DBPIA-NURIMEDIA

Original Article Journal of Apiculture 32(2) : 119~131 (217) DOI: 1.17519/apiculture.217.6.32.2.119 Development of Rapid Detection System for Small Hive Beetle (Aethina tumida) by using Ultra-Rapid PCR Jung-Min Kim, Su-Jin Lim, Truong A Tai, Ki-Jeong Hong 1 and Byoung-Su Yoon* Department of Life Science, Kyonggi University, Suwon 16227, Korea 1 Department of Plant Medicine, College of Life Science and Natural Resources, Sunchon National University, Sunchon 57922, Korea (Received 2 June 217; Revised 28 June 217; Accepted 28 June 217) Abstract For the Rapid detection of small hive beetle (SHB; Aethina tumida) and for the mass-survey against SHB invasion, SHB-specific ultra-rapid PCR system was developed. Three different pairs of Aethina tumida-specific primers were deduced from cytochrome oxidase subunit I (COI) gene in mitochondrial DNA of SHB. Using optimized SHB-specific ultra-rapid PCR, 2.1 1 1 molecules of COI gene belonged to SHB could be detected specifically and quantitatively within 18 minutes 4 seconds. For the purpose of the application in apiary field, a DNA extraction method from bee debris was separatedly developed. When 1 5 SHB-specific COI molecules (1/1 body of SHB larvae) are existed in 1g of bee debris, it could be verified inner 1 minutes as qualitative and quantitative manner. SHB-specific ultra-rapid PCR we proposed would be expected to apply widely, either in apiary field or laboratory, for the rapid detections and the control against SHBinvasion. Key words: Ultra-Rapid PCR, Small Hive Beetle, SHB, Parasites, Honeybee (Small Hive beetle; SHB; Aethina tumida Murray), 1998 (Thomas, 1998),.,,, 214,. Kodamaea ohmeri *Corresponding author. E-mail: bsyoon@kyonggi.ac.kr 119

12,, (Benda et al., 28). Deformed Wing Virus (DWV) (Eyer et al., 28). 216 9,, (, 217).,. 1998, 1, Trap., 216 9,,.,,,.,,,, (Murihas, 24; Spiewok and Neumann, 26)., PCR. PCR PCR (cycle) (step;,, ), 4 PCR 1, (Lee et al., 27; Han et al., 28; Yoo et al., 212)., COI(cytochrome oxidase subunit I) (Evans et al., 2; Evans et al., 22), 1, (mitochondrial DNA; mtdna), DNA(eDNA; environmental DNA), COI, 1 (Microenvironment)., Ward (27) PCR (Real-Time PCR),,.,., PCR, DNA SHB PCR. (SHB) SHB

121 Table 1. Recombinat DNA clone for Cytochrome Oxidase subunit I in Small Hive Beetle Target insect Recombinant DNA Identical sequence in GenBank Target gene Small hive beetle Cytochrome oxidase ptop-shb COI* KP134137 Aethina tumida subunit I (COI) Honey bee ptop-rps 828** AADG636 Ribosomal Protein S18 Apis mellifera *DNA sequences of this clone is identical to reported sequences of KP134137 (236-98), except only 563 position. **DNA sequences of this clone is 1% identical to reported sequences of AADG636 (26215-2742)., SHB 9% EtOH 2 C. COI SHB genomic DNA (gdna),, DNeasy Blood & Tissue kit (Qiagen Co, Germany). gdna COI mtdna, Biophotometer (Eppendorf Co, Germany) DNA 7 C. (Apis mellifera) gdna, SHB. SHB. SHB (Bee debris), 2g SHB 1 DNA. SHB DNeasy Blood & Tissue kit (Qiagen Co, Germany) gdna, 7 C. 2g SHB 1 gdna (mtdna ) 3.59µg, 37ng/µl. 3ng gdna COI. SHB SHB COI (molecular cloning) (Table 1). GenBank Database SHB COI, 683bp. primer SHB-COI F/R, (Table 2). SHB-COI F/R SHB gdna PCR 683 PCR ( ), T-vector cloning, ptop- SHB COI. ptop-shb COI ( ), GenBank KP134137( SHB, 214) (683 682 ; ). PCR primer 2, NCBI primer Blast, SHB, (Apis mellifera) (Apis cerena), (213) (Galleria mellonella L.), (Achroia grisella F.), (Vespa mandarinia Smith), (Anax parthenope Julius), (Tenodera aridifolia Stoll), (Lasius niger).

122 Table 2. Specific primer pairs for SHB-specific COI gene and Honeybee-specific genes Target genes Name of primers Sequence (5 3 ) Reference Aethina tumida, Cytochrome oxidase subunit I (COI) Honey bee (β-actin) Honey bee (RPS) SHB-COI F TGCGACCCTCAGGCATAACC SHB-COI R TGGGAATCATTGAACAAATCCGGC This study SHB27F TCTAAATACTACTTTCTTCGACCCATC SHB315R TCCTGGTAGAATTAAAATATAAACTTCTGG Ward et al., 26 SHB-DP-F1 TGATTCTTCGGACACCCAGA SHB-DP-R1 AGGCTCGAGTATCAACGTCT This study SHB-DP-F2 CTACTTTCTTCGACCCATC SHB-DP-R2 AATGCTCATAGAGTTACTGG This study β-actin151-f ATGCCAACACTGTCCTTTCTGG Yang and Cox-. β-actin151-r GACCCACCAATCCATACGGA Foster 25 RPS18 417 F1 GTCATGGGCACGAATATTGA RPS18 417 R1 TTACTTCTTTTTCGATACACCCAC This study RPS18 173 F1 CATGGCTAATCCTAGACAATACAAG RPS18 149 R CCCAATAATGACGCAAACCT This study, Ward (27) SHB primer ptop-shb COI PCR,, PCR,, β-actin 4s Ribosomal Protein S18 (RPS) primer. primer (Table 2). SHB COI, primer annealing. 55 C 1 C PCR, DNA ptop-shb COI 2.9 1 6 (.3 femtomole= 3 attomole), primer.5µm, 1µl 2xGreenstar qpcr Master Mix (Bioneer, Korea), 2µl PCR. PCR Exicycler TM Quantit ative Thermal Block (Bioneer, Korea), 95 C 5min, 95 C 3, annealing 55 C 1 C 3, 72 C 3 1 35. Ct (Threshold Cycles) primer annealing, Genechecker (Genesystem, Korea) PCR. PCR ptop-shb COI 2.9 1 6, primer 1µM, 5µl 2 RapiMix (Genesystem, Korea), 1µl PCR. PCR 95 C, 3, 95 C 5, annealing 52~6 C 5, 72 C 5 1 4. Ct annealing. SHB COI primer, primer 1µM, 5µM, 2µM, 1µM, 5nM PCR. 1µl, ptop- SHB COI 2.9 1 6, 2 RapiMix primer PCR.

123 95 C, 3, 95 C 5, annealing 52~6 C 5, 72 C 5 1 4. SHB ptop-shb COI 2.9 1 6 2.9 1 PCR, 2 RapiMix, primer Forward Reverse primer 1µl PCR. PCR 95 C, 3 5,,, annealing, 95 C( ), primer annealing (52~6 C), 72 C( ), 3, 2, 1 PCR. gdna SHB primer 1ng gdna, PCR SHB primer 3 PCR, rdna β-actin1 primer. 1, ptop- SHB COI 2.9 1 6 1ng gdna, primer, 2 RapiMix PCR. 95 C, 3, 95 C 2, annealing 52~6 C 2, 72 C 2 1 4. gdna SHB ptop- SHB COI, PCR,. gdna 1ng, ptop-shb COI 2.9 1 6 2.9 1. PCR 1ng gdna 2.9 1 6 2.9 1 DNA, 2 Rapi master Mix, Primer, 95 C, 3, 95 C 2, annealing 52~6 C 2, 72 C 2 1 5 PCR. SHB- PCR SHB. 2g 1 SHB gdna 3.59µg, 3ng SHB PCR. total DNA SHB PCR, SHB,. Ward (27) SHB PCR SHB-, Ct (29.69 cycles)., gdna RPS β-actin gene PCR. SHB primer 3 annealing PCR (Real-Time PCR) Exicycler TM Quantitative Thermal Block (Bioneer CO.,

124 Table 3. Ct and Tm values on each annealing temperature of three primer pairs Annealing temperature ( C) SHB-DP F1/R1 SHB-DP F2/R2 SHB 27F/315R Ct Final fluorescence Ct Final fluorescence Ct Final fluorescence 52 - - 17.61.36 189.33 28.22 - - 54 15.99.53 181.33 26.22 16.85.47 25. 37.33 19.46.68 172. 32.67 56 15.17.34 187. 27.33 17.89.78 182.67 25.11 19.1.99 178.67 26.89 58 16.89.18 156.67 17.11 - - 21.31 1.54 158. 32.67 6 16.96.56 157.67 22.44 - - 21.29.84 147. 26. Tm values ( C) 74.91.32 77.2.16 76.53.32 Table 4. Ct and Tm values for each concentration of three primer pairs Final concentration SHB-DP F1/R1 SHB-DP F2/R2 SHB 27F/315R Ct Final fluorescence Ct Final fluorescence Ct Final fluorescence 1µM 16.37.31 142.67 6.89 15.87.16 184. 6. 18.77.22 15.67 3.11 5µM 14.53.31 169.67 3.11 14.83.18 22.67 4.89 17.73.24 17.33 1.56 2µM 15.27.16 192.33 3.78 15.6.4 251.67 2.89 18.4.27 185.33 4.89 1µM 15.1.13 232.67 15.78 16.2.33 255. 2.67 2.2.33 25. 4.67 5nM 16.7.22 233.33 7.56 19.3.2 265.33 6.44 23.17.31 199.33 5.11 Tm values ( C) 74.91.33 76.69.16 76.86.32 Korea) PCR. SHB primer SHB-DP F1/R1, SHB-DP F2/R2, 45~65 C, SHB-DP F1/R1 primer 56 C 4 C, SHB-DP F2/R2 primer 54 C 4 C. SHB 27F/315R primer 57 C 3 C annealing, 3 primer 52~6 C annealing PCR annealing ( ). PCR Genechecker (Genesystem, Korea), 52~6 C annealing Ct (Table 3). SHB-DP F1/R1 primer 56 C Ct, 54 C. 74.91.33. SHB-DP F2/R2 primer 54 C Ct, 77.2.16 C. SHB 27F/315R primer Ct 54, 56 C Ct 56 C. PCR 3 SHB primer annealing, primer, 54 C. PCR primer 5nM(5pmole/µl), 1µM(1pmole/µl), 2µM(2pmole/µl), 5µM(5pmole/µl) 1µM(1pmole/µl) PCR (Table 4). primer 3, Ct Melting graph. SHB-DP F1, R1 primer 1µM Ct

125 Molecules of DNA (Log 1 ) 7 6 5 4 3 2 1 y=-.2615x + 1.619 R 2 =.9989 y=-.2621x + 11.454 R 2 =.9981 y=-.353x + 12.298 R 2 =.9935 15 2 25 3 35 4 Ct (Threshold cycle) DP1 DP2 SHB Fig. 1. Detection limit and Tm using three primer pairs for SHB COI specific Ultra-rapid quantitative PCR. Ultra-rapid PCR with each primer pairs was performed using optimal condition. ptop-shb COI was used as template in PCR that was diluted from 2.9 1 6 to 2.9 1 molecules /PCR, respectively. 2.9 1 1 molecules template were amplified successfully by UR-PCR using SHB DP F2/R2 pairs and SHB 27F/315R pairs, except SHB DP F1/R1 pairs. primer, SHB-DP F2, R2 primer 2µM Ct. SHB 27F, 315R primer 2µM Ct 1µM, 5µM Primer Ct, 1µM,.5µM Primer Ct 2cycle 2µM. 3 primer annealing primer DNA 2.9 1 8 2.9 1. PCR,, 2, 2, 2, 3 Ct Tm. PCR 5 18 4 (Fig. 1). SHB-DP F1/R1 primer PCR, 2.9 1 2, Ct, Ct (Regression equation) Y=-.2615X +1.619 (Regression coefficient; R 2 ).9989., 2.9 1 1 3 1. Tm (Temperature of mid-point) 76. C, 2.9 1 Tm 73.3 C, 73. C, Tm. SHB-DP F2/R2 primer PCR, 2.9 1 1, Y= -.2621X+11.454, R 2 =.9981. Tm 77.7 C, 2.9 1 Tm 74.3 C, 72.3 C, Tm. SHB 27F/315R primer PCR, 2.9 1 1, Y=-.353X+12.298, R 2 =.9935. Tm 77.6 C, Tm 69. C, Tm (Table 5). SHB 3 primer DNA primer 1ng gdna. RPS gdna, RPA- DNA PCR Tm, gdna. SHB 3 primer gdna PCR,

126 Table 5. Ct and Tm using three primer pairs for SHB-specific Ultra-rapid qpcr Molecules of recombinant DNA SHB-DP F1/R1 SHB-DP F2/R2 SHB 27F/315R Ct Tm Ct Tm Ct Tm 2.9 1 6 16.62.66 76.7.17 19.44.85 77.67.3 18.85.8 77.87.6 2.9 1 5 2.15 1.47 75.67.18 23.58 1.66 77.49.1 23.13.91 77.6.5 2.9 1 4 23.81 1.6 75.85.28 26.91.81 77.39.18 26.51 1.5 77.89.34 2.9 1 3 28.1.87 75.67.18 31.54 1.72 77.71.38 3.1.94 77.69.17 2.9 1 2 31.74 1.9 76.5.29 34.91 1.24 77.69.17 33.9 1.53 77.6.5 2.9 1 1 34.49 2.22 74.59 1.38 38.37.52 77.58.35 35.28 1.42 77.6.5 2.9 1 37.17 1.7 73.29 1.38 4.19 2.14 74.26 38.93.99 77.6.19 Negative 28.6 7.4 72.97.46 38.91 1.29 72.31 37.74 68.96 1.8 Average Tm value (Positive) 75.84.26 77.59.25 77.69.38 Average Tm value (Negative) 73.22.78 73.29 1.38 68.96 1.8 Total reaction time 18min 4sec / 5 cycles 9 6 3 9 6 3 RPS18 173 F1, 149R 6 7 8 9 SHB DP F2, R2 6 7 8 9 Fig. 2. Melting temperature analysis of ultra-rapid PCR with genomic DNA from honeybee and/or SHB-specific DNA using various primer pairs. RPS genes were successfully amplified by both ultra-rapid PCRs with gdna from honeybee and/or RPA-specific DNA. All ultra-rapid PCRs using SHB-DP F1/R1, -DP F2/R2, -27F/315R pairs with gdna from honeybee and/or SHB-specific DNA produced correct or incorrect product, respectively. 9 6 3 9 6 3 SHB DP F1, R1 6 7 8 9 SHB 27F,315R 6 7 8 9, Tm PCR 3 C. gdna SHB DNA, 2.9 1 2 SHB PCR (Fig. 2). gdna SHB DNA primer SHB. SHB ptop-shb COI 2.9 1 6 2.9 1, 1ng gdna PCR (Fig. 3; 1ng 3kb DNA.5 femtomole, 3 1 8 molecules ). Primer 3 SHB PCR 1ng gdna 2.9 1 1, Tm SHB DP F1/R1 primer 75.82 C.21 C, SHB DP F2/R2 primer 77.82 C.25 C, SHB 27F/315R primer 77.82 C.17 C

127 Molecules of DNA (Log 1 ) 7 6 5 4 3 y=-.265x + 11.118 R 2 =.9945 y=-.2447x + 11.839 R 2 =.9847 y=-.2757x + 12.145 R 2 =.995 2 1 15 2 25 3 35 4 45 Ct (Threshold cycle) DP1 DP2 SHB Fig. 3. Detection limit of ultra-rapid PCR using three SHB-specific primer pairs with SHB-specific DNA and honeybee gdna. Ultra-rapid PCR reactions were performed at optimal conditions for each primer pairs with 1ng honeybee gdna. All ultra-rapid PCRs using SHB DP1, SHB DP2, SHB 27F/315R primer pairs could be detected with 2.9 1 1 molecules of SHB-specific DNA under 1 ng honeybee gdna.. SHB DNA, 2.9 1 PCR,, primer Tm, 79.38 C.59 C (SHB DP F1/R1 primer ), 79.53 C 1.38 C(SHB DP F2/R2 primer ), 71.59 C.57 C(SHB 27F/315R primer ), Tm SHB- Tm., 3 SHB primer SHB- PCR SHB 2.9 1 1 SHB, (Table 6). SHB, gdna primer 2 (RPA, β-actin) SHB primer 3 (SHB-DP F1/R1, -DP F2/R2, -27F/315R) PCR (Fig. 4). β-actin RPS gdna, Tm, DNA ( ), gdna. SHB primer 3 PCR SHB COI, SHB Tm. Table 6. Ct and Tm values for each number of molecular copies with 3 primer pairs in 1ng Honeybee gdna Molecules of recombinant DNA SHB-DP F1/R1 SHB-DP F2/R2 SHB 27F/315R Ct Tm Ct Tm Ct Tm 2.9 1 6 18.38.83 76.6.12 23.8 1.4 77.88.18 2.9.76 77.78.19 2.9 1 5 21.22.72 75.69.26 25.31.22 77.7.33 24.16.98 77.6.15 2.9 1 4 25.65.41 75.76.13 31.92.45 77.91.16 29.17 1.37 77.68.28 2.9 1 3 3.26 1. 75.76.13 35.32.67 77.82.22 32.45 1.26 77.8.3 2.9 1 2 33.13 1.8 75.88.21 38.4.46 78.2.18 35.65.26 77.89.9 2.9 1 1 36.57.77 75.75.45 42.92.7 77.61.36 39.54.64 78.2.18 2.9 1 36.57.75 79.57.54 44.23.76 79.21 1.71 42.16 1.3 78.47. Negative 27.22 9.15 79.19.65 44.24 1.32 79.84 1.4 42.4 1.48 71.59.57 Average Tm value (Positive) 75.82.21 77.82.25 77.82.17 Average Tm value (Negative) 79.38.59 79.53 1.38 71.59.57 Total reaction time 18min 4sec

128 β-actin 151 F, R RPS18 173 F1, 149R 9 9 6 6 3 3 6 7 8 9 6 7 8 9 SHB DP F1, R1 SHB DP F2, R2 SHB 27F, 315R 9 9 9 6 6 6 3 3 3 6 7 8 9 6 7 8 9 6 7 8 9 Fig. 4. Melting temperature analysis of Ultra-rapid PCRs using 5 different primer pairs with total DNA from bee debris and SHB larvae. 3.59µg total DNA was isolated from 2 gram bee debris and 1 SHB larvae. Ultra-rapid PCRs using each primer pairs with 3ng total DNA were performed. All results demonstrated successful amplification according to primers-specificity., 2g SHB, total DNA 3.59µg, 3ng total DNA 8/1, SHB SHB mtdna, PCR. SHB SHB PCR. SHB total DNA 1/1 total DNA SHB PCR (Fig. 5). total DNA 3ng, 3ng,.3ng,.3ng, SHB-DP F1/R1, -DP F2/R2 primer PCR SHB. total DNA 3ng, 3ng,.3ng,.3ng 8/1,, 8/1,, 8/1,, 8/1,, SHB DNA, 1 1 SHB,,. SHB DNA,.8 SHB SHB 6.64 1 3,.8 SHB SHB 1.37 1 3,.8 SHB SHB 2.38 1 2,.8 SHB SHB

129 Amount of DNA (Log 1 ) 6 5 4 3 2 1 y=-.268x + 1.371 R 2 =.9971 15 2 25 3 35 4 Ct (Threshold cycle) Standard 8/1 8/1 8/1 8/1 Fig. 5. CTs of Ultra-rapid PCRs using serially diluted total DNA from bee debris and SHB larvae on regression equation using C T s and SHB-specific DNAs. Total DNA was isolated from 2 gram of bee debris and 1 SHB larvae. Serially diluted total DNA were measured as.8 (6.64 1 3 molecules),.8 (1.37 1 3 molecules),.8 (2.38 1 2 molecules),.8 (1.31 1 1 molecules) SHB larvae, respectively. Standard is indicated PCRs using serially diluted SHB-specific DNA. 8/1, 8/1, 8/1, 8/1 are indicated.8,.8,.8,.8 SHB larvae, respectively. 1.31 1 1. SHB, SHB. (SHB). SHB, SHB SHB, SHB. SHB, SHB, SHB (,,, SHB ), SHB (Murihas, 24; Spiewok and Neumann, 26).,., (micro-environment).. SHB, DNA (edna; environmental DNA) SHB DNA, PCR total DNA SHB DNA. Ward (27) PCR PCR, PCR, 5 C 2 95 C 1, 95 C 15, 6 C 1 4. PCR 2 / PCR, (5 C, 95 C, 6 C) (Ramping time), 62, PCR 79 Sequence Detection System (Applied Biosystems, USA) PCR 2., PCR, 95 C 3, 95 C 2, 56 C 2, 72 C 2 1 5 Genechecker (Genesystem, Korea). PCR 18 4, 4 (15 ) Ct (Table 6).

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