Zastosowanie metody Real-time PCR opartej na fluorescencji barwnika SYBR Green I do wykrywania genów zlokalizowanych w DNA plazmidów pXO1 i pXO2 oraz specyficznej sekwencji chromosomalnej rpoB szczepów Bacillus anthracis

• Autorzy: Budniak S. , Kedrak-Jablonska A. , Reksa M. , Szczawinska A. , Borkowska-Opacka B.

Warianty tytułu

EN

Application of the real-time PCR method with the intercalating dye SYBR Green I for the detection of genes located on plasmids pXO1 and pXO2 as well as the specific chromosomal rpoB sequence of Bacillus anthracis strains

• Języki publikacji: PL

Abstrakty

EN

The aim of the study was to apply the real-time PCR method with the intercalating dye SYBR Green I for the detection of genes of Bacillus anthracis located on plasmids pXO1 and pXO2 and the specific chromosomal rpoB sequence. The research was conducted on one vaccinal and four field strains of Bacillus anthracis. The assessment of the specificity of the tests involved isolates of other species of the genus Bacillus as well as strains of six other species of microorganisms. The studies were conducted with the PCR QuantiTect kit (Qiagen) and primers specific for the gene pag coding PA protein, gene cap coding capsule, and primers for the amplification of the specific chromosomal sequence. PCR enabled the detection of all genes under examination by the observation of amplification curves. The specificity of real-time PCR was confirmed by estimating melting temperatures of PCR products. It was shown that the melting temperatures of amplicons obtained in the reaction were 78°C, 79°C and 76°C in cases of detecting the chromosomal rpoB sequence, pag gene, and cap-C gene, respectively. The sensitivity and linearity of the reactions were determined using a regression coefficient. A high regression coefficient of 0.99 was demonstrated for all the reactions. The real-time tests were highly sensitive and specific.

Słowa kluczowe

PL

Bacillus anthracis; identyfikacja; laseczki waglika; metoda Real Time PCR; mikrobiologia; SYBR Green I; szczepy bakteryjne

• Wydawca: -
• Czasopismo: Medycyna Weterynaryjna
• Rocznik: 2010
• Tom: 66
• Numer: 09
• Opis fizyczny: s.630-634,rys.,tab.,bibliogr.

Twórcy

autor

Budniak S.

• Państwowy Instytut Weterynaryjny - Państwowy Instytut Badawczy, Al.Partyzantów 57, 24-100 Puławy

autor

Kedrak-Jablonska A.

autor

Reksa M.

autor

Szczawinska A.

autor

Borkowska-Opacka B.

Bibliografia

• 1.Adone R., Pasquali P., La Rosa G., Marianelli C., Muscillo M., Fasanella A., Francia M., Ciuchini F.: Sequence analysis of the genes encoding for the major virulence factors of Bacillus anthracis vaccine strain Carbosap. J. Appl. Microbiol. 2002, 93, 117-121.
• 2.Bode E., Hurtle W., Norwood D.: Real-Time PCR assay for a unique chromosomal sequence of Bacillus anthracis, J. Clin. Microbiol. 2004, 42, 5825-5831.
• 3.Bragg T. S., Robertson D. L.: Nucleoide sequence of the lethal factor gene from Bacillus anthracis. Gene 1989, 81, 45-54.
• 4.Dixon T. C., Meselson M., Guillemin J., Hanne P. C.: Antrax. N. Engl. J. Med. 1999, 341, 815-826.
• 5.Ellerbrok H., Nettermann H., Özel M., Beutin L., Appel B., Pauli G.: Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR. FEMS Microbiol. Lett. 2002, 214, 51-59.
• 6.Franz D. R., Jahrling P. B., Friedlander A. M., Clain D. J., Hooven D. L., Bryne W. R., Pavlin J. A., Christopter G. W., Eitzen E. B.: Clinical recognition and management of patients exposed to biological warfare agents. JAMA 1997, 278, 399-411.
• 7.Heid C. A., Stevens J., Livak K. J., Williams P. M.: Real time quantitative PCR. Genome Res. 1996, 6, 986-994.
• 8.Helgason E., Okstad O. A., Caugant D. A., Johansen H. A., Fouet A., Mock M., Hegna I., Kolstro A. B.: Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis - one species on the basis of genetic evidence. Appl. Environ. Microbiol. 2000, 66, 2627-2630.
• 9.Kaspar R. L., Robertson D. L.: Purification and physical analysis of Bacillus anthracis plasmids pXO1 and pXO2. Biochem. Biophys. Res. Commun. 1987, 149, 362-368.
• 10.Keim P., Klevystka A. M., Price L. B., Schupp J. M., Zinser G., Smith K. L., Hugh-Jones M. E., Okinaka R., Hill H. K., Jackson P. J.: Molecular divercity in Bacillus anthracis. J. Appl. Microbiol. 1999, 87, 215-217.
• 11.Makino S., Cheun H. I., Watarai M., Uchida I., Takeshi K.: Detection of anthrax spores from air by real-time PCR. Let. Appl. Microbiol. 2001, 33, 237-240.
• 12.Makino S., Uchida I., Terakado N., Sasakawa C., Yoshikawa M.: Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis. J. Bacteriol. 1989, 171, 722-730.
• 13.Mikesell P., Ivins B. E., Ristroph J. D., Dreier T. M.: Evidence for plasmid mediated toxin production in Bacillus anthracis. Infect. Immun. 1983, 39, 371-376.
• 14.Morrison T. B., Weis J. J., Wittwer C. T.: Quantification of low copy transcripts by continuons SYBR Green I monitoring during amplification. Biotechniques 1998, 24, 954-962.
• 15.Mullis K., Faloona F., Scharf S., Saiki R., Horn G., Erlich H.: Specific enzymatic amplification and detection of DNA in vitro: the polymerase chain reaction. Biotechnology 1992, 24, 17-27.
• 16.Okinaka R., Cloud K., Hampton O., Hoffmaster A. R., Hill K. K., Keim P., Koehler T. M., Lamke G., Kumano S., Mahillon J., Manter D., Martinez Y., Ricke D., Svensson R., Jackson P. J.: Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J. Bacteriol. 1999, 181, 6509-6515.
• 17.Okinaka R., Cloud K., Hampton O., Hoffmaster A. R., Hill K. K., Keim P., Koehler T. M., Lamke G., Kumano S., Manter D., Martinez Y., Ricke D., Svensson R., Jackson P.: Sequence assembly and analysis of pXO1 and pXO2. J. Appl. Microbiol. 1999, 87, 261-262.
• 18.Prince L. B., Hugh-Jones M., Jackson P. J., Keim P.: Genetic diversity in the protective antigen gene of Bacillus anthracis. J. Bacteriol. 1999, 181, 2358-2362.
• 19.Qi Y., Patra G., Liang X., Williams L. E., Rose S., Redkar R. J., DelVecchio V. G.: Utlization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis. Appl. Environ. Microbiol. 2001, 67, 3720-3727.
• 20.Ririe K. M., Rasmussen R. P., Wittwer C. T.: Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 1997, 245, 154-160.
• 21.Shafazand S., Doyle R., Ruoss S., Weinacker A., Raffin T. A.: Inhalation anthrax: epidemiology, diagnosis and management. Chest. 1999, 116, 1369-1376.
• 22.Stadejek T.: Postęp w rozwoju techniki cyklicznej polimeryzacji DNA in vitro - Real-Time PCR. Medycyna Wet. 2006, 62, 390-394.
• 23.Stadejek T., Janicka K., Pejsak Z.: Zastosowanie Real-Time PCR do wykrywania zakażeń pestiwirusowych. Medycyna Wet. 2006, 62, 165-169.
• 24.Turnbull P.: Antrax in Humans and Animals. WHO, Genewa 2008.
• 25.Turnbull P. C., Hutson R. A., Ward M. J., Jones M. N., Quinn C. P., Finnie N. J., Duggleby C. J., Kramer J. M., Melling J.: Bacillus anthracis but not always anthrax. J. Appl. Bacteriol. 1992, 72, 21-28.
• 26.Uchida I., Sekizaki T., Hashimoto K., Terakado N.: Association of the encapsulation of Bacillus anthracis whit a 60 megadalton plazmid. J. Gen. Microbiol. 1985, 131, 363-367.
• 27.Welkos S. L.: Plasmid - associated virulence factors of non-toxigenic (pXO1) Bacillus anthracis. Microb. Pathog. 1991, 10, 183-198.

Uwagi

Rekord w opracowaniu