Pulsed-Field Gel Electrophoresis (PFGE) standardization protocol for Proteus mirabilis

Year 2013, Volume: 4 Issue: 3, 306 - 312, 01.09.2013

Alper Karagöz Mehmet Reşat Ceylan Rıza Durmaz

https://doi.org/10.5799/ahinjs.01.2013.03.0290

Abstract

Objective: For the detection of outbreaks caused by Proteus mirabilis, strains clonal relations are determined methods as “pulsed-field gel electrophoresis (PFGE)”. The aim of this study was optimization of a pulsed-field gel electrophoresis for molecular typing of P. mirabilis. Methods: In this study, PFGE\' protocol is optimized for use in molecular typing of P. mirabilis. Phylogenetic analyzes of strains were evaluated with Bionumerics software system (version 6.01; Applied Maths, Sint-Martens-Latem, Belgium). Results: This protocol compared with Gram-negative bacteria PFGE protocols, NotI enzyme is suitable for this bacterium. Electrophoresis conditions should be revealed as; - block 1: initial pulse duration 1 sec, ending pulse duration 30 sec, striking angle 120°, the current 6 V/cm2, temperature 14°C, time 8 hours; - block 2: initial pulse duration 30 sec, ending pulse duration 70 sec, striking angle 120°, the current 6 V/cm2, temperature 14°C, time 16 hours; - TBE, pH=8.4. Conclusion: P. mirabilis strains were typed by PFGE and Bionumerics analysis program were determined clonal relationships. The procedure was simple, reproducible and suitable for these bacteria. Also it was evaluated, because of reducing time, the solution volumes and enzymes can be economically. Outbreaks of nosocomial infections due to bacteria studied assessment and the potential to provide useful information about the degree of prevalence. This optimized protocol is allowed different centers\' PFGE results to compare with other laboratories results. J Clin Exp Invest 2013; 4 (3): 306-312

Keywords

Proteus mirabilis, molecular typing, pulsed-field gel electrophoresis.

Proteus mirabilis’in moleküler tiplemesi için pulsed-field jel elektroforezinin optimizasyonu

Abstract

Amaç: Proteus mirabilis ile oluşan salgınların saptanması
için suşlar arasında klonal ilişkinin belirlenmesine yönelik
“pulsed-field jel elektroforezi (PFGE)” gibi yöntemlere
ihtiyaç vardır. Bu çalışmanın amacı; Proteus mirabilis’in
moleküler tiplemesi için pulsed-field jel elektroforezinin
optimizasyonudur.
Yöntemler: Bu çalışmada; Proteus mirabilis tiplemesinde
kullanılmak üzere optimize edilmiş bir pulsed-field jel
elektroforezi protokolü sunulmuştur. Suşların filogenetik
analizleri Bionumerics yazılım sistemi (version 6.01; Applied
Maths, Sint-Martens-latem, Belgium) ile değerlendirilmiştir.
Bulgular: Gram-negatif bakteriler için kullanılan PFGE
protokollerinden farklı olarak NotI enziminin bu bakteri
için uygun olduğu belirlendi ve elektroforez koşulları: -
blok 1 için başlangıç vuruş süresi 1 sn, bitiş vuruş süresi
30 sn, vuruş açısı 120°, akım 6 V/cm2, sıcaklık 14°C, süre
8 saat; - blok 2 için başlangıç vuruş süresi 30 sn, bitiş vuruş
süresi 70 sn, vuruş açısı 120°, akım 6 V/cm2, sıcaklık
14°C, süre 16 saat; - TBE pH=8.4 olarak saptandı.
Sonuç: Bu PFGE protokolünün; P. mirabilis suşlarının
tiplendirilmesi ve Bionumerics yazılım sistemi ile klonal
ilişkilerinin belirlenmesi için uygun olduğu anlaşıldı. Optimize
edilen yöntem basit, tekrarlanabilir ve bu bakteri
için kullanıma uygundur. Ayrıca süreyi kısaltması, kullanı-
lan çözelti ve enzimlerin düşük hacimleriyle çalışabilmesi
nedeniyle, ekonomik olarak değerlendirilmiştir. Çalışılan
bakterilere bağlı salgınları değerlendirme ve hastane enfeksiyonlarının
yaygınlık derecesi hakkında yararlı bilgiler
sunma potansiyeli vardır. Optimize edilen bu protokol,
farklı merkezlere ait PFGE sonuçlarının birbirleriyle karşılaştırılmasına da olanak sağlayacaktır

Keywords

Proteus mirabilis, moleküler tipleme, pulsed-field jel elektroforezi.

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