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Evaluation of Loop Mediated Isothermal Amplification Based Methods for the Detection of African Swine Fever Virus from Food Waste
Biomed Sci Letters 2022;28:334-339
Published online December 31, 2022;  https://doi.org/10.15616/BSL.2022.28.4.334
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Siwon Lee1,*, Junhwa Kwon2,**, Su Hyang Kim2,**, Jin-Ho Kim3,4,†,**, Jaewon Jung1,5,***, Kyung-Jin Lee4,***, Ji-Yeon Park4,***, Taek-Kyun Choi6,****, Jun-Gu Kang2,***** and Tae Uk Han2,†,******

1R&D Team, LSLK Co. Ltd., Gimpo 10111, Korea
2Waste-to-Energy Research Division, Environmental Resources Research Department, National Institute of Environmental Research, Incheon 22689, Korea
3Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Chungam 31116, Korea
4Department of Chemistry, College of Science and Engineering, Dankook University, Chungnam 31116, Korea
5Division of Biological Science and Technology, Yonsei University, Wonju 26493, Korea
6KOTITI Testing & Research Institute, Seongnam 13202, Korea
Correspondence to: Jin-Ho Kim. Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Chunam 31116, Korea.
Tel: +82-41-529-6256, Fax: +82-41-559-7860, e-mail: 12180374@dankook.ac.kr
Tae Uk Han. Waste-to-Energy Research Division, National Institute of Environmental Research, Incheon 22689, Korea.
Tel: +82-32-560-7530, Fax: +82-32-568-1658, e-mail: taeukhan@korea.kr
*Research director, **Researcher, ***Undergraduate student, ****General Manager, *****Director, ******Senior Researcher.
Received November 1, 2022; Accepted December 5, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
African swine fever virus (ASFV) is a highly contagious and lethal pathogen that poses a threat to the global pork industry. The World Organization for Animal Health (WOAH) has placed strict surveillance measures for ASFV. The possibility of long-term survival of ASFV in raw meat or undercooked pork has been reported. Accordingly, the problem of secondary infection in food waste from households or waste disposal facilities has emerged, raising the need for ASFV monitoring of food waste. However, most of the previously reported ASFV gene detection methods are focused on clinical monitoring of pigs. There are very few cases in which their application in waste has been verified. Since ASFV diagnosis requires rapid monitoring and immediate action, loop-mediated isothermal amplification (LAMP) may be suitable, but this requires conformity assessment for LAMP to be used as a diagnostic technique. In this study, six LAMP methods were evaluated, and two methods (kit and manual) were recommended for use in diagnosing ASFV in food waste.
Keywords : African swine fever virus, ASFV, Food waste, LAMP
Body

African swine fever virus (ASFV)뒗 Asfarviridae 怨쇰줈 遺꾨쪟릺뒗 씠以 媛떏 DNA 諛붿씠윭뒪濡, 넂 쟾뿼꽦怨 移섏궗쑉 諛 쟾 꽭怨꾩쟻 뤌吏怨좉린 궛뾽뿉 떖媛곹븳 쐞삊쑝濡 씤빐 꽭怨꾨룞臾쇰낫嫄닿린援[World Organization of Animal Health; OIE]쓽 뾼寃⑺븳 媛먯떆瑜 諛쏄퀬 엳뒗 蹂묒썝泥댁씠떎(Gaudreault et al., 2020). ASFV뿉 븳 뿰援ъ 愿떖 遺遺 뤌吏 엫긽뿉 吏묒쨷릺뼱 蹂닿퀬릺怨 엳떎(Agüero et al., 2003; Basto et al., 2006; O'Donnell et al., 2015). 洹몃윭굹, 議곕━븯怨 궓 깮怨좉린 삉뒗 遺덉땐遺꾪븯寃 議곕━맂 뤌吏怨좉린 벑뿉꽌 ASFV媛 옣湲곌컙 깮議 媛뒫꽦씠 蹂닿퀬맖뿉 뵲씪, 媛젙 삉뒗 泥섎━ 떆꽕쓽 쓬떇臾쇱벐젅湲 삉뒗 쓬떇臾쇱벐젅湲 湲곕컲쑝濡 옱깮궛맂 궗猷뚯뿉꽌 옱 媛먯뿼 媛뒫꽦 벑 씠李 媛먯뿼 臾몄젣媛 몢릺뿀怨, 씠뿉 뵲씪 援궡뿉꽌뒗 궓 쓬떇臾쇱쓽 吏곸젒泥섎━湲됱뿬瑜 젣븳븯뒗 룓湲곕Ъ愿由щ쾿 떆뻾洹쒖튃 媛쒖젙, 쓬떇臾쇰쪟룓湲곕Ъ쓽 뤌吏 궗猷뚰솕 젣븳 벑 젙遺쟻 쓳씠 씠猷⑥뼱議뚮떎[National Institute of Environmental Research (NIER), 2020]. 삉븳, 궗猷 삉뒗 눜鍮꾪솕瑜 쐞븳 泥섎━ 떆꽕 벑뿉꽌 쓬떇臾쇰쪟룓湲곕Ъ쓣 긽쑝濡 ASFV뿉 븳 紐⑤땲꽣留 븘슂꽦씠 젣湲곕릺뿀쑝굹, 湲곗〈 蹂닿퀬맂 ASFV 쑀쟾옄 寃異 諛⑸쾿뱾씠 쓬떇臾쇰쪟룓湲곕Ъ뿉꽌 쟻슜꽦씠 寃利앸맂 궗濡뒗 洹뱁엳 뱶臾쇰떎(Lee et al., 2022). 븳렪, 벑삩 利앺룺踰(Loop mediated isothermal amplification; LAMP) 以묓빀슚냼뿰뇙諛섏쓳怨 룞씪븳 빑궛 利앺룺踰 湲곕컲쓽 吏꾨떒 湲곗닠씠떎. LAMP뒗 떊냽븯怨, 슦닔븳 寃異 誘쇨컧룄瑜 媛吏 寃궗踰뺤쑝濡 듅닔븳 삩룄議곗젅옣移 뾾씠 룞씪븳 삩룄瑜 쑀吏떆耳 以 닔 엳뒗 빆삩 닔議곕굹 삤釉먭낵 媛숈 媛꾨떒븳 옣鍮꾧 엳쑝硫 쁽옣뿉꽌룄 諛섏쓳씠 媛뒫븯硫, 룞떆뿉 諛섏쓳븷 닔 엳뒗 諛섏쓳 닔媛 利앷븯湲 븣臾몄뿉 留롮 떆猷뚯쓽 떊냽븳 諛섏쓳뿉 옣젏씠 엳떎(Ahn et al., 2008; Cho et al., 2013; Kim et al., 2017). OIE 洹쒖젙뿉 뵲씪 떊냽븳 紐⑤땲꽣留곴낵 利됯컖 議곗튂媛 슂援щ릺뒗 ASFV 吏꾨떒뿉 엳뼱 LAMP 諛⑸쾿씠 쟻빀븯寃 솢슜맆 닔 엳쑝굹, 쓬떇臾쇰쪟룓湲곕Ъ 벑 솚寃 떆猷뚯쓽 珥 빑궛 엫긽 떆猷뚯뿉 鍮꾪빐 긽쟻쑝濡 궙 닔以쓽 蹂묒썝泥 냽룄, 떎뼇븯怨 넂 닔以쓽 븣 닔 뾾뒗 寃異 빐 臾쇱쭏 룷븿 벑쓣 썝씤쑝濡 吏꾨떒뿉 쁺뼢씠 엳쓣 媛뒫꽦씠 엳뼱(Lee et al., 2021, 2022), 吏꾨떒 湲곗닠 쟻빀꽦뿉 븳 룊媛媛 븘슂븯떎. 뵲씪꽌 蹂 뿰援ъ뿉꽌뒗 LAMP 湲곕컲쓽 湲곗〈 蹂닿퀬맂 吏꾨떒踰 諛 떆以 쑀넻릺뒗 kit뿉 븳 쓬떇臾쇰쪟룓湲곕Ъ 떆猷뚯뿉꽌쓽 솢슜꽦쓣 룊媛븯떎.

ASFV 빑궛 誘멸뎅 援由쎌깮臾쇱젙蹂댁꽱꽣(National Center for Biotechnology Information; NCBI) accession number MN207061.1 (P72-73 gene)쓣 湲곗쑝濡 Marcrogen Co. Ltd. (Seoul, Korea)뿉꽌 吏꾪빀꽦 븯떎. 삉븳 李멸퀬 諛붿씠윭뒪 二[Porcine circovirus 2 (PCV2), Porcine parvovirus (PPV) 諛 Porcine Pseudorabies virus (PPrV)]뒗 媛곴컖 PCV2 NC_005148 (1,034~1,283, 250 nt), PPV NC_001718 (2,387~2,636, 250 nt) 諛 PPrV NC_006151 (66,781~67,030, 250 nt)濡 빀꽦븯떎(Lee et al., 2022). LAMP瑜 썝由щ줈 븯뒗 5醫낅쪟 [諛⑸쾿 #1, Dokphut et al., 2021; #2, Shaanxi innolever biological technology Co., Ltd.; #3, James et al., 2010; #4 & #5, Center for animal disease control and prevention of Beijing (2醫낅쪟)]怨 떆以 쑀넻릺뒗 1媛쒖쓽 냽由쇰 뿀媛 kit [MmisoASFV detection kit (Mmonitor, Korea)]瑜 긽쑝濡 쓬떇臾쇰쪟룓湲곕Ъ뿉꽌 ASFV 寃異 꽦뒫쓣 룊媛븯떎(Table 1). 쓬떇臾쇰쪟룓湲곕Ъ 떆猷뚮뒗 以묎컙 媛怨, 諛붿씠삤媛뒪, 눜鍮꾪솕 벑쓽 쟾援 떆꽕濡쒕꽣 臾댁옉쐞 20媛 吏젏쓣 긽쑝濡 븯떎(NIER, 2020). Homogenize Kit (Innogenetech, Korea) MP FastPrep-24TM 5G (MP Biomedicals, USA)瑜 궗슜븯뿬 쓬떇臾쇰쪟룓湲곕Ъ 떆猷뚮 遺꾩뇙븯떎(Lee et al., 2022). 遺꾩뇙븸 140 μL濡 遺꽣 QIAamp® DNA Mini kit (Qiagen, Germany)쓽 留ㅻ돱뼹뿉 뵲씪 珥 빑궛쓣 異붿텧븯쑝硫, DS-11 Spectrophotometer (DeNOVIX, USA)濡 냽룄 諛 닚룄瑜 痢≪젙븳 寃곌낵, 룊洹 76.27 ng/μL쓽 냽룄 諛 1.93쓽 닚룄濡 遺꾩꽍뿉 쟻빀븳 닔以쑝濡 솗씤릺뿀떎.

List of LAMP based ASFV detecting methods

# Target gene Primer Location Product size (nt)* Condition References
Name Sequence (5'→3') Length (nt) Start End NCBI accession #
1 P72 FIP TGATAAAGCGCTCGCCGAAGGCGCTTTTGGTTTAATGAGAA 41 - - MH713612 212 60℃, 90 min Dokphut et al., 2021
BIP GCTTGCATCGCAAAAGGATTTGCTATAAAACGTGACTGGC 40 - -
Floop CTGAGGGAATAGCAAGGTTCAC 22 - -
F3 ACTATCAGCCCCCTCTTG 18 960 977
B3 GCGTATATTGCGTCTACTGG 20 766 785
2 P72 4FIP TGATCGGATACGTAACGGGATAGAGATACAGCTCTTC 37 - - MN207061.1 244 65℃, 40 min Wu et al., 2020 (Shaanxi innolever biological technology Co. Ltd.)
4BIP CCGTAACTGCTCATGGTACGTAGTGGAAGGGTAT 34 - -
4LoopF ATAGATGAACATGCGTC 17 - -
4LoopB AGTTCTGCAGCTCTTA 16 - -
4F3 ACGCAGAGATAAGCTT 16 1,508 1,523
4B3 AAGGTAATCATCATCGC 17 1,735 1,751
3 FIP GCAGAACTTTGATGGAAACTTATCGTTAAAAACATTTCCGTAACTGCT 48 - - AY261361 195 65℃, 50 min James et al., 2010
BIP GCTCTTACATACCCTTCCACTACGGTAATCATCATCGCACCCG 43 - -
F3 CGTTACGTATCCGATCACATT 21 102,297 102,317
B3 TATTCCTCCCGTGGCTTC 18 102,123 102,140
4 P72 B-FIP CATGAGCAGTTACGGAAATGTTTTTTGTTCATCTATATCTGATATTAGCC 50 - - MH910495.1 212 63℃, 50 min Center for animal disease control and prevention of Beijing
B-BIP AATTTCCATCAAAGTTCTGCAGCTTTTAATCGCATTGCCTCC 42 - -
B-LB GTAATGTGATCGGATACGTAACGG 24 - -
B-F3 TACAGCTCTTCCAGACGC 18 103,984 104,001
B-B3 TAATCATCATCGCACCCG 18 103,790 103,807
5 P72 A-FIP AGCTGCAGAACTTTGATGGAAATTTAAACATTTCCGTAACTGCTCA 46 - - MH910495.1 208 63℃, 50 min
A-BIP ACGGAGGCAATGCGATTAAAATATTCCTCCCGTGGCTTC 39 - -
A-LB CCGATGATCCGGGTGCGAT 19 - -
A-F3 CGTATCCGATCACATTACCT 20 103,932 103,951
A-B3 ATATGACCACTGGGTTGG 18 103,744 103,761
6 No data 63℃, 30 min & 80℃, 2 min MmisoASFV detection kit (Mmonitor, Korea)

*Amplicon sizes were using LAMP outer PCR primer set



룊媛 긽씤 6醫낅쪟 以 뿼湲곗꽌뿴씠 젣怨듬맂 5媛 諛⑸쾿(#1 ~#5) LAMP 븘슦꽣 봽씪씠癒몃 궗슜븳 PCR 諛섏쓳, 듅씠쟻 諛섏쓳, 떆猷 寃젙 諛 씤쐞쟻 삤뿼 湲곕컲 寃異 誘쇨컧룄瑜 遺꾩꽍븯떎. LAMP 븘슦꽣 봽씪씠癒몃 궗슜븳 PCR 諛섏쓳쓽 議곗꽦 AccuPower® HotStart PCR PreMix (Bioneer, Korea)瑜 궗슜븯뿬, 봽씪씠癒 2 μL(젙諛⑺뼢 諛 뿭諛⑺뼢 봽씪씠癒몃 媛곴컖 25 pmol 1 μL), 二쇳삎 빑궛 1 μL, 硫멸퇏 利앸쪟닔 17 μL濡 珥 20 μL濡 븯쑝硫, 諛섏쓳 議곌굔 珥덇린 蹂꽦 95℃뿉꽌 5遺, 35쉶 諛섎났[蹂꽦 95℃ 45珥, 寃고빀 52.5~ 65.0℃(뒠釉 蹂 2.5℃ 媛꾧꺽) 60珥 諛 떊옣 72℃ 60珥], 理쒖쥌 떊옣 72℃ 5遺 媛 諛섏쓳븯떎. 寃고빀 삩룄뿉 諛섏쓳릺吏 븡 諛⑸쾿 #3怨 #5뒗 寃고빀 삩룄瑜 40.0~52.5℃濡 븯뿬 異붽 닔뻾븯떎. 洹 寃곌낵, 媛곴컖 諛⑸쾿 #1 52.5~57.5℃, #2뒗 52.5~55.0, #3 40.0~50.0℃, #4뒗 55.0~57.5℃ 諛 #5뒗 40.0~50.0℃뿉꽌 븘슦꽣 봽씪씠癒몄뿉 븳 利앺룺 諛섏쓳씠 솗씤릺뿀떎(옄猷 誘 젣怨). 諛⑸쾿 #1~#5뿉 븳 LAMP 諛섏쓳쓣 쐞븳 議곗꽦 10X ThermoPol Buffer 2.5 μL(理쒖쥌 냽룄 1X), dNTP (10 mM) 3.5 μL(理쒖쥌 냽룄 1.4 mM), MgSO4 (100 mM) 1.5 μL(理쒖쥌 냽룄 6 mM), 봽씪씠癒(10 pmol) 媛곴컖 1 μL(珥 4.0~6.0 μL), Bst DNA Polymerase, Large Fragment (New England Biolabs, USA) 1.0 μL, 二쇳삎 빑궛 1.0 μL 諛 硫멸퇏 利앸쪟닔瑜 10.5~12.5 μL濡 븯뿬 珥 25 μL濡 븯떎. LAMP 諛섏쓳 議곌굔 蹂닿퀬맂 諛⑸쾿쓣 룷븿븯뿬 60, 62, 63 諛 65℃濡 븯떎. 諛섏쓳 썑 2% agarose gel뿉꽌 30遺 媛 쟾湲 쁺룞 썑 UV 븯뿉꽌 솗씤븯떎. 諛⑸쾿 #1~#3뿉꽌뒗 紐⑤뱺 諛섏쓳 議곌굔뿉꽌 ASFV 듅씠쟻 諛섏쓳씠 굹굹吏 븡븯떎(Table 2). 빐떦 諛⑸쾿뱾 씠濡좎쟻쑝濡 빀꽦맂 뿼湲곗꽌뿴뿉 遺李⑸맆 닔 엳뿀쑝굹(옄猷 誘 젣怨), kit濡 젣怨듬릺吏 븡 留ㅻ돱뼹 諛⑹떇쓽 LAMP 썝由 떆뿕踰뺤쓽 븳怨꾩젏쑝濡 LAMP諛섏쓳뿉 븳 議곌굔쓽 꽕젙뿉 븳 媛쒖꽑씠 븘슂븷 寃껋쑝濡 蹂댁씤떎. 諛섎㈃, #4뒗 63℃뿉꽌 #5뿉꽌뒗 62℃ 65℃뿉꽌 諛섏쓳씠 솗씤릺뿀怨, PCV2, PPV 諛 PPrV뿉 븳 鍮 듅씠쟻 諛섏쓳씠 굹굹吏 븡븯쑝硫, 쓬떇臾쇰쪟룓湲곕Ъ 떆猷 20媛쒖뿉꽌 紐⑤몢 쓬꽦쑝濡 솗씤릺뿀떎(Table 2). LAMP 諛섏쓳씠 굹궃 諛⑸쾿 #4 #5瑜 긽쑝濡 쓬떇臾쇰쪟룓湲곕Ъ 떆猷 궡 쟻슜꽦쓣 룊媛븯떎. 떆猷뚮줈遺꽣 異붿텧븳 빑궛뿉 ASFV 빑궛쓣 10-1 (100 pg/μL)뿉꽌 10-8 (10 ag/μL)濡 씤쐞쟻쑝濡 삤뿼 떆궓 썑 寃異 誘쇨컧룄瑜 遺꾩꽍븳 寃곌낵, 諛⑸쾿 #4 #5뒗 쓬떇臾쇰쪟룓湲곕Ъ 궡 ASFV瑜 媛곴컖 10-5 (10 fg/μL)怨 10-6 (1 fg/μL) 誘쇨컧룄 닔以뿉꽌 寃異쒕릺뿀떎(Fig. 1). 븳렪, 諛⑸쾿 #6 [MmisoASFV detection kit (Mmonitor)] 듅씠쟻 諛섏쓳, 떆猷 寃젙 諛 씤쐞쟻 삤뿼 湲곕컲 寃異 誘쇨컧룄瑜 遺꾩꽍븯떎. 젣뭹쓽 留ㅻ돱뼹뿉 뵲씪 premix type쑝濡 媛 뒠釉뚯뿉 떞寃⑥엳뒗 Lamp Premix 15 μL, control primer 2 μL, 二쇳삎 빑궛 1 μL(cf. 젣뭹쓽 留ㅻ돱뼹 2 μL씠吏留, 룊媛瑜 쐞빐 룞씪븳 뼇쓽 二쇳삎 빑궛쓣 룷븿), 硫멸퇏 利앸쪟닔 7 μL濡 븯뿬 珥 25 μL濡 븯뿬 63℃ 30遺, 80℃ 2遺 諛섏쓳 썑, 뙆깋 뼇꽦, 蹂대씪깋 쓬꽦쑝濡 뙋룆븯떎. 洹 寃곌낵, 諛⑸쾿 #6 ASFV 빑궛뿉 븳 듅씠쟻 諛섏쓳씠 솗씤 諛 3媛 李멸퀬 諛붿씠윭뒪 빑궛뿉 븳 鍮 듅씠쟻 諛섏쓳 굹굹吏 븡븯怨, 떆猷 20媛쒖뿉 븳 諛섏쓳 紐⑤몢 쓬꽦씠뿀쑝硫, 씤쐞쟻쑝濡 삤뿼 湲곕컲 寃異 誘쇨컧룄뒗 10-6 (1 fg/μL)濡 굹궗떎(Fig. 2).

Evaluation of LAMP based methods for detecting of ASFV from food waste sample types

Methods Evaluation items Evaluation (Advantages / Disadvantages)
PCR Reactiona Specificity Sample test Sensitivityb Reaction time (min) Cost (per perp., won)d Remark
1 Dokphut et al., 2021 Amplification ND ND ND 90c 2,000~3,000 Preparation and result analysis processes requires Cannot evaluate (Need to re-examine the reaction conditions)
2 Shaanxi innolever biological technology Co. Ltd. 40c
3 James et al., 2010 50c
4 Center for animal disease control and prevention of Beijing (1) Specific Negative 10-5 (1 fg/μL) 50c Available
5 Center for animal disease control and prevention of Beijing (2) 10-6 (1 fg/μL) 50c Recommended (ASFV detection in food waste samples at relatively low cost / about 3× slower to get results compared to method #6, relatively many preparations process requires)
6 MmisoASFV detection kit (Mmonitor, Korea) ND 10-6 (1 fg/μL) 32 10,313 User-friendly & excellent reproducibility estimated Recommended [Excellent sensitivity in food waste sample & Rapid (about 3 times) / expensive (Approximately 5 times) compared to manual methods]

aUsing outer primers. bArtificial infection-based sensitivity. cAdditional gel-electrophoresis time (about 40~60 minutes) required. dCalculation from LAMP reaction to electrophoresis confirmation



Fig. 1. Artificial infection-based sensitivity of two LAMP based methods. M, 100 bp Ladder marker (Enzynomics, Korea); N, negative control.

Fig. 2. Specific reaction, sample test and artificial infection-based sensitivity of LAMP based method #6 (MmisoASFV detection kit; Mmonitor, Korea). P1, kit positive control; N1, kit negative control; P2, ASFV nucleic acid 10-1 (100 pg/μL); N2, deionized distilled water (ddH2O); PCV2, Porcine circovirus 2; PPV, Porcine parvovirus; PPrV, Porcine Pseudorabies virus; 1~20, sample number.

씠踰 뿰援ъ뿉꽌뒗 떊냽븳 紐⑤땲꽣留곸씠 슂援щ릺뒗 蹂묒썝泥 ASFV 吏꾨떒뿉 엳뼱 빑궛 利앺룺踰 湲곕컲 以 媛옣 떊냽븳 LAMP瑜 湲곕컲쑝濡 븳 諛⑸쾿뿉 븳 쓬떇臾쇰쪟룓湲곕Ъ 떆猷 궡 쟻슜꽦쓣 룊媛븯떎. Pikalo et al. (2022)뒗 뤌吏 엫긽 떆猷뚯뿉꽌 OIE 沅뚯옣 諛⑸쾿씤 떎떆媛 젙웾 PCR (qPCR) 湲곕컲 12媛 kits뿉 븯뿬 ASFV 쑀쟾옄 寃異쒖쓣 쐞븳 꽦뒫怨 듅꽦쓣 鍮꾧탳븯쑝硫, 궗슜옄 移쒗솕꽦, 궡遺 넻젣, 냼슂 떆媛꾨룄 룷븿릺뿀떎. 紐⑤뱺 qPCR 꽌濡 떎瑜 留ㅽ듃由뒪뿉꽌 紐⑹쟻뿉 留욊쾶 ASFV 寃뚮냸쓣 寃異쒗븯怨, 쟻빀븳 듅씠룄 誘쇨컧룄媛 굹궗쑝硫, 씠뿉 뵲씪 궡遺 젣뼱 떆뒪뀥쓽 샇솚꽦 諛 슦꽑 닚쐞뿉 뵲씪 kit瑜 꽑깮븷 닔 엳떎怨 蹂닿퀬븯떎(Pikalo et al., 2022). 씠踰 뿰援ъ뿉꽌뒗 6醫낅쪟쓽 LAMP 湲곕컲 諛⑸쾿 以 3醫낅쪟뿉꽌 듅젙 諛섏쓳씠 굹궗쑝硫, 듅엳 諛⑸쾿 #5뒗 졃븳 湲덉븸쑝濡 쓬떇臾쇰쪟룓湲곕Ъ뿉꽌 ASFV 寃異쒖뿉 솢슜맆 닔 엳쓣 寃껋쑝濡 룊媛릺뿀怨, 諛⑸쾿 #4룄 솢슜씠 媛뒫븷 寃껋쑝濡 寃넗릺뿀떎(Table 2). 洹몃윭굹, 留ㅻ돱뼹 諛⑸쾿쑝濡 긽쟻쑝濡 留롮 以鍮 怨쇱젙怨 寃곌낵 遺꾩꽍쓣 쐞븳 쟾湲 쁺룞 怨쇱젙쑝濡 諛⑸쾿 #6뿉 鍮꾪빐 빟 3諛곗쓽 떆媛꾩씠 냼슂맆 寃껋쑝濡 異붿젙릺뿀떎. 洹몃윭굹 쟾湲 쁺룞 怨쇱젙 SYBR Green 삉뒗 hydroxynaphthol blue 벑쓣 궗슜븯硫 떆媛꾩쓣 떒異뺤떆궗 닔 엳怨, 諛⑸쾿 #5瑜 湲곕컲쑝濡 젣뭹솕븳떎硫 씠윭븳 臾몄젣젏 洹밸났맆 寃껋쑝濡 蹂댁씤떎(Goto et al., 2009; Lee et al., 2016). 삉븳, 젣뭹쑝濡 젣怨듬릺뒗 #6 留ㅻ돱뼹 諛⑸쾿뿉 鍮꾪빐 빟 3諛 떊냽븯寃 쓬떇臾쇰쪟룓湲곕Ъ뿉꽌 ASFV 寃異쒖뿉 솢슜맆 닔 엳쓣 寃껋쑝濡 룊媛릺뿀쑝硫, kit濡 젣怨듬릺뒗 留뚰겮 슦닔븳 옱쁽꽦씠 異붿젙릺怨, 웾쓽 떆猷뚮 寃젙븯뒗뜲 엳뼱 쟻빀븷 寃껋쑝濡 蹂댁씤떎. 洹몃윭굹 뒠釉 1媛 떦 빟 1留뚯썝씠 꽆뒗 媛寃⑹ 留ㅻ돱뼹 諛⑸쾿뱾뿉 鍮꾪빐 빟 5諛 닔以씠뿀떎(Table 2). 삉븳, LAMP 諛⑸쾿뱾쓽 궡遺 議곌뎔(Internal control)씠 룷븿릺吏 븡븘 떎뿕 꽕怨꾩옄뿉 쓽빐 蹂꾨룄濡 꽕怨꾨릺뼱빞 븳떎.

Lee et al. (2022) 쓬떇臾쇰쪟룓湲곕Ъ뿉꽌 ASFV 寃異쒖뿉 쟻슜맆 닔 엳뒗 conventional PCR 諛⑸쾿쓣 媛쒕컻 諛 湲곗〈 蹂닿퀬맂 5媛 諛⑸쾿怨 鍮꾧탳븯떎. 紐뉖챺 PCR 諛⑸쾿뿉꽌뒗 鍮 듅씠쟻 諛섏쓳씠 굹궗怨 씤쐞쟻 삤뿼 湲곕컲 寃異 誘쇨컧룄뒗 빟 10-5 ~ 10-7 닔以씠뿀떎(Lee et al., 2022). 씠踰 뿰援ъ뿉꽌뒗 諛⑸쾿 #5 #6뿉꽌 紐⑤몢 10-6 (1 fg/μL) 닔以쓽 寃異 誘쇨컧룄媛 굹궗떎. Lee et al. (2022)쓽 媛쒕컻 諛⑸쾿뿉 鍮꾪빐 빟 10諛 궙븯怨, 떎瑜 諛⑸쾿뱾뿉 鍮꾪빐꽌뒗 빟 10諛 슦닔븯嫄곕굹 룞벑 닔以 씠뿀떎. 洹몃윭굹 슦닔븳 寃곌낵瑜 蹂댁씤 Lee et al. (2022)쓽 conventional PCR 諛⑸쾿 쓬떇臾쇰쪟룓湲곕Ъ遺꽣 ASFV瑜 寃異쒗븯뒗뜲 떆猷뚯뿉꽌 珥 빑궛 異붿텧 씠썑遺꽣 빟 175遺(PCR 怨쇱젙 빟 135遺 諛 쟾湲 쁺룞 빟 40遺)씠 냼슂릺뒗 諛섎㈃, LAMP 湲곕컲쓽 諛⑸쾿 #5 #6 빟 100遺꾧낵 32遺꾩쑝濡 conventional PCR 湲곕컲 諛⑸쾿뿉 鍮꾪빐 理쒕 143遺 닔以쓽 諛섏쓳 떆媛꾩쓣 媛먯냼떆궗 닔 엳떎.

븳렪, 궗엺 肄붾줈굹 諛붿씠윭뒪媛 븷셿슜 룞臾쇰줈遺꽣 蹂닿퀬맂 궗濡媛 엳뒗 벑 궗엺怨 룞臾쇱뿉 긽샇 쟾뙆릺뒗 씤닔怨듯넻媛먯뿼蹂묒씠 떎닔 異쒗쁽릺怨 엳떎(Bae, 2021). ASFV뒗 臾쇰쟻吏꾨뱶湲(Ornithodoros erraticus) 벑 留ㅺ컻異(Basto et al., 2006) 쇅 떎瑜 醫 媛 쟾뙆媛 븘吏 솗씤릺吏 븡븯쑝굹, 솗궛 삁諛⑹뿉 븳 떊냽 吏꾨떒쓽 以묒슂꽦씠 뜑슧 떆궗맆 닔 엳떎. 理쒓렐, 援궡 솚寃쎈뿉꽌뒗 쓬떇臾쇰쪟룓湲곕Ъ뿉꽌 ASFV 寃異쒖쓣 쐞븯뿬 떆뙋슜 qPCR kit뿉 븳 룊媛媛 씠猷⑥뼱吏怨 엳떎(NIER, 2020, 2021). 씠踰 뿰援щ뒗 conventional PCR, qPCR 벑怨 븿猿 뼢썑 媛 떎뿕떎, 寃궗떎 벑쓽 궡遺 긽솴뿉 뵲씪 뼢썑 쓬떇臾쇰쪟룓湲곕Ъ 벑 솚寃 떆猷뚯뿉꽌 ASFV瑜 吏꾨떒뿉 솢슜맆 닔 엳쓣 寃껋쑝濡 蹂댁씤떎.

ACKNOWLEDGEMENT

This work was supported by a grant from the National Institute of Environmental Research (NIER) and the Ministry of Environment (MOE) of the Republic of Korea (NIER-2020-01-01-062).

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

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