Ocena przydatnosci elektroforetycznego rozdzialu jednoniciowego DNA z zastosowaniem stopniowanego profilu termicznego [Multitemperature -SSCP] do identyfikacji alleli wybranych genow bakteryjnych

• Autorzy: Gierczynski R. , Jagielski M. , Rastawicki W.
• Języki publikacji: PL

Abstrakty

PL

Stwierdzono, że elektroforetyczny rozdział jednoniciowego DNA z zastosowaniem stopniowanego profilu termicznego (MSSCP) jest przydatny do różnicowania i identyfikacji alleli genu ail pałeczek Y enterocolitica 03 i 08, zarówno wtedy gdy badaniu poddano odcinki DNA różniące się obecnością 15 jak i 11 mutacji punktowych, przy długości badanego fragmentu DNA wynoszącej odpowiednio 425 i 251 nukleotydów. Ponadto, porównując wybrane fragmenty genów blaCTX-M-3., i blaCTX-M-15 wykazano, że MSSCP może być przydatny do różnicowania odcinków DNA zawierąjących pojedynczą mutację punktową.

EN

Recently, the single strand conformation polymorphism (SSCP) analyses were shown to be useful for identification a variety of bacterial genes. Although, SSCP was successfully applied for detection of single nucleotide polymorphism (SNP), it was also considered a time consuming and insufficiently reliable technique. Therefore, the modified Multitemperature-SSCP method was introduced. It was shown to be reliable and time effec1 tive technique due to a stringent control of the gel temperature and utilization of a high voltage up to 1 kV In this study the usefulness of MSSCP for differentiation of gene variants and detection of the single point mutations was evaluated, using Yersinia enterocolitica 03 and 08 ail alleles or genes blaCTX-M-15 and blaCTX-M-3, which differs by a single point mutation. The 425 and 251 bp fragments of 03 and 08 ail alleles containing 15 and 11 point mutations respectively, as well as 277 and 208 bp fragments of both blaCTX-M differing in positions 243 and 62 were PCR amplified, denatured and loaded onto 7% and 9% Polyacrylamide nondenaturing gel. Electrophoresis was carried out in the DNA-Pointer apparatus (Kuchrczyk, Poland) at voltage ranging from 500 to 750V. The thermal profile consisted of 50 min. at 35°C, 40 min at 20oC and 40 min at 5°C. Obtained results showed, MSSCP was capable to differentiate ail alleles independently of the length of analyzed fragment. However, the 425 bp fragment profile consisted of three bands, whereas 251 bp revealed two bands. The single point mutation in blaCTM, genes was also successfully distinguished by MSSCP in both tested fragments. Surprisingly, 277 bp fragment profile showed differences more apparently than 208 bp. Summarizing, MSSCP was found to be useful, sensitive and time efficient tool for detection of multiple and single point mutations in DNA fragments ranging from 208-425 bp.

Słowa kluczowe

PL

metoda rozdzialu jednoniciowego DNA; adhezyna Ail; polimorfizm; Yersinia enterocolitica; metoda RFLP; lancuchowa reakcja polimerazy; biologia molekularna; gen ail; allele; metoda SSCP; patogeny bakteryjne; DNA

• Wydawca: -
• Czasopismo: Medycyna Doświadczalna i Mikrobiologia
• Rocznik: 2003
• Tom: 55
• Numer: 2
• Opis fizyczny: s.147-155,tab.,rys.,bibliogr.

Twórcy

autor

Gierczynski R.

• Zaklad Bakteriologii PZH, ul.Chocimska 24, 00-791 Warszawa

autor

Jagielski M.

autor

Rastawicki W.

Bibliografia

• 1. Baraniak A, Fiett J, Hryniewicz W i inni. Ceftazidime hydrolysing CTX-M15 extended-spectrum b-lactamase (ESBL) in Poland. J Antimicrob Chemother 2002; 50: 393-6.
• 2. Cockerill III FR. Genetic methods for assessing antimicrobial resistance. Antimicrob Agents Chemother 1999; 43: 199-212
• 3. Ellison J, Dean M, Goldman D. Efficacy of fluorescence-based PCR-SSCP for detection of point mutations. BioTechniques 1993; 15: 684-91.
• 4. Gierczyński R. Ocena przydatności wybranych markerów wirulencji do identyfikowania chorobotwórczych szczepów pałeczek Yersinia enterocolitica. II. Genotypowe markery związane z plazmidem pYV Med Dośw Mikrobiol 2000; 52: 35-49.
• 5. Gierczyński R, Jagielski M, Rastawicki W. The Presence of the ail gene in clinical strains of Yersinia enterocolitica isolated from stools in Poland and characteristics of gene variant. Acta Microbiol Pol 2001; 50: 19-25.
• 6. Gierczyński R., Szych J, Cieślik A i inni. The occurrence of the first two CTX-M-3 and TEM-lproducing isolates of Salmonella enterica serovar Oranienburg in Poland. Int J Antimicrob Agents 2003; 21: 498-99.
• 7. Kaczanowski R, Trzeciak L, Kucharczyk K. Multitemperature single-strand conformation polymorphism. Electrophoresis 2001; 22: 3539-45.
• 8. Liu Q, Scaringe WA, Sommer SS. Discrete mobility of single stranded DNA in non-denaturing gel electrophoresis. Nucleic Acids Res 2000; 28: 940-43.
• 9. McPherson M J, Moller SG. 2001. Single strand conformation polymorphism analysis (SSCP) str 244-247 w PCR, The Basics from background to bench. Springer Verlag New York Ine і BIOS Ltd.
• 10. M'Zali FH, Heritage J, Gascoyne-Binzi DM i inni. PCR single strand conformational polymorphism can be used to detect the gene encoding SHV-7 extended spectrum ?-lactamase and to identify different SHV genes within the same strain. J Antimicrob Chemother 1998; 41: 123-25.
• 11. Pitcher D, Sills M, Robertson JA. Simple method for determining biovar and serovar types of Ureaplasma urealiticum clinical isolates using PCR-single strand conformation polymorphism analysis. J Clin Microbiol 2001; 39: 1840-44.
• 12. Sambrook J, Fritsch E, Maniatis T. 1989. Molecular cloning. A laboratory manual. Cold Spring Harbor Lab.
• 13. Sayada C, Picard В, Krishnamoorthy R. A simple procedure to differentiate ailA and ailNA gene variants among human pathogenic Yersinia enterocolitica strains, Mol Cell Probes. 1994; 8: 187-92.
• 14. Speldooren V, Heym B, Labia G, Nicolas-Chanoine MH. Discriminatory detection of inhibitor resistant b-lactamases in Escherichia coli by single-strand conformation polymorphism - PCR. Antimicrob Agents Chemother 1998; 42: 879-84.
• 15. Takenouchi T, Sakagawa E., Sugawara M. Detection of gyrA mutations among 335 Pseudomonas aeruginosa strains isolated in japan and their susceptibilities to fluoroquinolones. Antimicrob Agents Chemother 1999; 43: 406-09.
• 16. Widjojoatmodjo MN, Fluit AC, Verhoef H. Rapid identification of bacteria by PCR-single-strand conformation polimorphism. J Clin Mikrobiol 1994; 32: 3002-3007.