Method for detection of enteroviruses in water

FIELD: medicine.

SUBSTANCE: enteroviruses are concentrated by introduction into an analysed water sample of a magnetic sorbent microparticles coated with polymeric silicon dioxide with amino poropyl groups in proportions 1:1000-3000 of water sample volume. It is incubated at constant stirring for 1-2 hours. The sorbent is collected with a magnet, a supernatant is removed, and a sorbent-enterovirus complex is produced. Enteroviruses are eluted by 0.5M NaCl and 0.05M Tris (pH-10.5) solution. Enteroviruses are identified by immunochemical, cultural and molecular methods.

EFFECT: high degree of virus concentration in the eluate, decreased amount of the eluating solution.

2 ex

 

The invention relates to the field of medicine, epidemiological surveillance and Virology, and can be used to detect enteric viruses in tap water, in the water, open water, freshwater and seawater, water from underground sources, bottled drinking water, in the water of swimming pools and waste water.

The level of technology.

At the present time to detect enteric viruses in water in the Russian Federation there are different ways, each of which applies to a specific type or volume of water. This method using filtration membranes using ion-exchange resins, two-step method (sorption on ion-exchange resin and precipitation using ammonium sulfate), the method of two-phase separation method using non-woven bags with makroporistom glass and using the kit for collection and concentration of viruses from drinking water using trap device [HOWTO No. 4.2.2029-05 "control Methods. Biological and microbiological factors. Sanitary and virological monitoring of water objects"].

There are also known methods, proposed by foreign authors. This concentration of viruses on membranes with ligands [US patent US 20080014625], negatively charged membranes [Katayama H., Shimasaki A., Ohgaki S. Development of a virus concentration method and its application to detection of nterovirus and Norwalk virus from coastal seawater. Appl. Environ. Environ., 2002, 68: 1033-1039].

Closest to the claimed method is a method of detection of enteric viruses, including the concentration of viruses by using non-woven bags with macroporous glass [Kontorovich V.B. have been, Ivanova PU, Eremeeva SO, Shearman GA, V.A. Kazantsev Method for the concentration of viruses in the water environment. The matters. virusol., 1996, 1: 40-42]. In the specified way time concentration of viruses from water surface water and waste water is 3-7 days. To improve the sorption properties of macroporous glass, representing the white powder, it is treated as follows: one volume of glass is poured into the flask one volume of a mixture (1:1) 3% solution of H2O2and 6M hydrochloric acid and boil in a fume hood for 1 h without tube, observing precautions. Washed a large amount of distilled water until neutral pH and dried at 100°C. In a batch of fabric measuring 5×7 cm was placed 3.0 cm3the prepared sorbent. Package sorbent is fixed by means of a scaffold for a motionless subject so that he was in the water flow. After exposure within 3-7 days package take out, put in some new plastic bag or sterile vial and transported to the laboratory in a refrigerator bag in the shortest with the OK (no more than 6 hours). Each sample is labeled indicating the point of selection, installation date, and time of exposure of the package. Before processing samples can be stored for days at 4°C. the Package with the sorbent is removed from the shipping container and placed in a sterile Petri dish. Cut the edge of the package, wash the glass with distilled water (5 ml) using a pipette in the same Petri dish and transferred by pipette or through the funnel into the column in a volume of 5-10 ml elute Viruses step three solutions in 3 ml each, collecting fractions in separate penicillin vials. Research expose each fraction separately (all 3 factions). As eluting solutions are used: 1) 0.05 M Tris-HCl pH 9,1; 2) 0.05 M Tris-HCl pH 9.1 and 0.5 M NaCl; 3) 3% beef extract in 0.05 M Tris-HCl pH to 9.1. For detection of viruses in the eluate choose culture, immunohistochemistry or molecular methods.

The disadvantages of the above method are its duration, a large number and volume fractions of the eluate, a small area of contact of the sorbent with water volume.

The present invention, which is directed technical result is to reduce the duration of the claimed method and its simplification. In addition, there is a high degree of concentration of viruses, decreases the volume of eluting solution and, consequently, increases the output is irusa in the eluate by using as adsorbent magnetic particles, polymer coated silicon dioxide with aminopropylene groups.

To achieve the technical result in the method of detection of enteric viruses, including the concentration of viruses from water samples on the sorbent, the elution of enteric viruses from sorbent, followed by immunochemical detection, culture or molecular methods, wherein the concentration of enteric viruses is carried out by depositing in the analyzed water sample sorbent on the basis of magnetic microparticles coated with the polymer of silicon dioxide with aminopropylene groups in the ratio of 1:1000-3000 the volume of the water sample, incubation with constant stirring for 1-2 hours, collecting the sorbent using a magnet, remove the supernatant and obtain complex sorbent with intestinal viruses, and the elution of enteric viruses carry out the solution of 0,5M NaCl and 0,05M Tris (pH of 10.5).

Features that distinguish the present invention from analogues are:

the time of incubation of the sorbent MagSi+ sample - 1-2 hours;

- sorbent MagSi+ presents suspension, which increases the probability of adsorption of viruses due to the uniform distribution of the sorbent in the whole volume of water;

- magnetic properties MagSi+ it is possible to collect the sorbent using the magnet in vials or flowing systems;

a positive charge MagSi+ in water which allows it to adsorb viruses not only due to hydrophobic interactions, but due to ion;

- elution of viruses from the sorbent is a small amount of eluting solution, which increases the concentration of viruses in the eluate;

composition and pH value of an eluting solution.

Disclosure of the invention.

The method of detection of enteric viruses in water is the concentration of water on magnetic microparticles coated with the polymer of silicon dioxide with aminopropylene groups, the removal of virions from the sorbent using a special buffer for elution and subsequent detection of enteric viruses in the eluate using molecular, immunochemical or cultural methods.

Sorbent MagSi+ was obtained according to the method described in U.S. patent US 20030148101 A1 with some modifications. The coated magnetite was carried out in two stages where the first stage was identical to that described in the patent (polymerization of tetraethoxysilane on the surface of particles of magnetite at pH 4.6 and a temperature of 90°C), and at the second stage the solution of tetraethoxysilane was replaced with 3-aminopropyltrimethoxysilane in the same concentration. The reaction on the surface of the magnetite formed polymer layers of silicon dioxide with aminopropylene groups that at pH lower than 7.0 have a positive charge. The sorbent is a particle dark brown color size 2-10 μm, not prone to the formation of aggregates is determined that same positive charge particles. The capacity of the sorbent MagSi+estimated according to the model protein BSA (bovine serum albumin)is 15-20 mg of protein per 1 cm3sorbent. In model experiments for concentrating viruses from 1 l of tap water efficiency concentration of adenovirus was 76%, enteroviruses - 81% and of hepatitis a virus - 88%.

The principle of adsorption of viruses on the sorbent MagSi+ based on two types of interactions: (1) hydrophobic interaction between the capsid proteins of viruses with polysiloxane bonds of the polymer silicon dioxide; (2) electrostatic interaction between the amine groups of the sorbent, which in aqueous conditions at pH lower than 7.0) have a positive charge, with virions, negatively charged at pH values above the 5.0. In addition, the same positive charge particles MagSi+ provides the distribution of the sorbent in the whole volume of water, thereby increasing the likelihood of exposure of virions with particles of sorbent and speeding up the process of sorption. Thus, to ensure the effective concentration of viruses due to electrostatic interactions it is necessary that the pH of the water was in the range of 5.0-7.0. This pH range corresponds to the average values of the pH of the water.

The method includes the following steps.

1. The introduction of sorbent in a water sample. Suspension of sorbent MagSi+ in a volume of 1 ml (1:1 by volume sorbent MagSi+ and 50 mm races the thief NaCl) is added to 1 l of the investigated water.

2. Incubation with constant stirring for 1-2 hours at room temperature. Mixing is carried out using a shaker or verhneprivodnaya stirrers, the entire sorbent must be suspended.

3. Collection of sorbent MagSi+ using magnet in vials or in special units for flow concentration of magnetic microparticles. Remove supernatant, obtaining complex virus + sorbent.

4. Elution of viruses is the solution of the following composition - 0,5M NaCl, 0,05 m Tris, pH of 10.5, based on 1 ml of the suspension of sorbent MagSi+ added 1 ml of an eluting solution.

5. Indication and identification of viruses using immunohistochemistry, culture or molecular methods. For the detection of rotavirus and hepatitis a virus applies the detection of viral antigens and enzyme-linked immunosorbent assay. Cultural methods, in particular, the neutralization reaction, used for the detection and typing of enteroviruses (ECHO, Coxsackie a and b, Polioviruses and Enteroviruses 68-71) and adenoviruses. Molecular techniques such as reverse transcription and subsequent PCR or PCR "real-time"can be used to identify all known enteric viruses [Svraka S et al. 2009, G. Shay Fout et al. 2003; guidelines No. 4.2.2029-05].

The implementation of the invention.

Prima is 1.

Carry out the concentration of enteric viruses from wastewater samples. To 1 l of pre-clarified waste water add 1 ml of the suspension of the sorbent MagSi+. Incubated with constant mixing on a shaker or using verhneprivodnaya stirrer for 1 h After exposure collecting the sorbent using a rare earth magnet, transfer the suspension into a 15-ml sterile polypropylene tube. Remove supernatant, holding magnet sorbent with adsorbed viruses on the inner wall of the tube, and hold the elution of viruses 1 ml of a solution of the following composition - 0,5M NaCl, 0,05M Tris, pH of 10.5. Intensively shake the test tube on a vortex for 15 minutes, the entire sorbent must be suspended. Precipitated sorbent by centrifugation at 3000 rpm for 10 minutes, or by using a magnet. The supernatant transferred to two 1.5-to 2.0-ml polypropylene tubes with screw cap. Indication and identification of viruses and their structural elements (viral proteins or nucleic acids), make use of immunohistochemistry, culture or molecular methods (various modifications of the PCR).

Example 2.

Carry out the concentration of enteric viruses from water (drinking) water. To 10 l water (drinking) water, add 3 ml of the suspension of the sorbent MagSi+. Incubate the ri constant mixing on a shaker or using verhneprivodnaya mixer 2 hours After exposure in water add 100 ml 5M NaCl and incubated with stirring for another 30 minutes. Collect the sorbent using a rare earth magnet, transfer the suspension into a 15-ml sterile polypropylene tube. Hold the sorbent magnet, remove the supernatant and spend the elution of enteric viruses in 3 ml of a solution of the following composition - 0,5M NaCl, 0,05 m Tris, pH of 10.5. Intensively shake the test tube on a vortex for 15 minutes, the entire sorbent must be suspended. Precipitated sorbent by centrifugation at 3000 rpm for 10 minutes, or by using a magnet. The supernatant in 1.5 ml transferred to two 1.5-to 2.0-ml polypropylene tubes with screw caps. Indication and identification of enteric viruses or their structural elements (viral proteins or nucleic acids), make use of immunohistochemistry, culture or molecular methods (various modifikacii PCR).

The method of detection of enteric viruses, including the concentration of viruses from water samples on the sorbent, the elution of enteric viruses from sorbent, followed by immunochemical detection, culture or molecular methods, wherein the concentration of enteric viruses is carried out by depositing in the analyzed water sample sorbent on the basis of magnetic microparticles coated with poly is a leader in silicon dioxide with aminopropylene groups, in the ratio of 1:1000-3000 the volume of the water sample, incubation with constant stirring for 1-2 hours, collecting the sorbent using a magnet, remove the supernatant and obtain complex sorbent with intestinal viruses, and the elution of enteric viruses carry out the solution of 0.5m NaCl and 0,05M Tris (pH of 10.5).



 

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