Dataset Information

Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas.

ABSTRACT: Ascochyta blight (AB) is a devastating fungal disease of chickpeas that has spread to nearly all of the chickpea cultivating regions of the world. The rapid diagnosis of Ascochyta rabiei L. (A. rabiei), the pathogen that causes AB, plays an important role in A. rabiei epidemic tracking and AB management. In this study, a group of loop-mediated isothermal amplification (LAMP) primers was designed to detect A. rabiei in chickpea plants and seeds via a LAMP method and a conventional PCR method based on an internal transcribed spacer (ITS) sequence analysis of A. rabiei. Compared with the conventional PCR method, the LAMP method not only exhibited greater sensitivity and specificity in the detection of A. rabiei but also used simpler equipment and required less operational time. The minimum detectable concentration of the A. rabiei genomic DNA solution with the LAMP method was 6.01?×?10(-6?)ng/?l, which was 100 times lower than that of the conventional PCR method with the same outer primers. The greatest advantage of the LAMP method is that results can be observed via the visualization of color changes in SYBR Green I dye with the naked eye, and it does not require expensive instruments, also with less time consumption.

SUBMITTER: Chen X

PROVIDER: S-EPMC4861956 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas.

Chen Xiaolu X Ma Lijuan L Qiang Song S Ma Deying D

Scientific reports 20160510

Ascochyta blight (AB) is a devastating fungal disease of chickpeas that has spread to nearly all of the chickpea cultivating regions of the world. The rapid diagnosis of Ascochyta rabiei L. (A. rabiei), the pathogen that causes AB, plays an important role in A. rabiei epidemic tracking and AB management. In this study, a group of loop-mediated isothermal amplification (LAMP) primers was designed to detect A. rabiei in chickpea plants and seeds via a LAMP method and a conventional PCR method base ...[more]

PMID: 27161564

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Project description:Purpose:Currently, thalassemia is commonly detected using gap-polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) reverse dot blot, which have high requirements of space, instruments, and personnel. Therefore, it is necessary to develop a new method for thalassemia detection with high sensitivity, low cost, and simple and fast operation. In this study, we aimed to design and evaluate a new method for detecting three ?-thalassemia genes including -Southeast Asian (SEA), -?3.7, and -?4.2 and five ?-thalassemia genes including 654M, 41/42M, -28M, 17M, and 27/28M based on loop-mediated isothermal amplification (LAMP). Methods:Primer sequences were designed using Primer Explorer V4 software. Blood samples (5 mL) were collected from all participants in EDTA. DNA was extracted using Chelex 100 and was subjected to LAMP. LAMP products were detected by fluorescence development in ultraviolet light. Results:We found that LAMP assays for positive samples of thalassemia reached a plateau before 60 minutes, whereas the negative control samples entered the plateau after 70 minutes or showed no amplification. The concentration range of positive reactions was between 20-60 pg/?L and 20-60 ng/?L. Additionally, there were no cross-reactivities among 8 thalassemia subtypes. For clinical samples, the positive sample tube showed strong green fluorescence, whereas the negative tube showed light green fluorescence. According to these results, the LAMP method has high sensitivity for detecting thalassemia (252/254). However, 43 false-positive results were obtained in the LAMP test. The LAMP assay was also of low cost and with simple and fast operation. Conclusion:The novel LAMP assay can be completed within 60 min using a heating block or a water bath, and the result can be read visually based on color change to detect thalassemia. The LAMP assay fulfills the requirements of field application and resource-limited areas, especially those with primary hospitals and rural areas.

Dataset Information

Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas.

Publications

Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas.

Similar Datasets

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