Korean journal of applied entomology (한국응용곤충학회지)

Korean Society of Applied Entomology (한국응용곤충학회)
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A Loop-mediated Isothermal Amplification Method for White-backed Planthopper-specific Detection

고리매개등온증폭법(LAMP)을 이용한 흰등멸구 특이 판별법

  • Seo, Bo Yoon (Crop Protection Division, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Park, Chang Gyu (Department of Industrial Entomology, Korea National College of Agriculture and Fisheries) ;
  • Jung, Jin Kyo (Crop Cultivation and Environment Research Division, National Institute of Crop Science, Rural Development Administration) ;
  • Cho, Jumrae (Crop Protection Division, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Lee, Gwan-Seok (Crop Protection Division, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Kim, Kwang-Ho (Crop Protection Division, National Institute of Agricultural Sciences, Rural Development Administration)
  • 서보윤 (국립농업과학원 작물보호과) ;
  • 박창규 (국립한국농수산대학 산업곤충과) ;
  • 정진교 (국립식량과학원 재배환경과) ;
  • 조점래 (국립농업과학원 작물보호과) ;
  • 이관석 (국립농업과학원 작물보호과) ;
  • 김광호 (국립농업과학원 작물보호과)
  • Received : 2018.11.05
  • Accepted : 2018.11.26
  • Published : 2018.12.01
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Abstract

A loop-mediated isothermal amplification (LAMP) primer set (WBPH-65) was designed for the species-specific detection of white-backed planthopper (WBPH) Sogatella furcifera based on the full-length sequence of the internal transcribed spacer 2 (ITS2) (KC417469.1). The WBPH-65 primer set consists of six primers (total 165 bp), F3 (18 bp), B3 (18 bp), FIP (43 bp), BIP (40 bp), LF (21 bp), and LB (25 bp). After the LAMP reaction of three rice planthoppers, S. furcifera, Nilaparvata lugens, and Laodelphax striatellus, with the WBPH-65 primer set for 60 min at $65^{\circ}C$, the LAMP products were observed in the genomic DNA of S. furcifera only. According to the DNA amount of S. furcifera and incubation duration at $65^{\circ}C$, the difference of fluorescence relative to the negative control (0 ng) was clearly observed in a 40-min incubation with 10 and 100 ng or in case of 60-min incubation with 0.01, 0.1, 1, 10, and 100 ng. There was little difference in fluorescence between the negative control and all the other DNAs tested in 20- and 30-min incubations. On the other hand, the WBPH-65 primer set without LF and LB primers showed little amplification in the genomic DNAs of the three rice planthoppers, S. furcifera, N. lugens, and L. striatellus in a 60-min incubation. These results suggest that all six primers (F3, B3, FIP, BIP, LF, and BF) are necessary for the WBPH-65 primer set to detect S. furcifera within a 60-min incubation, and is able to discriminate S. furcifera from at least N. lugens and L. striatellus.

고리매개등온증폭법(LAMP)으로 흰등멸구를 특이적으로 구별해낼 수 있는 프라이머 세트(WBPH-65)가 핵내 ITS2영역의 전체염기서열(KC417469.1)을 바탕으로 설계 제작되었다. WBPH-65는 총 6개의 프라이머, F3 (18 bp), B3 (18 bp), FIP (43 bp), BIP (40 bp), LF (21 bp), LB (25 bp)로 구성되는데, 전체 합한 길이가 165 bp이다. WBPH-65를 흰등멸구, 벼멸구 및 애멸구의 게놈 DNA와 $65^{\circ}C$에서 60분간 고리매개등온증폭 반응시켰을 때, 흰등멸구 시료에서만 증폭 산물들이 관찰되었다. $65^{\circ}C$에서 WBPH-65와 흰등멸구 게놈 DNA의 양과 반응시간을 달리하여 형광반응을 관찰하였을 때 40분 반응에서는 10과 100 ng DNA에서, 60분 반응에서는 0.01, 0.1, 1, 10, 100 ng DNA에서 발광여부가 명확히 구별되었다. 그러나 20분과 30분 반응에서는 준비된 모든 DNA 양에서 발광여부 구별이 어려웠다. 한편, WBPH-65에서 LF와 BF 프라이머를 뺀 경우 60분 반응에서는 벼멸구, 애멸구 뿐만 아니라 흰등멸구의 게놈 DNA에서도 발광되지 않았다. 본 연구 결과로부터 WBPH-65가 60분 이내 반응에서 흰등멸구를 특이적으로 구별하기 위해서는 6개의 프라이머가 모두 필요하며 최소한 벼멸구와 애멸구를 구별해낼 수 있음을 확인하였다.

Keywords

OOGCBV_2018_v57n4_393_f0001.png 이미지

Fig. 1. Agarose gel electrophoresis (left) and visual detections (right) after loop-mediated isothermal amplification (LAMP) reaction of DNA samples with the LAMP primer set (WBPH-65). In the agarose gel electrophoresis, various size of bands, such as ladder, were observed in the PC and 100 ng of WBPH DNA only, but no band in the NC and single bands in 100 ng of BPH DNA and 100 ng of SBPH DNA were observed. This indicates that the WBPH-65 LAMP primer set designed in this study can amplify the target region in the genomic DNA of WBPH specifically, but not in the genomic DNA of BPH and SBPH. Visual detections showed that the WBPH-65 LAMP primer set was also able to specifically amplify WBPH genomic DNA with a sensitivity of 0.01 ng in minimum, but not in the two rice planthoppers, BPH and SBPH. For LAMP reaction, all samples were incubated at 65℃ for 60 min and enzyme was inactivated at 80℃ for 5 min. †M, 100 bp ladder marker; PC, positive control (LAMP primer set and DNA, both were provided by manufacturer); NC, negative control (WBPH-65 LAMP primer set without DNA); WBPH, white-backed planthopper Sogatella furcifera; BPH, brown planthopper Nilaparvata lugens; SBPH, small brown planthopper Laodelphax striatellus.

OOGCBV_2018_v57n4_393_f0002.png 이미지

Fig. 2. Visual detections after loop-mediated isothermal amplification (LAMP) reaction with the LAMP primer set (WBPH-65) according to the incubation time (20, 30, 40, and 60 min) and the amount of genomic DNA (0, 0.01, 0.1, 1, 10, and 100 ng) of white-backed planthopper (WBPH) Sogatella furcifera. After the LAMP reaction, enzyme within the tube was inactivated at 80℃ for 5 min.

OOGCBV_2018_v57n4_393_f0003.png 이미지

Fig. 3. Loop-mediated isothermal amplification (LAMP) reaction results on five individuals of three rice planthoppers, white-backed planthopper (WBPH; Sogatella furcifera), brown planthopper (BPH; Nilaparvata lugens), and small brown planthopper (SBPH; Laodelphax striatellus) with two LAMP primer sets of WBPH-65, [F3 + B3 + FIP + BIP + LF + LB] and [F3 + B3 + FIP + BIP], respectively. The incubation time was 60 min at 65℃ and the amount of genomic DNA of each planthopper was > 100 ng. After the LAMP reaction, a Bst polymerase was inactivated at 80℃ for 5 min.

Table 1. Loop-mediated isothermal amplification (LAMP) primer sets designed in this study to discriminate Sogatella furcifera from other planthopper species

OOGCBV_2018_v57n4_393_t0001.png 이미지

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