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Application of Loop-Mediated Isothermal Amplification (LAMP) Assay to Rapid Detection of Methicillin-Resistant Staphylococcus aureus from Blood Cultures
Biomed Sci Letters 2019;25:75-82
Published online March 31, 2019;  https://doi.org/10.15616/BSL.2019.25.1.75
© 2019 The Korean Society For Biomedical Laboratory Sciences.

Yun-Hee Baek1,§,*, Mi-Young Jo2,3,§,*, Min-Suk Song1,*, Seung-Bok Hong4,* and Kyeong-Seob Shin2,†,*

Department of Microbiology, Chungbuk National University College of Medicine, Cheongju 28644, Korea,
Laboratory Medicine, Chungbuk National University College of Medicine, Cheongju 28644, Korea,
Department of Nursing Science, Kyungbuk College of Natural Sciences, Yeongju 36133, Korea,
Department of Clinical Laboratory Science, Chungbuk Health & Science Unviersity, Cheongju 28150, Korea
Correspondence to: Kyeong-Seob Shin. Department of Laboratory Medicine, Chungbuk National University College of Medicine, Cheong-ju 28644, Korea. Tel: +82-43-269-6240, Fax: +82-43-271-5243, e-mail: ksshin@chungbuk.ac.kr
Received February 8, 2019; Revised March 17, 2019; Accepted March 19, 2019.
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

We developed the multiplex LAMP assay using 16S rRNA, femA and mecA genes for direct detection of the methicillin resistance in Staphylococci from positive blood culture. To simultaneously recognize Staphylococci genus, S. aureus and methicillin resistance, three sets of six primers for 16S rRNA, femA and mecA were designed, respectively. The performance of LAMP assay was affirmed using VITEK system for the phenotypic methods of identification and for oxacillin and cefoxitin antimicrobial susceptibility. The optimal condition for LAMP assay was obtained under 64꼦 for 50 min. The detection limit was determined to be of 20 copies and CFU/reaction (104 CFU/mL). For clinical application of comparison with phenotypic methods, the sensitivity and specificity of the LAMP with femA gene for detecting S. aureus was 95.31% and 100%, respectively. The sensitivity and specificity of the LAMP with mecA gene for detecting methicillin resistance was 98.46% and 100%, respectively. The multiplex LAMP assay with femA and mecA gene successfully detected all of MRSA (38 isolates) isolates from 103 Staphylococci in blood cultures. The LAMP assay developed in this study is sensitive, specific, and of excellent agreement with the phenotypic methods.

Keywords : Blood culture, Methicillin-resistant Staphylococcus aureus, 16S rRNA, femA, mecA, Loop-mediated isothermal amplification
꽌 濡

Staphylococcus aureus뒗 쓽猷뚭린愿 뿰怨 媛먯뿼(hospital associated infection) 肉먮쭔 븘땲씪 吏뿭궗쉶 뿰怨 媛먯뿼(community associated infection)쓽 二쇱슂븳 썝씤 洹좎쑝濡 紐⑤궘뿼(folliculitis)씠굹 遊됱吏곸뿼(cellulitis)怨 媛숈 援냼 媛먯뿼遺꽣 떖궡留됱뿼(endocarditis), 뙣삁利(sepsis) (Lowy, 1998), 洹몃━怨 룆꽦 눦 利앺썑援(toxic shock syndrome) (Shands et al., 1980)怨 媛숈 쟾떊꽦 媛먯뿼룄 씪쑝궓떎. Methicillin-resistant Staphylococcus aureus (MRSA)뒗 mecA 쑀쟾옄瑜 쉷뱷븯뿬 penicillin binding protein 2a (PBP2a)瑜 諛쒗쁽븿쑝濡쒖뜥 methicillin쓣 룷븿븳 紐⑤뱺 β-lactam 빆洹좎젣쓽 寃고빀젰쓣 媛먯냼떆耳 궡꽦쓣 굹궦떎(Hartman and Tomasz, 1984). 삉븳 MRSA뒗 β-lactam 빆洹좎젣쇅뿉룄 뿬윭 醫낅쪟쓽 빆洹좎젣뿉 궡꽦쓣 굹굹寃 릺뼱 궗슜븷 닔 엳뒗 빆洹좎젣媛 젣븳릺湲 븣臾몄뿉 MRSA뿉 媛먯뿼맂 솚옄쓽 쑀蹂묐쪧(morbidity)怨 궗留앸쪧(mortality) methicillin-susceptible S. aureus (MSSA) 媛먯뿼솚옄 蹂대떎 넂떎(Cosgrove et al., 2003). 뵲씪꽌 삁븸諛곗뼇뿉꽌 MRSA쓽 鍮좊Ⅴ怨 젙솗븳 寃궗뒗 씠뱾 솚옄쓽 쟻젅븳 移섎즺뿉 留ㅼ슦 以묒슂븯떎.

삁븸諛곗뼇 뼇꽦 寃泥댁뿉꽌 MRSA쓽 寃異쒖쓣 쐞빐 옄룞솕湲곌린瑜 씠슜븳 룞젙씠굹 媛먯닔꽦 寃궗뒗 寃궗떎뿉꽌 媛옣 쓷엳 씠슜븯뒗 諛⑸쾿씠吏留, 씠 諛⑸쾿 18~36떆媛 씠긽씠 냼슂릺誘濡 移섎즺떆湲곌 뒭뼱吏 닔 諛뽰뿉 뾾떎. 븳렪 PCR怨 媛숈 遺꾩옄쑀쟾븰쟻 諛⑸쾿 鍮좊Ⅴ怨 젙솗븳 寃異쒖씠 媛뒫븯吏留 thermocycler굹 利앺룺궛臾쇱쓣 寃異쒗빐빞 븯뒗 듅蹂꾪븳 옣鍮꾧 븘슂븯硫 寃쏀뿕씠 엳뒗 寃궗옄媛 븘슂븯떎. Notomi 벑(Notomi et al., 2000)씠 Bst polymerase뿉 쓽빐 媛떏 蹂쐞빑궛 옄媛빀꽦(autocycling strand displacement nucleic acid amplification)뿉 湲곗큹븳 loop-mediated isothermal amplification(LAMP) 諛⑸쾿쓣 媛쒕컻븳 씠썑 씠 諛⑸쾿쓣 씠슜븳 寃궗媛 떎뼇븳 洹좎쓽 룞젙뿉 씠슜릺怨 엳떎(Iwamoto et al., 2003; Hara-Kudo et al., 2005; Ito et al., 2006; Curtis et al., 2008; Hara-Kudo et al., 2008; Yamazaki et al., 2008). LAMP 諛⑸쾿 4~6媛쒖쓽 primer瑜 씠슜븯뿬 PCR 蹂대떎 듅씠븯硫, 벑삩 議곌굔(isothermal condition)뿉꽌 利앺룺븯湲 븣臾몄뿉 利앺룺떆媛꾩씠 뜑 鍮좊Ⅸ 옣젏씠 엳떎. 寃뚮떎媛 利앺룺궛臾쇰룄 留ㅼ슦 留롮븘 利앺룺 궛臾쇱쓣 寃異쒗븯뒗 듅蹂꾪븳 諛⑸쾿씠 븘슂移 븡쑝硫 pH 吏떆옄(indicator)굹 삎愿묒쓣 씠슜븯硫 늿쑝濡 利앺룺궛臾쇱쓽 솗씤씠 媛뒫븯떎(Li et al., 2017).

씠 뿰援ъ뿉꽌 옄뱾 삁븸諛곗뼇뿉꽌 S. aureus methicillin 궡꽦쓣 吏곸젒 寃異쒗븯湲 쐞빐 3媛吏 쑀쟾옄瑜 씠슜븳 떎以(multiplex) LAMP 諛⑸쾿쓣 媛쒕컻븯怨 씠 諛⑸쾿쓽 꽦뒫쓣 룊媛븯怨좎옄 븯떎.

옱猷 諛 諛⑸쾿

긽洹좎<

異⑸턿吏뿭 븳 븰蹂묒썝쓽 誘몄깮臾 寃궗떎뿉꽌 삁븸諛곗뼇 뼇꽦 寃泥대 긽쑝濡 븯떎. 삉븳 16S rRNA쓽 듅씠룄瑜 룊媛븯湲 쐞빐 10媛쒖쓽 洹몃엺뼇꽦 몴以洹좎<(Staphylococcus aureus [ATCC 25923, ATCC 25913], Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus bovis, Streptococcus pneumoniae, Streptococcus salivarius, Enterococcus faecalis, Enterococcus casseliflavus), 6媛쒖쓽 洹몃엺쓬꽦 몴以洹좎<(Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, Acinetobacter baumannii), 洹몃━怨 1媛쒖쓽 吏꾧퇏(Candida albicans) 벑 珥 17媛쒖쓽 몴以洹좎<瑜 씠슜븯떎.

삁븸諛곗뼇 諛 삁븸諛곗뼇 뼇꽦洹좎<쓽 쟾泥섎━

솚옄濡쒕꽣 10 mL쓽 삁븸쓣 臾닿퇏쟻쑝濡 梨꾪삁븯뿬 BacT/ALERT SA standard aerobic bottle (bioMérieux Inc., Marcy-I Étoile, France)怨 BacT/ALERT SN standard anaerobic bottle뿉 媛곴컖 5 mL뵫 꽔뿀떎. 諛곗뼇 BacT/ALERT 3D 옄룞삁븸諛곗뼇湲(bioMérieux Inc., Durham, NC, USA)뿉꽌 5씪源뚯 떆뻾븯떎. 諛곗뼇 뼇꽦 떊샇媛 굹삱 寃쎌슦 洹몃엺뿼깋쓣 떆뻾븳 썑 삁븸슦臾대같吏(Blood agar plate, Asan, Korea) 諛 MacConkey 슦臾대같吏(Asan, Korea)뿉 怨꾨諛곗뼇븯뿬 洹좎쓽 룞젙 諛 빆洹좎젣 媛먯닔꽦 寃궗뿉 씠슜븯떎. 洹몃엺뿼깋뿉꽌 Staphylococcus species媛 愿李곕맆 寃쎌슦 삁븸諛곗뼇 蹂묒뿉꽌 삁븸 5 mL瑜 痍⑦븯뿬 삁泥遺꾨━愿(serum separator tube)뿉 삷湲 썑 3,000 rpm뿉꽌 10遺꾧컙 썝떖遺꾨━븯뿬 긽痢듭븸쓣 踰꾨━怨 寃 諛붾줈 쐞뿉 엳뒗 洹좎묠궗瑜 硫대큺쑝濡 eppendorf 뒠釉뚯뿉 삷寃 떆뿕쓣 븷 븣源뚯 -20꼦뿉 蹂닿븯떎.

洹좎쓽 룞젙 諛 methicillin 媛먯닔꽦 寃궗

洹좎쓽 룞젙 깮솕븰쟻 寃궗濡 catalase, coagulase, mannitol 寃궗瑜 룷븿븯怨 VITEK 2 옄룞誘몄깮臾쇰텇꽍湲(bioMérieux Inc., Hazelwood, MO, USA)濡 솗씤븯떎. Methicillin 빆깮젣 媛먯닔꽦 寃궗뒗 Vitek system (bioMérieux)쓣 씠슜븯뿬 oxacillin怨 cefoxitin 媛먯닔꽦 寃궗瑜 떆뻾븯떎(CLSI, 2018).

LAMP primer쓽 꽕怨 諛 理쒖쟻 諛섏쓳 議곌굔

Staphylococcus species, S. aureus 諛 methicillin 궡꽦 쑀쟾옄瑜 寃異쒗븯湲 쐞빐 16S rRNA, femAmecA 쑀쟾옄瑜 GenBank뿉꽌 寃깋븯怨 媛곴컖쓽 뿼湲곗꽌뿴쓣 寃곗젙븯떎. 씠썑 LAMP assay瑜 쐞븳 primer 꽕怨꾪봽濡쒓렇옩씤 Primer Explorer V5 software (http://primerexplore.jp/e/index.html: Eiken chemical Co. Ltd, Japan)瑜 궗슜븯뿬 媛 쑀쟾옄 蹂꾨줈 2醫낆쓽 쇅遺 primer (F3 B3), 2醫낆쓽 궡遺 primer (FIP BIP) 諛 2醫낆쓽 loop primer (F loop primer (LF) B loop primer (LB))벑 珥 6醫낆쓽 primer瑜 꽕怨꾪븯떎(Table 1 & Fig. 1).

The sequences of primers for LAMP assay used in this study

Target genePrimerSequence (5’ → 3’)
16S rRNAF3*TGGAATTCCATGTGTAGCGG
B3*AGGCGGAGTGCTTAATTGC
FIPTCGCACATCAGCGTCAGTTACA-ATGCGCAGAGATATGGAGGA
BIPAGATACCCTGGTAGTCCACGCC-CACTAAGGGGCGGAAACC
LFCCAGAAAGTCGCCTTCGCCACT
LBAAACGATGAGTGCTAAGTGTTAGG

femAF3*CAGAATCAAAAGCTTTTGCTG
B3*AAGTTATCTCGCTTGTTGTG
FIPCTAAAGGTACTAACACACGGTCTTT-TCGTGATGACAAATTTTACTACA
BIPAAGAACTAAACGAAGAGCGTGAT-CAGGACGTTTTTCAATATCCTT
LFGTAATATTTTAAGCGAT
LBTAAAGATTTAAATAAAGCGT

mecAF3*TGATGCTAAAGTTCAAAAGAGT
B3*GTAATCTGGAACTTGTTGACC
FIPAGGTGTGCTTACAAGTGCTAATAAT-CAACATGAAAAATGATTATGGCT
BIPTGACGTCTATCCATTTATGTATGGC-GAGGTTCTTTTTTATCTTCGGTTA
LFTGAGGGTGGATAGCAGTACC
LBTGAGTAACGAAGAATAT

*Two outer primers F3 and B3 were also used as primers for PCR of 16S rRNA, femA and mecA.

Abbreviations: loop-mediated isothermal amplification; FIP, forward inner primer; BIP, backward inner primer; LF, forward loop primer; LB, backward loop primer


Fig. 1.

Primers designed for 16S rRNA, femA, mecA loop-mediated isothermal amplification (LAMP) assays. Nucleotide sequences of 16S rRNA (A), femA (B), mecA (C) and the location of LAMP primers. The forward and backward inner primers are F1c-F2 and B1c-B2 sequences, respectively. The forward and backward outer primers are F3 and B3, respectively.


LAMP assay 諛섏쓳 primer (FIP & BIP, 40~80 pmol; F3 & B3, 5~10 pmol; LF & LB, 20 pmol) 3 μL, genomic DNA 2 μL, WarmStart colorimetric LAMP master mix (New England Biolabs Inc., MA, USA) 5 μL瑜 뜑븯뿬 理쒖쥌 슜웾쓣 10 μL濡 븯뿬 떆뻾븯떎. LAMP assay쓽 諛섏쓳 理쒖쟻 떆媛 諛 삩룄瑜 솗씤븯怨좎옄 떎뼇븳 떆뿕 議곌굔쑝濡 븯쑝硫, T100™ Thermal cycler (Bio-Rad Laboratories Inc., Foster City, CA, USA)瑜 씠슜븯뿬 利앺룺쓣 븳 썑 슚냼솢꽦쓣 젣嫄고븯湲 쐞빐 80꼦뿉꽌 5遺꾧컙 諛섏쓳떆궓 썑 醫낅즺븯떎. 諛섏쓳씠 걹궃 썑 뒠釉뚯쓽 깋 蹂솕瑜 쑁븞쑝濡 愿李고븯뿬 뼇꽦뿬遺瑜 뙋룆븯怨, 룞떆뿉 2% agarose gel뿉꽌 쟾湲곗쁺룞쓣 븯뿬 LAMP assay뿉꽌 듅씠쟻쑝濡 굹굹뒗 궗떎由 紐⑥뼇쓽 利앺룺 쑀쟾옄 諛대뱶瑜 솗씤븯뿬 뼇꽦뿬遺瑜 뙋젙븯떎.

LAMP, PCR 諛 RT-PCR쓽 寃異 븳怨

MRSA 遺쑀븸 (1.0 × 107 CFU/mL)쓣 1.0 × 101 CFU/mL源뚯 10諛곗닔 떒怨 씗꽍븯뿬 媛 냽룄뿉꽌 16S rRNA, femAmecA뿉 븳 LAMP 諛 PCR쓣 떆뻾븯떎. 寃異 븳怨 떎뿕 3쉶뵫 諛섎났 떎뿕븯떎.

LAMP assay뒗 蹂 뿰援ъ뿉꽌 솗由쎈맂 몴以 議곌굔濡 떆뻾븯쑝硫, PCR F3 諛 B3 primer瑜 씠슜븯쑝硫 DNA2 μL瑜 PCR kit (Enzymomics, Daegeon)뿉 꽎뼱 理쒖쥌 슜웾 10 μL媛 릺寃 븳 썑 떆뻾븯떎. PCR 諛섏쓳 T100™ Thermal cycler (Bio-Rad)瑜 씠슜븯뿬 denaturation (95꼦) 30珥, annealing (58꼦) 30珥, elongation (72꼦) 60珥덈줈 35쉶 떆뻾븯怨 72꼦뿉꽌 7遺꾧컙 理쒖쥌 諛섏쓳쓣 떆뻾븯떎. 利앺룺궛臾쇱 2% agarose gel뿉꽌 쟾湲곗쁺룞 븯떎.

16S rRNA LAMP assay쓽 듅씠룄

17媛쒖쓽 몴以洹좎<瑜 씠슜븯뿬 16S rRNA LAMP assay쓽 듅씠룄瑜 룊媛븯떎.

엫긽洹좎<瑜 씠슜븳 LAMP assay쓽 꽦뒫 룊媛

삁븸諛곗뼇씠 쓽猶곕맂 寃泥 以 洹좎씠 利앹떇븯怨 洹몃엺뿼깋뿉꽌 Staphylococcus species媛 愿李곕맂 103媛쒖쓽 엫긽 寃泥대 긽쑝濡 Vitek 2 system뿉 쓽븳 洹좎쓽 룞젙 洹몃━怨 媛먯닔꽦 寃궗瑜 떆뻾븯怨 룞떆뿉 16S rRNA, femAmecA뿉 븳 multiplex LAMP assay瑜 떆뻾븯뿬 2媛吏 寃궗媛꾩쓽 꽦뒫쓣 鍮꾧탳븯떎.

寃 怨

MSSA MRSA 洹좎쓣 긽쑝濡 60~65꼦 30, 40, 50, 60遺꾩쓽 諛섏쓳떆媛꾩쑝濡 LAMP assay瑜 떆뻾븯뿬 64꼦 50遺꾩뿉꽌 媛옣 슌졆븳 寃곌낵瑜 蹂댁쑝誘濡 씠썑쓽 紐⑤뱺 떎뿕 64꼦뿉꽌 50遺꾩쑝濡 吏꾪뻾븯떎(Fig. 2).

Fig. 2.

Visual (A) and agarose gel (B) images of the 16S rRNA, mecA, femA loop-mediated isothermal amplification (LAMP) product of MSSA and MRSA on various reaction times. The yellow color change of pH indicator was interpreted positive for amplification of DNA (A). The electrophoresis was performed at 2% agarose gel and the amplified products typically showed the ladder like shape (B). The best reaction was obtained at 64꼦 and 50 min. Abbreviations: NEG, negative; MSSA, methicillin-susceptible Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; M, molecular size marker


LAMP assay 寃異 誘쇨컧룄 痢≪젙

S. aureus瑜 씠슜븳 16S rRNA, femA, mecA gene뿉 븳 LAMP怨 conventional PCR뿉 븳 寃異 誘쇨컧룄뒗 Table 2怨 媛숈븯떎(Table 2 & Fig. 3). Conventional PCR 16S rRNA뿉꽌뒗 105 CFU/mL 씠긽뿉꽌 利앺룺릺뿀怨 femA mecA뒗 106 CFU/mL 씠긽뿉꽌 利앺룺릺뿀吏留 LAMP 諛⑸쾿 3媛쒖쓽 쑀쟾옄 紐⑤몢 104 CFU/mL 냽룄뿉꽌 利앺룺릺뼱 conventional PCR뿉 鍮꾪빐 10~100諛 誘쇨컧븯떎.

Detection limits of LAMP assay, conventional PCR for 16S rRNA, femA and mecA genes of Staphylococcus aureus

 GeneDetection limit CFU/mL (copies/run)

LAMPPCR
16S rRNA104 (20 copies)105 (200 copies)
femA104 (20 copies)106 (2,000 copies)
mecA104 (20 copies)106 (2,000 copies)

Abbreviation: CFU, colony forming unit


Fig. 3.

Visual (A) and agarose gel (B) images of the 16S rRNA, mecA, femA loop-mediated isothermal amplification (LAMP) product of clinical MRSA isolate at serial diluted concentration (101 ~ 107 CFU/mL). The yellow color change of pH indicator was interpreted positive for amplification of DNA (A). The electrophoresis was performed at 2% agarose gel and the amplified products typically showed the ladder like shape at 104 CFU/mL except faint amplification products of 16S rRNA (B). Abbreviation: Neg, negative


16S rRNA LAMP assay쓽 듅씠룄

17媛쒖쓽 몴以洹좎<(洹몃엺뼇꽦븣洹 10二, 洹몃엺쓬꽦媛꾧퇏 6二, C. albicans 1二)瑜 긽쑝濡 븳 16S rRNA LAMP 寃궗뿉꽌 紐⑤몢 씪移섑븯뒗 寃곌낵瑜 蹂댁떎(Table 3).

Specificity of LAMP assay for detecting the 16S rRNA gene of Staphylococci in reference strains

  MicroorganismsReference strainsResult for 16S rRNA LAMP assay
Gram positive bacteria
Staphylococcus aureusATCC 25923Positive
Staphylococcus aureusATCC 25913Positive
Staphylococcus epidermidisATCC 12228Positive
Streptococcus pyogenesATCC 19615Negative
Streptococcus agalactiaeATCC 12386Negative
Streptococcus bovisATCC 49147Negative
Streptococcus pneumoniaeATCC 49619Negative
Streptococcus salivariusATCC 40412Negative
Enterococcus faecalisATCC 29212Negative
Enterococcus casseliflavusATCC 700327Negative
Gram negative bacteria
Escherichia coliATCC 25922Negative
Klebsiella pneumoniaeATCC 700603Negative
Enterobacter cloacaeATCC 700323Negative
Citrobacter freundiiATCC 8090Negative
Pseudomoas aeruginosaATCC 27853Negative
Acinetobacter baumanniiATCC 19606Negative
Fungus
Candida albicansTIMM 3316Negative

엫긽 寃泥대 쟻슜븳 LAMP assay쓽 誘쇨컧룄뿉 듅씠룄 룊媛

삁븸諛곗뼇뿉꽌 遺꾨━맂 103媛쒖쓽 룷룄븣洹좎쓣 긽쑝濡 븳 16S rRNA LAMP 寃궗뿉꽌 紐⑤몢 뼇꽦쓣 蹂댁떎. femA LAMP 寃궗뒗 3媛쒖쓽 MSSA瑜 寃異쒗븯吏 紐삵븯쑝굹 굹癒몄 100洹좎<뒗 젙솗븳 寃곌낵瑜 蹂댁뿬 깮솕븰쟻 룞젙 諛 媛먯닔꽦 寃궗 寃곌낵瑜 湲곗쑝濡 룊媛븳 삁誘쇰룄 듅씠룄뒗 媛곴컖 95.31% (61/64) 100% (39/39)씠뿀떎. mecA LAMP 寃궗뒗 1媛쒖쓽 MRCoNS瑜 寃異쒗븯吏 紐삵븯쑝굹 굹癒몄 102媛쒖쓽 寃泥대뒗 젙솗븳 寃곌낵瑜 蹂댁뿬 삁誘쇰룄 듅씠룄뒗 媛곴컖 98.46% (64/65) 100% (38/38)씠뿀떎(Tables 4 & 5).

Results of LAMP assay for detecting femA and mecA genes in 103 clinical isolates from positive blood culture

 OrganismsMultiplex LAMP assayNumber of clinical isolates

femA+mecA+femA+mecA-femA-mecA+femA-mecA-
MRSA38---38
MSSA-23-326
MRCoNS--26127
MSCoNS---1212

Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus; MRCoNS, methicillin-resistant coagulase negative Staphylococci; MSCoNS, methicillin-susceptible coagulase negative Staphylococci


Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP assay with femA and mecA genes for detection of MRSA 103 clinical isolates from positive blood cultures

 GenesSensitivity (%)Specificity (%)PPV (%)NPV (%)
femA61/64 (95.31)39/39 (100)61/61 (100)38/41 (92.68)
mecA64/65 (98.46)38/38 (100)64/64 (100)38/39 (97.43)

怨 李

쟾넻쟻쑝濡 삁븸諛곗뼇뿉꽌 MRSA쓽 寃異쒖 몴쁽삎쟻 諛⑸쾿(phenotypic method)쑝濡 洹좎쓣 룞젙븯怨 methicillin 媛먯닔꽦 寃궗瑜 븯뒗 寃껋씠 李멸퀬 諛⑸쾿쑝濡 릺뼱 엳吏留 씠 諛⑸쾿 洹좎쓽 怨꾨諛곗뼇, 룞젙 諛 媛먯닔꽦 寃궗뿉 36떆媛 씠긽 냼슂맂떎. 寃궗떆媛꾩쓣 떒異뺥븯湲 쐞빐 삁븸諛곗뼇 뼇꽦 寃泥댁뿉꽌 吏곸젒 遺꾩옄쑀쟾븰쟻 寃궗 諛⑸쾿 삉뒗 MALDI-TOF 벑쓣 씠슜븯뒗 뿬윭 寃궗뱾뿉 븳 蹂닿퀬뱾씠(Stamper et al., 2007; Stevenson et al., 2010; Clerc et al., 2014; Verroken et al., 2015) 엳떎. 洹몃젃吏留 遺꾩옄쑀쟾븰쟻 諛⑸쾿 DNA쓽 利앺룺 諛 寃異쒖옣鍮꾧 븘슂븯硫 利앺룺떆媛꾩씠 3떆媛 씠긽 냼슂릺硫 batch 諛⑹떇쑝濡 떆뻾빐빞 븳떎. 洹몃━怨 MALDI-TOF 諛⑸쾿 mass spectrophotometry媛 븘슂븯硫, methicillin 궡꽦쓣 寃異쒗븯湲 쐞빐꽌뒗 듅蹂꾪븳 떒怨꾧 異붽濡 븘슂븯떎(Opota et al., 2015).

理쒓렐뿉 냼媛쒕맂 LAMP 寃궗 諛⑸쾿 6~8媛 듅씠 遺쐞瑜 씤吏븯뒗 6媛쒖쓽 primer瑜 씠슜븯뿬 Bst DNA 以묓빀슚냼뿉 쓽븳 autocycling strand displacement DNA 빀꽦쓣 넻븯뿬 쟻 뼇쓽 DNA룄 젙솗븯怨 鍮좊Ⅴ寃 利앺룺떆궗 닔 엳떎. 삉븳 60꼦 젙룄쓽 벑삩뿉꽌 利앺룺븯湲 븣臾몄뿉 삩룄媛 蹂솕븯뒗뜲 븘슂븳 떆媛꾩쓣 젅빟븷 닔 엳쑝硫 PCR뿉 鍮꾪빐 寃泥댁뿉 쓽븳 뼲젣 슚怨쇰룄 嫄곗쓽 뾾떎. 寃뚮떎媛 利앺룺궛臾쇱쓣 샎긽룄(turbidity)굹 깋 蹂솕(pH indicator 삉뒗 SYBRE Green I)瑜 쑁븞쑝濡쒕룄 솗씤븷 닔 엳뒗 옣젏씠 엳떎(Li et al., 2017). 븳렪 紐뉖챺 쑀쟾옄瑜 씠슜븳 LAMP 寃궗媛 S. aureus 諛 MRSA瑜 寃異쒗븯뒗뜲 씠슜릺뿀떎. spa (Misawa et al., 2007), nuc (Chen et al., 2017), femA (Xihong et al., 2013)媛 S. aureus瑜 寃異쒗븯뒗뜲 씠슜릺뿀怨, methicillin 궡꽦쓣 寃異쒗븯뒗뜲 mecA媛 씠슜릺뿀떎. 삉븳 orfXS. aureus MRSA瑜 寃異쒗븯뒗뜲 씠슜릺湲곕룄 븯떎(Su et al., 2014). 蹂 뿰援ъ뿉꽌뒗 16S rRNA, femAmecA 쑀쟾옄瑜 씠슜븳 떎以 LAMP 寃궗瑜 씠슜븯뿬 삁븸諛곗뼇 뼇꽦 寃泥댁뿉꽌 Staphylococcus species, S. aureus 諛 methicillin 궡꽦쓣 룞떆뿉 寃異쒗븯怨좎옄 븯떎.

LAMP assay쓽 寃異 삁誘쇰룄瑜 conventional PCR怨 鍮꾧탳븳 寃곌낵, Table 2怨 媛숈씠 LAMP assay뿉꽌 3媛쒖쓽 쑀쟾옄뿉 빐 寃異 삁誘쇰룄뒗 104 CFU/mL (20 copies)떎. 븳렪 104 CFU/mL 냽룄쓽 MRSA뿉꽌 femA mecA쓽 쑀쟾옄뿉 븳 LAMP 寃궗뿉꽌 紐낇솗븳 궗떎由 紐⑥뼇쓽 利앺룺궛臾쇱씠 愿李곕릺뿀吏留 16S rRNA LAMP 寃궗뒗 104 CFU/mL뿉꽌뒗 씗誘명븯吏留 3쉶 諛섎났 떎뿕 紐⑤몢뿉꽌 솗떎븯寃 利앺룺릺뼱 3媛쒖쓽 쑀쟾옄뿉 븳 LAMP쓽 寃異 삁誘쇰룄瑜 104 CFU/mL濡 寃곗젙븷 닔 엳뿀떎. 寃곕줎쟻쑝濡 LAMP 諛⑸쾿 conventional PCR쓽 16S rRNA뒗 10諛, femA mecA뒗 100諛 젙룄 誘쇨컧븯떎. 씠뒗 LAMP assay뿉꽌 strand displacement activity媛 넂怨 DNA 넀떎瑜좎씠 쟻뼱 利앺룺슚쑉씠 넂떎뒗 寃껋쓣 쓽誘명븳떎.

103媛쒖쓽 엫긽 寃泥대 긽쑝濡 깮솕븰쟻 룞젙 諛 빆洹좎젣 媛먯닔꽦 떆뿕 寃곌낵 鍮꾧탳븳 femA mecA LAMP 諛⑸쾿쓽 誘쇨컧룄 듅씠룄뒗 Table 4 Table 5 媛숇떎. 64媛쒖쓽 S. aureusfemA뿉 빐 쓬꽦씤 洹좎씠 3洹좎<濡 femA쓽 誘쇨컧룄뒗 95.31% (61/64)쑝硫 39媛쒖쓽 CNS뒗 紐⑤몢 femA뿉 쓬꽦씠뼱꽌 듅씠룄뒗 100%씠뿀떎. femA 쓬꽦씤 S. aureus媛 3洹좎<媛 議댁옱븯湲 븣臾몄뿉 S. aureus쓽 媛먮퀎뿉 femA 씠쇅뿉 nuc 벑 쑀쟾옄瑜 異붽빐 蹂 븘슂媛 엳쓣 寃껋씠떎. 65媛쒖쓽 MRSA MRCoNS 以 1洹좎<瑜 젣쇅븯怨 紐⑤몢 mecA LAMP 뼇꽦쓣 蹂댁怨 38 MSSA MSCoNS뒗 쓬꽦쓣 蹂댁뿬 삁誘쇰룄 듅씠룄뒗 媛곴컖 98.46% 100%씠뿀떎. 쐞쓬꽦씤 MRCoNS쓽 寃쎌슦 methicillin 궡꽦뿉 愿뿬븯뒗 쑀쟾옄媛 mecA 肉먮쭔 븘땲씪, mecA1, A2, mecB, mecC 媛숈 mec 쑀쟾옄 쑀궗泥(homologues)쓽 寃쎌슦 넻긽쟻씤 mecA 利앺룺뿉 쓽빐 寃異쒕릺吏 븡븯쓣 媛뒫꽦씠 엳떎(Wu et al., 2001). 븳렪 삁븸諛곗뼇뿉꽌 MRSA non-MRSA쓽 媛먮퀎씠 移섎즺뿉 媛옣 以묒슂븳뜲, 蹂 뿰援ъ뿉꽌 femA mecA瑜 룞떆뿉 寃궗븷 븣 MRSA 씠쇅쓽 洹좎< 媛먮퀎 100% 媛뒫븯떎(Tables 4 & 5).

LAMP assay 肉먮쭔 븘땲씪 遺꾩옄寃궗 諛⑸쾿쓽 떒젏 삁븸諛곗뼇뿉꽌 MSSA MRCoNS媛 룞떆뿉 議댁옱븯硫 MRSA쓽 쐞뼇꽦(false positive) 寃곌낵媛 굹삱 닔 엳뒗 寃껋씠떎(Becker et al., 2006). 삁븸諛곗뼇뿉꽌 洹몃윴 寃쎌슦뒗 留ㅼ슦 뱶臾쇱留 븵쑝濡 MRSA뿉 븳 듅씠 marker쓽 媛쒕컻 삉뒗 蹂댁셿씠굹 LAMP assay 씠썑 洹좎쓽 諛곗뼇 寃곌낵瑜 솗씤빐빞 븷 寃껋씠떎.

寃곕줎쟻쑝濡 쐞 媛숈 遺꾩옄깮臾쇳븰쟻 떒젏뿉룄 遺덇뎄븯怨 삁븸諛곗뼇 뼇꽦蹂묒뿉꽌 MRSA쓽 吏곸젒 寃異쒗븯뒗뜲 LAMP assay쓽 쟻슜 MRSA 洹좏삁利 솚옄쓽 떊냽븳 移섎즺瑜 媛뒫耳 븯뒗뜲 留ㅼ슦 룄씠 맆 寃껋쑝濡 궗猷뚮맂떎.

ACKNOWLEDGEMENT

씠 끉臾몄 2018뀈룄 젙遺(誘몃옒李쎌“怨쇳븰遺)쓽 옱썝쑝濡 븳援뿰援ъ옱떒쓽 吏썝쓣 諛쏆븘 닔뻾맂 湲곗큹뿰援ъ궗뾽엫(No. NRF-2018M3A9H4055769).

CONFLICT OF INTEREST

The authors have no conflicts of interest to disclose.

Acknowledgments
씠 끉臾몄 2018뀈룄 젙遺(誘몃옒李쎌“怨쇳븰遺)쓽 옱썝쑝濡 븳援뿰援ъ옱떒쓽 吏썝쓣 諛쏆븘 닔뻾맂 湲곗큹뿰援ъ궗뾽엫(No. NRF-2018M3A9H4055769).
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