreement with RT‐PCR detection for 52 samples. This novel Cas12a‐based method is ideal for rapid and reliable detection of apple viruses in the orchard without the need to send samples to a specialized laboratory.

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Evaluating two approaches for using positive control in standardizing the avian influenza H5 reverse transcription recombinase polymerase amplification assay

Cited by 6 publications

References 25 publications

Evolutionary conservation of the DRACH signatures of potential N6-methyladenosine (m6A) sites among influenza A viruses

Evolutionary conservation of the DRACH signatures of potential N6-methyladenosine (m6A) sites among influenza A viruses

New and rapid visual detection assay for Trogoderma granarium everts based on recombinase polymerase amplification and CRISPR/Cas12a

Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay

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