Molecular Technologies for Detection of Anthrax Causative Agent by PCR of Different Formats

Автор: O.Yu. Limanskaya, L.A. Murtazaeva, S. Klee, and A.P. Limanskii

Страница: 86-96

Molecular Technologies for Detection of Anthrax Causative Agent by PCR of Different Formats

Biotekhnologiya, 2013, N 3, P. 86-96

UDC 577.2:573.6

Section:  “Metrology, Standardization, and Control”

O.Yu. Limanskaya ^{1, 2, *},  L.A. Murtazaeva ¹,  S. Klee ³,  and  A.P. Limanskii ¹

¹  The Mechnikov Institute of Microbiology and Immunology, Natl. Acad. Med. Sci. of Ukraine,  61057, Kharkov Ukraine

²  The National Scientific Center Institute of Experimental and Clinical Veterinary Medicine, Natl. Acad. Agrar. Sci. of Ukraine,  61023, Kharkov Ukraine

³  Robert Koch-Institut,  D-13353, Berlin Germany

e-mail:  olga.limanskaya@mail.ru.

Accurate identification of Bacillus anthracis bacteria remains a challenge in discrimination of anthrax causative agent from closely related species of B. cereus and B. thuringiensis due to the high level of similarity of nucleotide sequences among the members of the Bacillus cereus sensu lato(Bacillus cereus s.l.) group, to which all the above bacteria belong. In this work, we have developed primer sets and probes for real-time PCR (RT-PCR) with fluorescence and hybridization detection for the discrimination of B. anthracis from B. cereus and B. thuringiensis and primer sets for conventional PCR with the electrophoretic detection. For the members of the Bacillus cereus s. l. group, the nucleotide sequences of the chromosomal rpoB, plcr, and ssp genes, which may be molecular genetic markers for their typing were analyzed. The fragment of the chromosomal ssp gene that was only characterized by a hexanucleotide insertion in B. anthracis isolates was identified as a target for the primers and probes. The conventional PCR with electrophoretic detection and designed primer set can reliably discriminate B. anthracis bacilli from the closely related B. cereus and B. thuringiensis species. Application of real-time PCR with TaqMan and molecular beacon probes also permits to carry out an accurate discrimination of B. anthracis from the closely related bacilli: the fluorescence signal for hairpin molecular beacon probes was only positive for the B. anthracis strains, and it was negative for other members of the Bacillus cereus s. l. group. Using the TaqMan linear probes, high-­intensity fluorescence signal was observed for all the B. anthracis isolates, whereas the signal of much lower intensity was fixed for the other members of the Bacillus cereus s. l. group.The developed approaches can be useful in clinical, epidemiological and epizootiological studies.

Key words:  anthrax causative agent,  Bacillus anthracis,  conventional PCR,  molecular beacon,  real-time PCR.

The full English version of the article was published in “Biotechnology in Russia”, 2013, Issue 3, pp. 86-96 as O.Yu. Limanskaya, L.A. Murtazaeva, S. Klee, and A.P. Limanskii “Molecular Technologies for Detection of Anthrax Causative Agent by PCR of Various Formats”.

It is contained at the Russian Scientific Electron Library website:  http://elibrary.ru/item.asp?id=22952564

09.04.2015, 1691 просмотр .