Recombinant plasmid psp64 dna-s, providing transcription in vitro slice s segment rna of the virus is the causative agent of hemorrhagic fever with renal syndrome serotype hantaan and positive rna control for the diagnosis of hemorrhagic fever with renal syndrome rt-pcr based on it

 

The invention relates to biotechnology, in particular genetic engineering and medical Virology. The proposed recombinant plasmid pSP64 DNA-S, with the promoter SP6 DNA-dependent RNA polymerase and the fragment of the cDNA of the S-segment of the virus is the causative agent of hemorrhagic fever with renal syndrome (HFRS) serotype Hantaan. Plasmid effectively carries out the transcription of the S-segment negative strand of RNA that is identical to the fragment of the RNA viral genome. Also offered positive RNA control for the diagnosis of virus is the causative agent of hemorrhagic fever with renal syndrome. To obtain the proposed positive RNA control as a matrix using plasmid pSP64-S. the Proposed invention allows to simplify and improve the safety of work to obtain a positive RNA control for the diagnosis of virus is the causative agent of hemorrhagic fever with renal syndrome, and improve quality control. 2 S. p. f-crystals., 1 Il.

The invention relates to medical Virology, in particular for the diagnosis of hemorrhagic fever with renal syndrome (HFRS).

In everyday laboratory diagnosis of HFRS use the methods of detection of antibodies in the blood, such as different variants of the methodology enzyme immunoassay, and changesnow chain reaction (RT-PCR) - a relatively new method that has several advantages such as high sensitivity. The method is not received until a wide distribution in regions endemic for this disease, in the absence of amplication of diagnostic systems. In the development of diagnostic systems one of the main problems is the choice of the positive control. As such can be used RNA isolated from cell cultures infected with laboratory strains of the virus is the causative agent of hemorrhagic fever with renal syndrome, or from organs of infected rodents.

The main disadvantages of receipt of such control are the following: 1) the need for re-Biosafety level during operations; 2) the diversity of genetic material extracted from organs of rodents.

Known recombinant plasmid DNA pF1R2 and pF1R2+118, which allows to obtain in vitro positive RNA control for the detection method of amplification of Hantavirus serotype Sin Nombre [1, prototype].

But the virus serotype Sin Nombre causes another disease, Hantavirus pulmonary syndrome that differs from HFRS.

Known methods and kits for generification diagnostic methods PCR and RT-PCR of hepatitis b and C, HIV-diagnostiki, and automate their execution.

However, such systems are not described for the diagnosis of HFRS.

A known method of detecting Hantavirus serotype Sin Nombre RT-PCR [1, prototype].

However, this method is used to determine virus serotype Sin Nombre, causing pulmonary syndrome, which is different from the virus serotype Hantaan causing HFRS.

An object of the invention is to simplify and improve the safety of work to obtain a positive RNA control for the diagnosis of virus is the causative agent of hemorrhagic fever with renal syndrome, as well as improving quality control due to its homogeneity.

The problem is solved by constructing the plasmid pSP64-S with the promoter for SP6 DNA-dependent RNA polymerase and the fragment of the cDNA of the S-segment of the virus is the causative agent of hemorrhagic fever with renal syndrome serotype Hantaan, the coding sequence nucleocapside protein. With this piece now is the transcription of the negative RNA strand that is identical to the fragment of the RNA viral genome.

Recombinant plasmid pSP64 DNA-S, providing transcription in vitro slice S segment RNA of the virus is the causative agent of hemorrhagic fever with renal syndrome serotype Hantaan, characterized by the following features: - has molecular the nom resistance to ampicillin, promoter for SP6 DNA-dependent RNA polymerase [3] ; - cDNA fragment of the S-segment of the virus is the causative agent of hemorrhagic fever with renal syndrome serotype Hantaan size 1310 p. O.; contains: - a unique recognition sites of restriction endonucleases: PvuI, SeaI, SalI, XbaI, AvaI, SmaI, EcoRI.

DNA pSP-64 is treated with a restriction enzyme in the appropriate buffer. For preparation of DNA for the ligation reaction hydrolyzate applied to 1% agarose gel, followed by purification of the DNA using a set of Ultra Clean ("Mo Bio Lbrtries"). The cDNA fragment of the S-segment cut by SacI sites of the plasmid pMOS-S, constructed earlier, and thus distinguish it from the 1% agarose gel. The resulting product is used to transform cells of E. coli strain XL-2Blue. Ampicillin-resistant colonies perekusyvayut on nitrocellulose filters with subsequent lysis and immobilization on them plasmid DNA. Then hold the reaction hybridization using as a probe drug in the same fragment of the S-segment, radiolabelled phosphorus. Plasmid DNA isolated from hybridization of colonies analyzed using restricted HindIII, PST, NcoI and subsequent distillation in a 1% agarose gel. Selected clones containing the insert cDNA in the correct orientation, providing a negative transcription dedicated DNA is treated with restriction enzyme EcoRI, then a mixture of phenol-chloroform, planted with alcohol and dissolved in water. The linear form suitable for use in the reaction of transcription.

In response transcription take pre-linearized plasmid DNA, add a mixture rNTP, the inhibitor RNA-AZ and SP6 RNA polymerase. After the reaction RNA transcription spend processing DNA Asai, not containing RNA-AZ. The obtained RNA precipitated, washed with ethanol and dissolved water, free from DNA AZ and RNA-AZ. The RNA concentration and purity determined spectrophotometrically. The specificity of the RNA test method RT-PCR and visualization of bands of the appropriate size in 1% agarose gel.

The list of graphical materials.

The drawing shows a physical map of the recombinant plasmid pSP64.

The invention is illustrated by the following examples.

Example 1. Construction of plasmid pSP64-S. To 10 µg DNA pSP64 add 15 E. A. restrictase SacI, incubated in the appropriate buffer in a volume of 50 μl for 1 h at 37oC. For the preparation of vector DNA for ligation reaction hydrolyzate applied to 1% agarose gel, followed by purification of the DNA using a set of Ultra Clean ("Mo Bio Laboratories). The cDNA fragment of the S-segment cut by SacI sites of the plasmid pMOS-S, scons μl for 16 hours at a temperature of 4oWith a molar ratio of vector: fragment 1:1. The resulting product is used to transform cells of E. coli strain XL-2Blue. Ampicillin-resistant colonies perekusyvayut on nitrocellulose filters with subsequent lysis and immobilization on them plasmid DNA. Then hold the reaction hybridization using as a probe drug in the same fragment of the S-segment, radiolabelled phosphorus. Plasmid DNA isolated from hybridization of colonies analyzed in 1% agarose gel using restricted HindIII, PST and NcoI. Selected clones containing the insert cDNA in the correct orientation, providing the transcription of the negative RNA strand. Plasmid DNA pSP64-S accumulate in the cells of E. coli in 50 ml LB-medium, followed by separation according to standard methods. The selected DNA is treated with restriction enzyme EcoRI, and then with a mixture of phenol-chloroform, planted with alcohol and dissolved in water. The linear form suitable for use in the reaction of transcription.

Example 2. Transcription of RNA. In response transcription take 1 µg pre-linearized plasmid DNA, add rNTP mix (10 mM each), 50 E. A. inhibitor RNA-AZ and 30 E. A. SP6 RNA polymerase. The final volume of the mixture is 50 ál. Time readuse the reaction mixture and incubated for 15 min at 37oC. In the future, add 1 volume (50 ál) of TE-saturated phenol-chloroform, shaken, centrifuged, selected the top phase. RNA is precipitated with a mixture of 1 volume of isopropanol and 1/10 volume of 3M NaAc. After twice washing the precipitate first 75% and then 96% ethanol, dissolve it in 100 ál of water, free from DNA AZ and RNA-AZ.

To determine the concentration of RNA take an aliquot and determine spectrophotometric absorption at the wavelength of 260 nm. Calculate the concentration of RNA based on the fact that 1 O. E. corresponds to 40 µg/ml the purity of the RNA is also determined spectrophotometrically. To do this, determine the ratio of absorption at wavelengths of 260 and 280 nm. Attitude a/A should be not less than 1.7. The specificity of the RNA test method RT-PCR and visualization of bands of the appropriate size in 1% agarose gel.

Thus, the obtained positive RNA control is a necessary component of amplication test systems, allowing to diagnose the virus is the causative agent of hemorrhagic fever with renal syndrome in extensive laboratory practice.

References
1. M. Terajima, J. D. Hendershot III, H. Kariwa and et. all. High levels of viremia in patients with the Hantavirus pulmonary syndrome. The Journal of Infection Diseases, 1990; 180:2030-2034.

2. RF patent 2164532 "Methods and kits for genapple the kers and nucleic acids, 1996, p.105.


Claims

1. Recombinant plasmid pSP64 DNA-S, providing transcription in vitro slice S segment RNA of the virus is the causative agent of hemorrhagic fever with renal syndrome serotype Hantaan, characterized by the following features: has a molecular mass of 2843,9; kd is the size 4309 base pairs; consists of the plasmid pSP-64 (Promega), size 2999 p. O., ori region, the gene for resistance to ampicillin, promoter for SP6 DNA-dependent RNA polymerase; - cDNA fragment of the S-segment of the virus is the causative agent of hemorrhagic fever with renal syndrome serotype Hantaan, size 1310 p. O. contains a unique recognition sites of restriction endonucleases, PvuI, ScaI SalI, XbaI, AvaI, SmaI, EcoRI.

2. Positive RNA control for the diagnosis of virus is the causative agent of hemorrhagic fever with renal syndrome RT-PCR, characterized in that as a matrix for obtaining in vitro positive RNA control use of recombinant plasmid pSP64 DNA-S, providing transcription in vitro slice S segment RNA of the virus is the causative agent of hemorrhagic fever with renal syndrome serotype Hantaan.

 

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