The method of determining gene of thermostable enterotoxin the representatives of the family enterobacteriaceae

 

(57) Abstract:

The invention relates to biotechnology, medical Microbiology. The method is carried out by dot-blot hybridization using oligonucleotide probe. As an oligonucleotide probe use 30-membered oligonucleotide homologous to the highly conserved site of the famous ST - enterotoxins Enterobacteriaceae installed nucleotide sequence: 5'-TGCTGTGAANTNTGTTGTAATCCTGCCTGT-3'. Using oligonucleotide probe is used to detect ST-genes, regardless of their species. 5 Il., table 1.

The present invention relates to the field of medicine, namely, medical Microbiology, and can be used for analysis of clinical samples in the decryption process of the etiology and characteristics of pathogenesis, Department for industrial environment associated with infectious processes.

There is a method of detecting ST-enterotoxigenic Escherihia coli by the method of blot-hybridization using a 23-membered of the oligonucleotide, complementary to the gene STa-toxin E. coli (Echeverria P., Taylor, D. N., Seriwatana J., Brown E. , Lexomboon U. Examination of colonies and stool blots for detection of enteropathogens by DNA hybridization with eight DNA probes. // J. of Clin. Environ. - 1989. - v. 27, No. 2. - p. 331 - 334.) Used a synthetic 23-membered has liberali in hybridization solution of the following composition: 50% formamide, Hssc (hssc of 0.15 M NaCl and 0.15 sodium citrate), 0.1% of SDS, 1 mm EDTA and denhardt's solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.2% bovine serum albumin). Then put in fresh hybridization solution containing denatured by heating DNA and similarly treated DNA calf thymus at a concentration of 75 μg/ml, and then incubated overnight at 37oC. Hybridization with labeled -32P-oligonucleotide probe was carried out overnight at 50oC, dried in air and was radioautography within 24 hours at -70oC.

This uses less specific 23-membered oligonucleotide is detected only one type of known ST-genes Escherihia coli, and is not determined by genes ST-toxins among strains of the Department for industrial environment other species. Possible in principle proposed by the authors of the approach relative to other representatives of Enterobacteriaceae in each case requires experimental selection of the appropriate oligonucleotide, which leads to an increase in the cost of research and not always feasible.

The aim of the invention is the extension of the spectrum detected by the method of the dot-blot hybridization ST-genes and the possibility of their detection in the representatives of informative and diagnostic value of research.

In Fig. 1 shows the results of comparative analysis of amino acid sequences currently known thermostable enterotoxins.

Performed alignment of nucleotide sequences of genes coding for heat-stable enterotoxins of E. coli, by the Clustal method using the program Megaline of the software package Lasergene (DNA Star"), analyzed the sequences of genes estA1, estA2, estA3, estA4, est1a and the degree of sequence homology 30-membered oligonucleotide probe to the conservative site genes.

In Fig. 2 - 5 shows radioautographic the results of dot-blot hybridization using the proposed oligonucleotide with enterobacteria different types.

The method definition is as follows.

DNA isolation.

Cells were cultured at 37oC in liquid LB medium overnight, 1 ml of the cell suspension is then precipitated at 12000 rpm in a centrifuge of the type "Eppendorf". Sediment resuspended and diluted in buffer containing 50 mm KCl, 10 mm Tris-HCl (pH 8,4), 2.5 mm MgCl20.01% of gelatin to a final concentration of 108CFU/ml. Lizirovania carry lysozyme ("Serva") at a concentration of 1 mg/ml for 15 min at room temperantiam for 30 min at 60oC. inactivate the Enzyme by boiling 15 minutes sample Storage prior to use is carried out at 4oC.

Dot-blot hybridization.

DNA is applied on the nitrocellulose filter, while its number matches the contents of the 105-106CFU/ml Fixation of DNA on the filter is carried out at 80oC under vacuum. As a genetic probe is used to 30-membered oligonucleotide probe is radioactively labeled at the 5'-end ( -32P). The hybridization of the DNA with the above oligonucleotide probe and subsequent washing of the filter is carried out at a temperature of 55oC according to the method (Maniatis, T., F. F. Fritsch, J. Sambrook (1982) Molecular cloning. A Laboratory Manual. Cold Spring Harbor Lab.). The dried filter radioautographic during the night.

To determine the gene of thermostable enterotoxin using dot-blot hybridization as a genetic probe is used to 30-membered oligonucleotide (5'-TGCTGTGAANTNTGTTGTAATCCTGCCTGT-3'), designed by us on the basis of the analysis of amino acid and nucleotide sequences corresponding to a highly conserved area of all currently known ST enterotoxins Enterobacteriaceae and Vibrionaceae (Fig. 1, 2).

As an example, explored the Museum and clinical statutoty shown in the table.

Example 1.

Allocated DNA 20 clinical strains of Citrobacter spp., which was examined by the method of the dot-blot hybridization using 30-membered oligonucleotide probe with the following nucleotide sequence: (5'-TGCTGTGAARRRTGTTGTAATCCTGCCTGT-3'). The results of autoradiography is shown in Fig. 2.

Example 2.

Allocated DNA 5 clinical strains of E. coli, which was examined by the method of the dot-blot hybridization using 30-membered oligonucleotide probe with the following nucleotide sequence: (5'-TGCTGTGAAATATGTTGTAATCCTGCCTGT-3'). The results of autoradiography is shown in Fig. 3.

Example 3.

Allocated DNA 10 Museum of strains of E. coli, which was examined by the method of the dot-blot hybridization of the probe with the following nucleotide sequence: (5'-TGCTGTGAACTCTGTTGTAATCCTGCCTGT-3'). The results of autoradiography is shown in Fig. 4.

Example 4.

Allocated DNA 3 Museum of strains of Y. enterocolitica, which was examined by the method of the dot-blot hybridization using 30-membered oligonucleotide probe with the following nucleotide sequence (5'-TGCTGTGAAGTGTGTTGTAATCCTGCCTGT-3'). The results of radiography shown in Fig. 5.

Studies have shown that the proposed is designed by us oligonucleotide probe is effective, is universal and allows to detect ST-genes, Department for industrial environment, regardless of their species.

The method of determining gene of thermostable enterotoxin the representatives of the family Enterobacteriaceae, including dot-blot hybridization using oligonucleotide probe, characterized in that the oligonucleotide probe used 30-membered oligonucleotide homologous to the highly conserved site of the famous ST - enterotoxins Enterobacteriaceae, with the following nucleotide sequence: 5'-TGCTGTGAANTNTGTTGTAATCCTGCCTGT-3'.

 

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