Primers and visualization of LAMP: A rapid molecular identification method for Liposcelis entomophila (Enderlein) (Psocodea: Liposcelididae)

Zeng, Lingyu; Su, Yun; Stejskal, V.; Opit, George P.; Aulický, Radek; Li, Zhihong

doi:10.1016/j.jspr.2021.101855

Cited by 6 publications

(6 citation statements)

References 26 publications

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“…Furthermore, identification results cannot be observed by the naked eye, which makes them limited to the laboratory. LAMP technique provides a rapid and visual identification of booklice by using hydroxy naphthol blue (HNB) or cresol red to judge the identification results (Zeng et al 2021, Zhou et al 2022b). However, this technique requires a long reaction time of 2 h, and needs a relatively high temperature of 65°C to react.…”

Section: Discussionmentioning

confidence: 99%

“…The molecular identification methods mentioned above for booklice are time-consuming, require specialized equipment and professional operations. Recently, based on COI gene, rapid and visual molecular identification methods using loop-mediated isothermal amplification (LAMP) were developed for L. entomophila (Zeng et al 2021), L. brunnea (Zhou et al 2022b) and L. bostrychophila (Zhou et al 2022a). However, this technique is prone to false positives, and requires a long reaction time of 2 h. The primer design principles of LAMP are so complex that they require 6 specific regions of the target gene to design 4–6 special primers, forming a primer group.…”

Section: Introductionmentioning

confidence: 99%

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An advanced approach for rapid visual identification of Liposcelis bostrychophila (Psocoptera: Liposcelididae) based on CRISPR/Cas12a combined with RPA

Deng

Feng

Stejskal

et al. 2023

Journal of Economic Entomology

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Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae) is a booklouse pest that is a threat to commodity storage security worldwide. Accurate and sensitive methods of L. bostrychophila on-site identification are essential prerequisites for its effective management. Evidence suggests that L. bostrychophila contains 3 intraspecific biotypes that are morphologically indistinguishable but can be discriminated at the level of mitochondrial genome organization and sequences. The traditional molecular identification methods, such as DNA barcoding and PCR-RFLP, are instrumentally demanding and time-consuming, limiting the application of the identification in the field. Therefore, this study developed a new CRISPR/Cas12a-based visual nucleic acid system based on the mitochondrial gene coding for NADH dehydrogenase subunit 2 (nad2), combined with recombinase polymerase amplification (RPA) to accurately identify L. bostrychophila from 4 other common stored-product booklice, and also differentiate 3 biotypes of this species at the same time. The entire identification process could be completed at 37 °C within 20 min with high sensitivity. The system could stably detect at least 1 ng/μl of DNA template. The green fluorescence signal produced by the trans-cleaving of the single-stranded DNA reporter could be observed by the naked eye under blue light. Additionally, the suggested system combined with the crude DNA extraction method to extract DNA rapidly, enabled identification of all developmental stages of L. bostrychophila. With crude DNA, this novel diagnostic system successfully identified an unknown booklouse by holding the reaction tubes in the hand, thus can be considered as an accurate, rapid, highly sensitive, and instrument-flexible method for on-site visual identification of L. bostrychophila.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An advanced approach for rapid visual identification of Liposcelis bostrychophila (Psocoptera: Liposcelididae) based on CRISPR/Cas12a combined with RPA

Deng

Feng

Stejskal

et al. 2023

Journal of Economic Entomology

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“…In terms of visualization detection, previous studies have more often combined calcium yellow-green and HNB with LAMP products to achieve the identification of unknown species by color change visible by the naked eye (Sial et al 2020, Zeng et al 2021). In positive reactions, the color changes from brownish-yellowish green to violet-sky blue.…”

Section: Discussionmentioning

confidence: 99%

Loop-mediated isothermal amplification of economically important Ceratitis species (Diptera: Tephritidae)

Zhang,

Li,

Virgilio

et al. 2023

Journal of Economic Entomology

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Ceratitis is an economically important genus of fruit flies that originated in Africa, has a wide host range, and causes serious economic losses due to its invasive damage. As a result, it is critical to identify them accurately and quickly in the world. Loop-mediated isothermal amplification (LAMP), as one of the representatives of isothermal amplification technology, has been widely used in the rapid nucleic acid detection of human pathogens and has shown its advantages in the identification of insect agricultural pests. In this study, using the mitochondrial cox1 and cob genes as target genes, the rapid molecular identification of the Ceratitis FARQ complex, C. cosyra, and C. capitata was realized based on LAMP. The experimental conditions optimization results showed that F3/B3:FIP/BIP = 1:8 was the optimal primer concentration ratio and 63 °C was the optimal reaction temperature. The sensitivity of the primers obtained in this study can reach up to 0.01 ng/μl DNA. A loop-mediated isothermal amplification identification technology system was established based on rapid, rough DNA extraction and visual detection of Ceratitis economically important fruit flies. The positive reaction system changed from pink to khaki by visual detection. The identification flow can be completed within 1 hour, including sample processing, DNA extraction, and LAMP visual detection.

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“…Although the existing polymerase chain reaction (PCR) and quantitative real‐time PCR (qRT‐PCR) achieve accurate identification, 13–15 the testing is time‐consuming and expensive equipment is required, being inappropriate for application in the grassroots sector. Despite the fact that rapid loop‐mediated isothermal amplification (LAMP) needs no special instruments and is easy to operate, the amplification temperature is ≈65 °C, requiring access to a medium temperature heater 7,16 . Also, the design of LAMP primers is rather difficult, 17 and false positive results emerged even when six primers (FIP, BIP, F3, B3, Floop and Bloop sequences) were designed, 18 which leads to misidentification of certain pest species.…”

Section: Introductionmentioning

confidence: 99%

“…Despite the fact that rapid loop-mediated isothermal amplification (LAMP) needs no special instruments and is easy to operate, the amplification temperature is ≈65 °C, requiring access to a medium temperature heater. 7,16 Also, the design of LAMP primers is rather difficult, 17 and false positive results emerged even when six primers (FIP, BIP, F3, B3, Floop and Bloop sequences) were designed, 18 which leads to misidentification of certain pest species. Therefore, for the notorious khapra beetle, rapid and accurate molecular identification allowing field testing at room temperature (RT) need to be developed.…”

Section: Introductionmentioning

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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Primers and visualization of LAMP: A rapid molecular identification method for Liposcelis entomophila (Enderlein) (Psocodea: Liposcelididae)

Cited by 6 publications

References 26 publications

An advanced approach for rapid visual identification of Liposcelis bostrychophila (Psocoptera: Liposcelididae) based on CRISPR/Cas12a combined with RPA

An advanced approach for rapid visual identification of Liposcelis bostrychophila (Psocoptera: Liposcelididae) based on CRISPR/Cas12a combined with RPA

Loop-mediated isothermal amplification of economically important Ceratitis species (Diptera: Tephritidae)

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

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