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| Establishment of Nested-PCR and LAMP Assays for Detection of Peronophythora litchii |
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Abstract Litchi downy blight, caused by Peronophythora litchii, is one of the most destructive diseases affecting litchi (Litchi chinensis). Rapid and accurate detection of P. litchii is essential for early diagnosis and timely controlling the disease in the field. Based on the GTP-binding protein gene (Ypt1) of P. litchii, nested PCR and loop-mediated isothermal amplification (LAMP) primers were designed in this study. The reaction conditions were optimized, and the specificity and sensitivity were also assayed. For specificity testing, DNA extracted from 21 P. litchii isolates, 34 isolates of 13 closely related species of different oomycetes and 8 isolates of other fungal species were used in this study. The results showed that the PCR specific primer pair PvYF1/PvYR1 amplified an unique DNA fragment of 249 bp in all P. litchii isolates from geographically distinct counties in China, but there was no amplification product in any other non-P. litchii isolates. In addition, optimization of LAMP reaction conditions (temperature and time) revealed that the ideal settings for the primer set were 63 ℃ for 60 min, and a positive color (green) was only observed in the presence of P. litchii with calcein as a fluorescence indicator and the typical ladder-like banding pattern were observed on 2.0% gel electrophoresis in all DNA products of P. litchii. The identities of the amplified products of LAMP were also confirmed by direct sequencing, in which the sequences obtained were perfectly matched with the expected DNA sequences. The results also showed that the detection sensitivity was creased by 1000-fold to 100 fg/25 μL genomic DNA by developed a nested PCR, which amplified using Phytophthora genus Ypt1 universal primer pair Yph1F/Yph2R as the first round primers and PvYF1/PvYR1 as the second round primers. The detection sensitivity of LAMP assay was 10 fg/25 μL genomic DNA, which was up to 10 times higher than that of nested-PCR. Positive detection rates of P. litchii in diseased litchi samples collected from naturally field was also evaluated using conventional PCR, nested PCR and LAMP assays, and the isolations of P. litchii from these samples were verified using conventional isolation methods. Purified DNA was used as a positive control while sterile distilled water was used as a negative control. Finally, 30 infected and healthy looking but infested litchi leaves or fruits naturally and 3 healthy litchi leaves or fruits from Zhangzhou county were examined, the positive detection rate were 17/22 (77.3%) by conventional PCR, 20/22 (90.9%) by nested PCR, 21/22 (95.5%) by LAMP assay and 22/22 (100%) by conventional isolation method; 25 infected and healthy looking but infested litchi leaves or fruits naturally and 3 healthy litchi leaves or fruits from Putian county, the positive detection rate were 15/17 (88.2%) by conventional PCR, 16/17 (94.1%) by nested PCR, 17/17 (100%) by LAMP assay and 17/17 (100%) by conventional isolation method. Healthy litchi samples were inspected using conventional PCR, nested PCR, LAMP assay and traditional isolation methods, and all samples were negative for P. litchii. All reactions were repeated twice with consistent results. We conclused that LAMP is more sensitive, more specific and simpler than PCR, and the results can be visualized with the naked eye for the detection of P. litchii in resource-poor settings.
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Received: 26 November 2015
Published: 06 May 2016
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Corresponding Authors:
WENG QiYong
E-mail: wengqy@faas.cn
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| [1] |
LI Hua-Wei, LIU Zhong-Hua, ZHANG Hong, XU Yong-Qing, LI Guo-Liang, LIN Zhao-Miao, XU Guo-Chun, QIU Yong-Xiang, JI Rong-Cang, LUO Wen-Bin, TANG Hao QIU, Si-Xin. Establishment and Application of a LAMP Method for the Rapid Detection of Sweet potato leaf curl virus[J]. 农业生物技术学报, 2019, 27(6): 1133-1140. |
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