The rapid detection of erm(41) functions in Mycobacterium abscessus complex by loop-mediated isothermal amplification-lateral flow test = LAMP-LFT를 활용한 Mycobacterium abscessus complex의 erm(41) 유전자 기능의 신속 검출 방법 개발

국문 초록 (Abstract)

전 세계적으로 Non-tuberculosis mycobacteria (NTM)에 의한 감염은 증가하고 있으며 만성 폐쇄성 폐질환과 같은 기저질환이 있는 취약 환자에게서 쉽게 발병한다. M. abscessus complex는 흔하게 분리되는 NTM중 rapidly growing mycobacterium (RGM)이며, 자연적으로 광범위한 항생제 저항성을 가지고 있고 치료가 매우 어렵다. M. abscessus complex는 M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, M. abscessus subsp. massiliense 3가지 아종으로 나누어 진다. NTM 감염병에서 1차 치료약물로 macrolide를 사용하지만 M. abscessus complex는 23S rRNA methylation을 통해 macrolide에 유도성 내성을 부여하는 erm(41)유전자를 갖는다. 항생제에 대한 내성을 나타내는 균주는erm(41) T28을 갖고 있으며 erm(41) C28 SNP를 갖는 strain에서는 감수성이 나타난다. M. massiliense는 염기쌍 결손으로 인한 truncated erm(41)을 가지고 있어macrolide에 대해 감수성을 나타낸다. 따라서 M. abscessus complex 감염의 치료방법을 결정하는데 full-length/truncated erm(41)이나 T28C sequevar를 평가하여 macrolide resistance를 구별하는 것이 중요하다. 그러나, macrolide 감수성에 영향을 미치는 sequence를 PCR 이외에 평가할 수 있는 간단하고 직접적인 방법은 없었다. 본 연구는 M. abscessus complex 폐 감염의 치료 방법을 결정하는데 도움이 될 수 있도록 erm(41)을 표적으로 truncated erm(41)이나 erm(41) C28 SNP를 갖는 macrolide에 감수성인 균주를 평가하는 것을 목표로 하였다. 이 연구에서 사용되는LAMP는 전체적인 erm(41)유전자를 증폭하게 디자인되었다. LAMP반응과 동시에 진행되도록 특이적 검출을 위해 full length erm(41), truncated erm(41)와 같은 specific primer를 디자인하였다. 또한 SNP를 구별하기 위해서 allele specific primer인 erm(41) T28, erm(41) C28을 디자인하여 적용하였다. 표지 된 specific primer를 포함한 product들은 Single Stranded Tag Hybridization-Printed Array Strip (STH-PAS)방법을 이용하여 특이적으로 검출되었다.
Base LAMP assay가 성공적으로 증폭된 것을 실시간 탁도 측정기를 통해 확인되었으며, 임상분리주에서도 정상적으로 증폭되는 것을 확인하였다. Specific primer를 포함한 Loop-Mediated Isothermal Amplification-Lateral Flow Test (LAMP-LFT)를 통해 특이적으로 full length erm(41), truncated erm(41), erm(41) T28, erm(41) C28를 검출하였다. 그리고 총91개의 type strains와 isolates, non-abscessus mycobacterium, non-mycobacterium를 통해 특이성과 유효성을 검증하였다. LAMP-LFT의 검출한계는 반응 당 100 pg의 DNA template이다. 이 연구는 LAMP assasy를 통해 erm(41)유전자를 직접적으로 검출하며 T28C sequevar를 평가할 수 있다는 것에 있어 다른 연구와의 차별성이 있다. 이 assay를 point-of care (POC) kit로 개발한다면 M. abscessus complex infection에 대한 빠른 진단과 적절한 치료를 통해 공중보건에 기여할 수 있을 것이다.

다국어 초록 (Multilingual Abstract)

Globally, non-tuberculosis mycobacteria (NTM) infections are on the rise, particularly among susceptible individuals with underlying health conditions. M. abscessus complex is commonly isolated species among rapidly growing mycobacteria (RGM) of NTMs that exhibits multiple antibiotic resistance, making it difficult to treat. M. abscessus complex is further divided into three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Macrolides are often used as first-line therapy for NTM infections, however, the M. abscessus complex often possesses the erm(41), which confers inducible macrolide resistance via methylation of 23S rRNA. Susceptibility to macrolide antibiotics was also observed in strains that contain an SNP (erm(41) C28) in erm(41), while macrolide-resistant strain harbor erm(41) T28. In addition, M. massiliense harbors a truncated erm(41) due to a large deletion in the erm(41), resulting in susceptible phenotype to macrolide antibiotics. Therefore, discrimination of macrolide- resistance by assessing full-length/truncated erm(41) and/or T28C sequevar is crucial for determination of treatment methods in M. abscessus complex lung infection. However, there have been no simple and direct methods, other than PCR, to assess sequences affecting macrolide susceptibility. This study aimed to assess macrolide-susceptible strains possessing truncated erm(41) or erm(41) C28 SNP, by directly targeting erm(41), to aid the rapid determination of treatment options for lung infection. The loop-mediated isothermal amplification(LAMP) assay used in this study was designed to amplify the full-length erm(41). The erm(41) functionality specific primers for full-length erm(41) and truncated erm(41) were designed to effectively detect specific regions of interest simultaneously with LAMP reaction. To distinguish the SNPs, allele-specific primers; erm(41)-T28 and erm(41)-C28 were designed and evaluated for effectiveness. Products containing tagged specific primers were individually detected using Single Stranded Tag Hybridization- Printed Array Strip (STH-PAS) method.
The successful amplification of the Base-LAMP assay was verified using a real-time turbidity meter. Full-length-erm(41), truncated-erm(41), erm(41)-T28, and erm(41)-C28 were specifically detected using LAMP-lateral flow test (LAMP-LFT) with erm(41) functionality specific primers. The specificity of the assay was assessed using a total of 91 bacterial strains (20 M. abscessus T28, 20 M. abscessus C28, 5 M. bolletii T28, 20 M. massiliense, 20 non-abscessus mycobacterium, and 6 non-mycobacterium). The detection limit of LAMP-LFT was 100 pg of DNA template per reaction. This study has merits compared to previous studies in that a direct and simultaneous detection method of variants of erm(41) has been developed. Development of this assay into a kit suitable for point-of care (POC) could contribute to public health by facilitating rapid diagnosis and appropriate treatment of M. abscessus complex infection.

목차 (Table of Contents)

• CHAPTER 1. INTRODUCTION 1
• CHAPTER 2. MATERIALS AND METHODS
• 2.1 Bacterial strains 6
• 2.2 Preparation of chromosomal DNA 9
• 2.3 Preparation of DNA samples by boiling method 10
• 2.4 LAMP primer and erm(41) functionality specific primer design 11
• 2.5 LAMP reaction 15
• 2.6 The erm(41) functionality specific primer reaction 17
• 2.7 LFT detection system 21
• 2.8 Optimal temperature test 23
• 2.9 Specificity of LAMP-LFT 24
• 2.10 Limit of detection (LOD) 25
• 2.11 Retest criteria for experiments 26
• CHAPTER 3. RESULTS
• 3.1 The erm(41) whole LAMP assay
• 3.1.1 Base-LAMP test of type strains 27
• 3.1.2 Test of clinical isolates and boiling samples 29
• 3.2 Specific LAMP–LFT assay
• 3.2.1 Test of erm(41) functionality specific primer 31
• 3.2.2 Optimization of reaction
• 3.2.2.1 Optimal temperature test 33
• 3.2.2.2 Primer concentration test 36
• 3.2.3 Test of fixed condition 38
• 3.3 Specificity of LAMP- LFT
• 3.3.1 M. abscessus complex test 40
• 3.3.2 Non-abscessus mycobacterium and non-mycobacterium test 42
• 3.4 Sensitivity of LAMP-LFT 44
• CHEPTER 4. DISCUSSION 46
• CHEPTER 5. REFERENCES 49
• ABSTRACT IN KOREAN 55