AT509276B1 - DEVICE FOR DETERMINING MICROORGANISMS - Google Patents

Abstract

The present invention relates to an apparatus for the determination of microorganisms, in particular chlamydia, gonococci and fungi, in a sample comprising a liquid-permeable solid medium defining a path for a liquid flow, the medium having spaced apart areas along the liquid path, said regions being in sequential order Task area, a reaction area, a detection area and a control area are divided, characterized in that in the task area a buffer, preferably a neutralization buffer, is provided in a dried state.

Description

Austrian Patent Office AT509 276B1 2014-08-15

Description: The present invention relates to a device, a method and a kit for the detection of microorganisms.

Microorganisms can be detected by various methods and devices. Probably the most sensitive and precise method for the determination of microorganisms is the polymerase chain reaction, with which even the smallest amounts of microorganisms can be specifically detected. However, such methods require expensive equipment that can be operated only by skilled persons.

Alternative methods for the determination of microorganisms take advantage of antibodies that are able to specifically bind to one or more epitopes of a microorganism. Although less sensitive than molecular biological methods (such as polymerase chain reaction), these methods are easier to handle and much faster. In order to allow the use of antibodies for the determination of microorganisms even untrained persons, devices such as test strips have been developed. With the help of test strips on which microorganism-specific antibodies are immobilized, anyone can easily ascertain the presence of microorganisms in a sample.

Depending on the microorganism to be determined, the samples must be processed on a test strip before use. In the course of sample preparation, the sample may be prepared by adding basic or acidic solutions to the microorganisms, e.g. be lysed, diluted. As a result, the sample has a pH that does not allow binding of antigen to antibody so that this sample can not be applied directly to a test strip. Therefore, before applying the sample, it must be neutralized by adding a buffer. The addition of solutions to the sample changes their volume, resulting in further dilution of the sample, significantly reducing the sensitivity of the assay.

It is an object of the present invention to provide a device which u.a. avoids the disadvantages of the prior art.

The present invention relates to a device for the determination of microorganisms, namely chlamydiae or gonococci, in a sample comprising a liquid-permeable solid medium, which defines a path for a liquid flow, which medium along the liquid path having spaced areas, wherein the areas are subdivided in the following order into a feed area, a reaction area, a detection area and a control area, characterized in that a neutralization buffer in the dried area is provided in the feed area, wherein a neutralization buffer in the dried area is provided in the feed area, the buffer being a basic Buffer having a pH of 11 to 13, in particular 12.7, or an acidic buffer having a pH of 2.5 to 6.5.

It has surprisingly been found that it is possible to adjust the pH of an untreated or pretreated sample to a value that allows binding of antigen to antibody in the test strip to provide a buffer in the dried state in the application area. After contacting the sample with the dried buffer, it is dissolved in the liquid-permeable solid medium so that its buffering effect can unfold and the sample is neutralized on the medium. The neutralized sample eventually moves due to the capillary action of the medium in this further to the other areas of the device (reaction area, detection area and control area).

The provision of a buffer in the dried state in the task area has the advantage that the user of the device no or less expensive sample preparation must perform, as an important step (the neutralizing, if necessary) takes place directly in the task area of the device according to the invention.

The device according to the invention may, in principle, be any test strip which is known in the art in Austrian Patent Office AT509 276B1 2014-08-15.

The liquid-permeable solid medium or the liquid-permeable solid support which can be used according to the invention may in principle be made of any material which has a certain porosity, more preferably porous supports comprising nitrocellulose, nylon, PTFE, polystyrene , Latex, glass fibers, silica, alumina, cellulose, dextran, agarose, polyvinyl alcohol. Of course, the device according to the invention can also comprise, in addition to one or more liquid-permeable solid medium (s), other media or materials which have different properties. So it is e.g. possible additionally to provide a non-liquid-permeable support in the device on which the liquid-permeable solid medium rests and stabilizes it.

As used above, "in the feed region" means that the buffer is either directly in the liquid-permeable solid medium or that another liquid-permeable solid medium is located after the feed region.

The term "dried-state buffer" means that the medium in the feed area comprises a buffer that has dried. Such a medium is obtainable by contacting the medium with a liquid buffer, followed by a drying process to remove the feed or solvent. By doing so, a feed area is available that includes a buffer in the dried state.

It has been shown according to the invention that the device according to the invention is particularly well suited to detect Chlamydia.

Chlamydia cause various acute and sometimes chronic diseases in humans. Typical diseases caused by Chlamydia are venereal genitourinary infections, eye infections and atypical pneumonia. The genus Chlamydia contains the species Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia psittaci.

Chlamydia trachomatis is primarily pathogenic to humans. In Chlamydia trachomatis, 18 serotypes are distinguished and it is the most common bacterial agent of sexually transmitted infections.

Chlamydia trachomatis, for example, depending on the manifestation of the serotype lead to eye infections to blindness.

In order to diagnose a Chlamydia infection in good time and, as a result, initiate therapy in time to prevent complications, it is critically important to rapidly diagnose such an infection.

The detection of chlamydia by means of test strips requires, according to the prior art, disruption of the microorganisms with an acidic or basic buffer followed by a neutralization step in which a corresponding basic or acidic buffer is used. By neutralizing the pH of the sample, the detection of Chlamydia in a sample is made possible.

In addition to Chlamydia, there are a variety of other microorganisms that are of importance to humans. Examples of such microorganisms are gonococci responsible for gonorrhea and fungi.

The usual on the market diagnostic tests for the determination of microbial infections are kits that need to be applied at least partially by professionals or which are used exclusively in the laboratory. Therefore, the rapid determination of whether a microbial infection occurs, for the affected person is not possible.

The device of the invention may also be part of a kit for the determination of microorganisms, namely chlamydia or gonococci, in a sample, the following Austrian Patent Office AT509 276B1 2014-08-15

Component comprises: a) at least one means for sampling, preferably at least one Watteup fer or a tampon, b) a container comprising a buffer for digesting chlamydia or

Gonococci and suitable for receiving the means for sampling, preferably the cotton swab or tampon, c) a device according to any one of claims 1 to 7. The inventive

Kit is due to the simple handling of the individual components for the first time possible to diagnose such a microbial infection.

The provided means for sampling, preferably a cotton swab or a tampon, which are ideally added to the kit in sterile form, serve to take the sample to be examined. For example, smears such as vaginal smears can be made with these agents.

In order to better determine the possible microorganisms occurring in the sample, the sampling means are brought into contact with a buffer, which unlocks the cells for the most part. This digestion makes the epitopes of the cells, for example, for antibodies, more accessible, allowing the cells to be detected with increased efficiency.

The container which is suitable to receive the sampling means preferably comprises a minimum amount of buffer of 50-1000 μΙ, preferably 100-500 μΙ, in particular 300 μΙ, which is sufficient to unlock the cells and to dissolve from the agent, so-that the cells or cell fragments remain in the solution.

Since the digestion of the microorganisms is carried out either in the basic or acidic pH range, it is necessary to neutralize the sample solution with either an acidic or basic buffer before it is brought into contact with a means for detecting microorganisms. Namely, when the means for detecting microorganisms is e.g. For antibodies, too acidic or too basic a pH value can lead to a strong reduction of the binding of antibodies to antigens, so that a reliable and reproducible determination is no longer possible. It is essential that a basic neutralization buffer be used when using an acid digestion reagent, and vice versa.

Means for the determination of microorganisms, in particular chlamydia, gonococci and fungi, are well known in the art. Due to the field of application of the kit according to the invention and in particular due to the group of people applying the kit according to the invention or the device according to the invention, it is of advantage that such means are as simple and uncomplicated as possible and can be used without the use of expensive equipment. Such agents are well known in the art and are already widely used. The majority of these detection means is based on the binding of antibodies to surface antigens of the microorganisms to be determined.

According to the present invention, the container b) comprises a basic buffer having a pH of 11 to 13, especially 12.7, or an acidic buffer having a pH of 2.5 to 6.5, wherein the kit each comprises a basic and an acidic buffer.

The inventive kit comprises - as mentioned above - a container with a digestion buffer. Depending on the type of digestion selected (acidic or basic), the dried neutralization buffer should be selected (acidic or basic).

The basic buffer preferably comprises 3 to 5 g, preferably 4 g, NaOH, 3 to 4 g of ethylenediaminetetraacetic acid (EDTA), 1 to 3 g, especially about 2 g, sodium salt of the cholhydrate in 1 L of water.

The acidic buffer preferably comprises 3 to 5 g, preferably 4 to 4.5 g, KH 2 PO 4, 5 9 Austrian Patent Office AT 509 276 B1 2014-08-15 to 9 g, preferably 7 to 8 g, Na 2 HPO 4, 2 to 8 ml, preferably 3 to 6 ml, in particular 5 ml, of a mixture of 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one (Proclin) in 1 L Water, wherein the pH is adjusted to pH 6 to 6.5.

The pH of this buffer can be adjusted to the appropriate pH by the addition of acid, preferably using HCl.

The acidic buffer preferably comprises 3 to 5 g, preferably 4 to 4.5 g, KH 2 PO 4, 5 to 9 g, preferably 7 to 8 g, Na 2 HPO 4, in 1 L of water, the pH being at pH 1 to 3, in particular to pH 2.5, is set.

The test strip preferably comprises a solid carrier of an absorbent medium, which medium is subdivided into a task area, into a reaction area, into at least one detection area and into at least one control area.

In the reaction region, preferably antibodies are mobile, which are capable of binding to an epitope of a surface antigen of the microorganisms to be determined, the antibodies preferably with a dye, colored, fluorescent or magnetic particles, in particular latex particles, or with colloidal gold are marked.

According to a preferred embodiment of the present invention antibodies are immobilized in the detection area, which are able to bind to an epitope of a surface antigen of the microorganisms to be determined, wherein the epitope of the antigen is capable, or is not able to bind to the mobile in the reaction area arranged antibody. According to the invention, antibodies can be immobilized in the detection region which bind to the same or another epitope of an antigen of the microorganisms to be determined. It has been found that the test strips described in US 5,073,484 and US 5,654,162 are most suitable for use in the method of the invention.

In the control region preferably antibodies are immobilized, which are able to bind to the mobile in the reaction region arranged antibodies.

According to a preferred embodiment of the present invention, the container b) comprise 50-1000 μΙ, preferably 100500 μΙ, in particular 300 μΙ, of the respective buffer.

In order to test whether the means for detecting the microorganisms are functioning properly, the kit preferably further comprises a positive control.

According to a particularly preferred embodiment of the present invention, buffer b) comprises a pH indicator, preferably bromothymol blue, preferably in an amount of 0.001 to 0.005 g, in particular in an amount of 0.003 g.

The addition of an indicator allows the user to determine whether the pH of the reagent meets the requirements and can be considered neutralized.

A further aspect of the present invention relates to a method for diagnosing a microbial infection, namely a chlamydial or gonococcal infection, comprising the steps: a) provision of a means for sampling, preferably at least one wetting swab or a tampon, [0045] 0046] b) sampling, c) transferring the sample-collecting sample to a container comprising a buffer for digesting chlamydiae or gonococci, d) incubating the sample with the buffer for 5 seconds to 30 minutes, preferably for 30 seconds to 15 minutes, more preferably for 1 to 10 minutes, in particular for 5 minutes, e) transfer of the neutralized buffer to a device according to the present invention

Invention for detecting one or more microorganisms, and f) diagnosing a microbial infection when microorganisms are detected in step e).

The buffers and agents used in the process according to the invention have already been described above.

Another aspect of the present invention relates to the use of a kit according to the invention for the determination of a microbial infection, namely a chlamydia or gonococcal infection.

The object of the present invention will be explained in more detail with reference to the following example, but without being limited thereto.

This example shows the detection of Chlamydia trachomatis, one of the three known species from the family of Chlamydia bacteria, infections with Chlamydia trachomatis are the most common sexually transmitted infections. For Germany alone, the number of new infections is estimated at 300,000 to 500,000 per year. Younger adults (15-25 years), persons with frequently changing sexual partners, sexual partners (also unnoticed and asymptomatic) of infected persons as well as newborns of infected mothers are most frequently affected. Infections without sexual contact (e.g., in the pool) are rare but can not be ruled out.

Chlamydia trachomatis infections often cause no symptoms. However, Chlamydia trachomatis can also cause discomfort such as eye inflammation, arthritis and inflammation of the urinary tract. Furthermore, untreated infections often lead to infertility and increase the risk of ectopic pregnancies and premature births. In developing countries, infections with Chlamydia trachomatis are the most common cause of neonatal blindness.

Particularly important is a treatment of Chlamydieninfektionen in pregnancy. Around 50% of infected mothers pass the infection on to the newborn. Common neonatal complications include conjunctivitis and pneumonia.

Preparations Test components: Sample container with 300 μM buffer A, small bottle with 400 μM buffer B, swab (= SWAB) and foil packaging with test strips and desiccant

Buffer A

NaOH 4g EDTA 3.722g

Sodium cholhydrate 2 g

Water 1 L

pH 12.7 buffer B KH2PO4 4.47 g

Na2HP4 7.87 g

Proclin 5 ml pH Adjustment with HCl to pH 6.4 The swab (= SWAB) was gently introduced to the cervix (end of the vaginal canal). The swab was evenly rotated for about 30 seconds and slowly pulled out. Since there is little liquid in the buffer bottle, it had to be collected by shaking it on the bottom of the bottle. AT509 276B1 2014-08-15

The swab was immersed with the sample to the bottom of the bottle. The swab was rotated in the liquid and squeezed several times on the sidewall to allow its sample material to disperse well in the buffer solution.

The swab was left in the bottle for about 5 minutes.

From the small buffer bottle B 6 drops were dropped into the large buffer bottle A.

The buffer bottle A was closed and pivoted.

Two drops of the sample were applied to the sample application panel of the test strip (US 5,073,484, US 5,654,162, Glad et al., Acta Chem. Scand. 6: 449-450 (1980), Glad et al., Anal. Bioch. 1978), Norgard-Pederson, Clinica Chemica Acta 48: 345 (1973) and Laureil et al., Methods in Enzymology, 73, Part B, 339 et seq. (1981)).

After about 15 minutes, the test result could be read on the test strip.

Example 2: Comparison of the standard cassettes chlamy- CARE-C (see Example 1) with modified cassettes, which comprise an additional membrane soaked in the buffer B of Example 1.

For carrying out a chlamydia test, as described in Example 1, the use of buffer A and B in 2 steps is necessary.

It will now be examined whether a prepared and soaked in buffer B membrane can be installed in the cassette and thus a step in the test implementation can be spared.

It is compared in this example whether the results of the newly prepared cassettes are the same as results of the normal cassettes. 4 different batches are compared.

First, a membrane was prepared, which was immersed in Buffer B and allowed to dry overnight in the dry room. The thus-prepared membrane was attached to and fixed to the sample application area of the test strip.

The unmodified test strips were marked with the letter N + number and the prepared with the letter P + number.

As a positive control Chlamydia Trachomatis Serovar LGV (4.21 mg / ml) from BIODESIGN was used.

For the conventional test strips, the stock solutions (SL) were first prepared as follows: 84.2 pg of antigen were dissolved in 500 μL of distilled water.

The controls were then prepared: Negative Control: Buffer A + B for conventional cassettes and for prepared cassettes Buffer A diluted 1: 1 with distilled water.

Control 1 Control 2 Control 3 Control 4 Control 5

SL 1: 1 + buffer A + B SL 1: 2 + buffer A + B SL 1: 4 buffer A + B SL 1: 8 + buffer A + B SL 1:16 + buffer A + B

For the prepared cassettes, the stock solution (SL) was prepared as above and then the positive controls were used according to the scheme as above, but instead of buffer A + B, only buffer A (diluted 1: 1 with distilled water) , 6/9 Austrian Patent Office AT509 276 B1 2014-08-15 [0082] Results:

Sample Lot .: 08619 Lot .: 01720 Lot .: 03722 Lot .: 06723 conventionally pre-assembled conventional pre-assembled conventional pre-assembled traditional pre-assembled Neg. Control Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Item Check 1 Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Check 2 Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Check 3 Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Check 4 Item Pos. Pos. Pos. Pos. Pos. Pos. Pos. Check 5 Weak Pos Weak Pos Weak Pos Weak Pos Weak Pos Weak Pos Weak Pos Weak Pos [0083] Results: [0084] All cassettes showed the same Results. For the prepared cassettes, the lines were a little bit stronger. 7.9

Claims (17)

Austrian Patent Office AT509 276 B1 2014-08-15 Patentansprüche 1. A device for the determination of chlamydia or gonococci, in a sample comprising a liquid-permeable solid medium, which defines a path for a liquid flow, which medium has spaced-apart areas along the liquid path, the regions being subdivided in the following order into a feed area, a reaction area, a detection area and a control area, characterized in that a neutralization buffer in the dried state is provided in the feed area, the buffer being a basic buffer with a pH of 11 to 13 , especially 12.7, or an acid buffer with a pH of up to 6.5. 2. Device according to claim 1, characterized in that in the reaction region are arranged mobile antibodies which are capable of binding to an epitope of a surface antigen of the microorganisms to be determined, the antibodies preferably with a dye, colored, fluorescent or magnetic particles, especially latex particles, or are marked with colloidal gold. 3. A device according to claim 1 or 2, characterized in that antibodies are immobilized in the detection area, which are capable of binding to an epitope of a surface antigen of the microorganisms to be determined, wherein the epitope of the antigen is capable or not in a position is to bind to the mobile in the reaction area arranged antibody. 4. Device according to one of claims 1 to 3, characterized in that antibodies are immobilized in the control region, which are capable of binding to the mobile in the reaction region arranged antibodies. 5. Device according to one of claims 1 to 4, characterized in that the basic buffer 3 to 5 g, preferably 4 g, NaOH, 3 to 4 g ethylenediaminetetraacetic acid (EDTA), 1 to 3 g, in particular about 2 g, the sodium Salt of cholic acid hydrate in 1 L of water. 6. Device according to one of claims 1 to 5, characterized in that the acidic buffer 3 to 5 g, preferably 4 to 4.5 g, KH2P04, 5 to 9 g, preferably 7 to 8 g, Na2HP04, 2 to 8 ml , preferably 3 to 6 ml, especially 5 ml, of a mixture of 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one (Proclin) in 1 L of water, wherein the pH is adjusted to pH 6 to 6.5. 7. Device according to one of claims 1 to 5, characterized in that the acidic reagent 3 to 5 g, preferably 4 to 4.5 g, KH2P04, 5 to 9 g, preferably 7 to 8 g, Na2HP04, in 1 L of water wherein the pH is adjusted to pH 1 to 3, in particular to pH 2.5 8. Kit for the determination of chlamydiae or gonococci, in a sample comprising: a) at least one sampling means, preferably at least one cotton swab or one tampon, b) a container comprising a buffer for chlamydia or gonococcal digestion and suitable for uptake the means for sampling, preferably the cotton swab or tampon, c) a device according to any one of claims 1 to 7. 9. Kit according to claim 8, characterized in that the container b) a basic buffer having a pH of 10 to 14, preferably 11 to 13, in particular 12.7, or an acidic buffer having a pH of 1 to 6.5, preferably from 2.5 to 6.4, the kit each comprising a basic or an acidic buffer for digesting chlamydiae or gonococci. 8/9 Austrian Patent Office AT509 276B1 2014-08-15 10. Kit according to claim 8 or 9, characterized in that the basic buffer 3 to 5 g, preferably 4 g, NaOH, 3 to 4 g ethylenediaminetetraacetic acid (EDTA), 1 to 3 g, in particular about 2 g, the sodium Salt of cholic acid hydrate in 1 L of water. 11. Kit according to claim 8 or 9, characterized in that the acidic buffer 3 to 5 g, preferably 4 to 4.5 g KH2P04, 5 to 9 g, preferably 7 to 8 g, Na2HP04, 2 to 8 ml, preferably 3 to 6 ml, in particular 5 ml, of a mixture of 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one (Proclin) in 1 L of water, the pH Value is adjusted to pH 6 to 6.5. 12. Kit according to claim 8 or 9, characterized in that the acid buffer comprises 3 to 5 g, preferably 4 to 4.5 g, KH2P04, 5 to 9 g, preferably 7 to 8 g, Na2HP04, in 1 L of water, wherein the pH value is adjusted to pH 1 to 3, in particular to pH 2.5 13. Kit according to one of claims 8 to 12, characterized in that the container b) 50-1000 μΙ, preferably 100-500 μΙ, in particular 300 μΙ, of the buffer comprises. 14. Kit according to any one of claims 8 to 13, characterized in that the kit further comprises a positive control. 15. Kit according to any one of claims 8 to 14, characterized in that buffer b) comprises a pH indicator, preferably Bromthymolblau, preferably in an amount of 0.001 to 0.005 g, in particular in an amount of 0.003 g. 16. A method of diagnosing a chlamydial or gonococcal infection comprising the steps of: a) providing a means for sampling, preferably at least one cotton swab or tampon, b) sampling, c) transferring the sample to the sampling means to a container comprising one Buffers for disrupting chlamydiae or gonococci, d) incubating the sample with the buffer for 5 seconds to 30 minutes, preferably for 30 seconds to 15 minutes, more preferably for 1 to 10 minutes, especially for 5 minutes, e) transferring the neutralized buffer in an apparatus according to any one of claims 1 to 7 for detecting chlamydiae or gonococci; and f) diagnosing a chlamydial or gonococcal infection when chlamydiae or gonococci are detected in step e). 17. Use of a kit according to any one of claims 8 to 15 for the determination of a Chlamydien- or Gonokokkeninfektion. For this no drawings 9/9