Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya

Tsai, Chun-Chin; Shih, Huei-Chuan; Ko, Ya-Zhu; Wang, Ren-Huang; Shu-ju, LI; Chiang, Yu–Chung

doi:10.3390/ijms17101630

Cited by 12 publications

(7 citation statements)

References 43 publications

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“…Therefore, isothermal amplification can be used to measure a small number of RNAs. Isothermal amplification has been used for virus detection, SNP detection, sex detection, and RNA amplification [57,58,59]. Examples of isothermal amplification are loop-mediated isothermal amplification (LAMP) [60], rolling circle amplification (RCA) [61], transcription-mediated amplification (TMA) [62], nucleic acid sequence-based amplification (NASBA) [62], strand displacement amplification (SDA) [63], isothermal multiple displacement amplification (IMDA) [64], helicase-dependent amplification (HAD) [65], single primer isothermal amplification (SPIA) [66], and SmartAmp [58,67].…”

Section: Isothermal Amplificationmentioning

confidence: 99%

Challenges for Accurate Quantification of RNA

Hasegawa

Hapsari

Iwahashi

2021

RAS

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Ribonucleic acid (RNA) quantification is an essential technique in biology. There has been remarkable progress in RNA quantification techniques over the recent years; however, the specificity of these techniques to quantify a very small amount of RNA is doubtful because of factors which can inhibit precise quantification. To develop a technique that leads to the most sensitive RNA quantification, these problems must be overcome. In this article, we first review the factors that inhibit precise quantification of RNA: the quality of RNA, secondary structure of RNA, efficiency of the enzyme reaction, annealing conditions, limitations of the experimental protocol and equipment, and detection sensitivity of the equipment. Next, we discuss the possible methods which contribute to these factors: RNA quality control focused on target RNA degradation, isothermal amplification, techniques for avoiding amplification errors, RNase H-dependent PCR, targeting using a fluorescent-labeled probe, targeting using a padlock probe, bridged/locked nucleic acid (BNA/LNA) and peptide nucleic acid (PNA), and the clustered regularly interspaced short palindromic repeat (CRISPR) system. One of the goals for the development of an ultrasensitive RNA quantification technique is the absolute quantification of RNA. Here, we discuss the techniques used for this type of RNA quantification.

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Section: Isothermal Amplificationmentioning

confidence: 99%

Challenges for Accurate Quantification of RNA

Hasegawa

Hapsari

Iwahashi

2021

RAS

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“…Researchers also attempted to completely avoid nucleic acid isolation and instead apply the sample directly into the isothermal amplification mix. This has been demonstrated for plants using the example of papaya [ 15 ] and for the fecal indicator bacteria E. coli and E. faecalis [ 16 ]. The authors concluded that the elevated temperature of 63 °C could lead to increased cell permeability, allowing direct amplification of the target DNA.…”

Section: Case Study: Towards Implementing a Field-applicable Workflowmentioning

confidence: 99%

Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis

et al. 2019

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Molecular diagnostic tools in the field of food and water quality analysis are becoming increasingly widespread. Usually, based on DNA amplification techniques such as polymerase chain reaction (PCR), these methods are highly sensitive and versatile but require well-equipped laboratories and trained personnel. To reduce analysis time and avoid expensive equipment, isothermal DNA amplification methods for detecting various target organisms have been developed. However, to make molecular diagnostics suitable for low-resource settings and in-field applications, it is crucial to continuously adapt the working steps associated with DNA amplification, namely sample preparation, DNA extraction, and visualization of the results. Many novel approaches have been evaluated in recent years to tackle these challenges, e.g., the use of ionic liquids for the rapid isolation of nucleic acids from organisms relevant for food and water analysis or the integration of entire analytical workflows on microfluidic chips. In any event, the future of applications in the field of isothermal amplification will probably lie in ready-to-use cartridges combined with affordable handheld devices for on-site analysis. This trend article aims to make prospective users more familiar with this technology and its potential for moving molecular diagnostics from the laboratory to the field. Graphical abstract

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“…LAMP has been used in many field studies as a molecular technique, such as the detection of malaria parasites (Plasmodium falciparum; Paris et al, 2007), the detection of Fusarium graminearum, the cause of Downy Mildew in wheat (Triticum aestivum L.), and also for the identification of human DNA (Kanchanaphum, 2018). Moreover, LAMP has been used to determine the sex of various plants, including papaya (Tsai et al, 2016), asparagus (Shiobara et al, 2011), and spinach (Fujita et al, 2017). In raptor species, sex identification based on LAMP also has been developed (Centeno-Cuadros et al, 2016).…”

Section: Introductionmentioning

confidence: 99%