Comparison of loop-mediated isothermal amplification, multiplex PCR, and REP- PCR techniques for identification of carbapenem-resistant Acinetobacter baumannii clinical isolates.

IF 1.3 Q4 MICROBIOLOGY

Iranian Journal of Microbiology Pub Date : 2023-10-01 DOI:10.18502/ijm.v15i5.13871

Aysar Abbood Al Jebur, Neda Soleimani, Seyed Masoud Hosseini

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Abstract

Background and objectives: Acinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-detecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique.

Materials and methods: LAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the bla _SHV-1 , bla _PER-1 , bla _TEM-1, AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic identification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilutions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR.

Results: Among one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL producers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were bla _SHV-1 positive, (40%) bla _PER-1, (85%) aac (6')-1, AMPC (67%), bla _TEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla _SHV-1 positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, bla _TEM-1 , and bla _PER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates.

Conclusion: Wherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detection rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. baumannii in these clinical isolates showed frequent commonality of genotypes.

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环介导等温扩增、多重PCR和REP-PCR技术鉴定耐碳青霉烯鲍曼不动杆菌临床分离株的比较。

背景和目的：鲍曼不动杆菌是一种机会性病原体，与医院获得性感染和死亡率增加有关。本研究旨在开发环介导等温扩增（LAMP）试验，用于快速检测鲍曼不动杆菌分离株，并通过REP-PCR技术确定这些分离株的遗传相关性。材料和方法：设计LAMP引物和多重PCR引物，用于识别携带bla SHV-1、bla PER-1、bla TEM-1、AMPC、qnr和aac（6）-1基因的鲍曼不动杆菌分离株，从伊朗德黑兰Shahid Motahari医院收集（2020年10月至2021年2月）。组合圆盘试验（CDT）结果用于评估ESBL生产商分离株的表型鉴定。LAMP方法的灵敏度使用一系列基因组DNA稀释液进行评估。将LAMP技术和多重PCR的结果进行比较。结果：在100份鲍曼不动杆菌样本中，经联合纸片检验，56%的分离株为ESBL产生菌。LAMP技术鉴定鲍曼不动杆菌的灵敏度为4.06ng/ml，而多重PCR的灵敏度为（16.2ng/ml）。关于多重PCR，（68%）的分离株分别为bla SHV-1阳性、（40%）bla PER-1、（85%）aac（6’）-1、AMPC（67%）、bla TEM-1（63%）和（15%）qnr。在LAMP中，（69%）的分离株为bla SHV-1阳性，（86%）为aac（6'）-1阳性，（20%）为qnr阳性。AMPC、bla TEM-1和bla PER-1基因的结果显示多重PCR和LAMP测定之间100%的兼容性。REP-PCR结果表明，共有17个克隆，14%的克隆A是最常见的分离株。结论：在设备和资金限制至关重要的地方，LAMP检测比多重PCR对鲍曼不动杆菌分离株的鉴定提供了更好、更有效的检测率。此外，鲍曼不动杆菌在这些临床分离株中的遗传多样性表现出频繁的基因型共性。

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来源期刊

Iranian Journal of Microbiology MICROBIOLOGY-

CiteScore

2.40

自引率

7.10%

发文量

审稿时长

12 weeks

期刊介绍： The Iranian Journal of Microbiology (IJM) is an international, multi-disciplinary, peer-reviewed journal that provides rapid publication of the most advanced scientific research in the areas of basic and applied research on bacteria and other micro-organisms, including bacteria, viruses, yeasts, fungi, microalgae, and protozoa concerning the development of tools for diagnosis and disease control, epidemiology, antimicrobial agents, clinical microbiology, immunology, Genetics, Genomics and Molecular Biology. Contributions may be in the form of original research papers, review articles, short communications, case reports, technical reports, and letters to the Editor. Research findings must be novel and the original data must be available for review by the Editors, if necessary. Studies that are preliminary, of weak originality or merely descriptive as well as negative results are not appropriate for the journal. Papers considered for publication must be unpublished work (except in an abstract form) that is not under consideration for publication anywhere else, and all co-authors should have agreed to the submission. Manuscripts should be written in English.