WO2003046213A2

WO2003046213A2 - Method for concentrating and detecting hiv

Info

Publication number: WO2003046213A2
Application number: PCT/EP2002/011931
Authority: WO
Inventors: Uwe Michelsen; Achim SCHWÄMMLE; Willi Roth
Original assignee: Merck Patent Gmbh
Priority date: 2001-11-26
Filing date: 2002-10-25
Publication date: 2003-06-05
Also published as: AU2002351787A8; AU2002351787A1; WO2003046213A3; DE10157624A1

Abstract

The invention relates to a method and a test kit for enriching and detecting lentiviruses, especially HIV (Human Immunodeficiency Viruses) in complex biological samples e.g. body fluids. The viruses are bonded in suitable conditions to a solid phase, isolated, lysed and subsequently the viral nucleic acids are separated and optionally detected.

Description

Methods for HIV enrichment and detection

The invention relates to a method and a test kit for the enrichment and detection of lentiviruses in complex biological samples such as e.g. Body fluids. The viruses are bound to solid phases and isolated under suitable conditions. The viruses can then be lysed and detected.

Viruses in body fluids can be detected by highly sensitive detection based on the viral nucleic acids or alternatively with the help of specific antibodies. For the detection of viral nucleic acids e.g. known nucleic acid amplification techniques, e.g. Polymerase chain reaction (PCR), branched-DNA detection (bDNA) or nucleic acid sequence based amplification (NASBA) are used.

A prerequisite for successful and sensitive detection of viral particles in body fluids using the nucleic acid amplification techniques mentioned is the efficient, quantitative isolation of the viral nucleic acids (DNA or RNA) in as pure and native a form as possible.

While there are various effective methods for the isolation of viruses and the subsequent detection of viral RNA / DNA from water (e.g. Pallin et al .; Journal of Virological Methods 67 (1997) 57-67 or Leisinger et al .; Zentralblatt für Hygiene and Umweltmedizin 200 (1997), 283-296), the isolation of viral nucleic acids from complex biological starting materials such as blood or plasma with a high proportion of proteins, lipids and other low-molecular substances has so far been very complex and not very effective.

Classic methods for the isolation of nucleic acids from complex samples start directly with the lysis of the biological material by means of a Detergent and / or a chaotropic salt. These substances enable the inactivation and denaturation of proteins and enzymes. The nucleic acids are subsequently purified using organic solvents, for example phenol and / or chloroform, ethanol precipitation and centrifugation steps. Not only are these methods very labor intensive and time consuming, they are also difficult to automate.

Other methods based on the use of solid phases have recently been proposed. No. 5,234,809 describes a method in which the nucleic acids are bound to a silica solid phase in the presence of chaotropic agents such as guanidinium salts. WO 91/12079 describes a method for precipitating long-chain nucleic acids onto the surface of solid phases. Here alcohols and salts are used as precipitation reagents.

While solid phase extraction methods such as those described increase the simplicity and the automatability of the nucleic acid separation process, there is still a need to isolate the viruses themselves quickly and easily, but also as quantitatively as possible from a sample. In particular, if the sample is of biological origin and contains only very few of the viruses to be detected or, accordingly, very few of the nucleic acids to be detected, as is the case for some viral diseases.

For all isolation and detection methods that want to isolate viral nucleic acids from body fluids, it is essential to record all virus particles in a sample in order to achieve high sensitivity. Currently, cells or viruses from a sample are enriched for this by filtration, ultra-high centrifugation or by affinity binding to antibodies that are bound to a solid phase. After this concentration step, the DNA or RNA is purified. The previously known concentration processes are very labor intensive and / or they use very expensive and sensitive solid phases, such as antibody beads.

The detection limit of the commercially available test systems is around 100 to 400 RNA copies per ml of plasma. Most commercial test systems are only for one virus type and are designed for the use of relatively small volumes (100-200 μl plasma).

In this context, the detection limit of the known tests poses a problem in particular for two important fields of application: 1.) Blood banks, which due to the high sample volume and cost pressure, form so-called mini pools (from up to 100 donor samples), which must potentially be from a single one Record serum viruses in the pool.

2.) The therapy of AIDS patients requires the highly sensitive monitoring of the virus concentration in the blood, since this virus can persist in very small copies despite therapy and its detection is crucial for the continuation of therapy.

Accordingly, there is a need, in particular for the detection of the AIDS pathogen, the human immunodeficiency virus (HIV), for improved methods for the detection of these viruses in complex biological materials. The method should be highly sensitive, if possible record all viral DNA or RNA molecules present in a sample and make it possible to use a large sample volume as the starting material for the detection reaction.

It has now been found that HIV viruses from complex biological samples, despite the disruptive influence of other substances contained in these samples, such as proteins, lipids, carbohydrates, under certain conditions, ie at a certain pH value and addition a certain amount of binding buffer, can be bound to a solid phase and can thus be separated from the rest of the sample. The isolated viruses can then be lysed and the nucleic acids released can be purified and detected under suitable conditions.

The present invention makes it possible to isolate a small number of viruses from a large volume of a complex sample and to detect them in a suitable manner with relatively simple and inexpensive manipulations. For example, after lysis of the virus envelope, the nucleic acid can be extracted in a native, highly pure state for the detection reaction by means of PCR, NASBA and other methods.

The present invention therefore relates to a method for the detection of lentiviruses in complex biological samples, characterized by the following steps a) acidifying the sample with a virus binding buffer, the pH being adjusted to a value of less than or equal to 4 and the sample with the virus binding buffer is mixed in a volume ratio (sample: binding buffer) between 1: 0.3 and 1: 2, adding a solid phase and incubating the mixture; b) separation of the solid phase with the bound viruses c) detection of the viruses.

In a preferred embodiment, a pH of between 1 and 3 is set in step a) with the virus binding buffer, i.e. the sample is acidified to a pH between 1 and 3 with the binding buffer.

In a preferred embodiment, in step a) the sample is mixed with the virus binding buffer in a volume ratio of sample to binding buffer of 1: 0.5 to 1: 1.5. In a particularly preferred embodiment, aqueous hydrochloric acid is used as the virus binding buffer in step a).

In another preferred embodiment, the incubation time in step a) is 1 to 5 minutes.

In a preferred embodiment, magnetic silica particles are used as the solid phase in step a).

In a further preferred embodiment, the solid phase with the bound virus particles is separated off by centrifuging, filtering or applying a magnetic field.

In a preferred embodiment, the detection of

Viruses by d) lysis of the viruses; c2) isolation of the viral nucleic acids from the lysate; c3) detection of the viral nucleic acids;

In a preferred embodiment, the viral is isolated

Nucleic acids in step c2) by binding to a solid phase.

In a particularly preferred embodiment, the viral nucleic acids are isolated in step c2) by binding to the same type of solid phase as was used in step a).

In a preferred embodiment, the viral nucleic acids are detected in step c3) by nucleic acid amplification techniques. The present invention also relates to a test kit for carrying out the method according to the invention at least comprising a solid phase and a virus binding buffer.

More detailed explanations of Figures 1 to 3 can be found in the

Examples 2 to 4.

According to the invention, the term viruses includes all lentiviruses, including defective or incomplete viruses. Lentiviruses are a subfamily of retroviruses. The main representatives are the SIV and HIV viruses that cause immunodeficiency.

The method according to the invention is particularly suitable for isolating or detecting HIV.

Complex biological samples in the sense of the present invention are all samples of biological origin, in particular human and animal body fluids such as blood, plasma, serum, urine, feces and cerebrospinal fluid. The present invention is particularly suitable for the detection of lentiviruses, in particular HIV, in blood, plasma or serum or for the purification of nucleic acids of the lentiviruses from blood, plasma or serum.

According to the invention, a material which carries metal hydroxide groups on the surface can be used as the solid phase. Suitable materials are, for example, silica gel, silicates, metal oxides such as iron hydroxides, hydroxyapatite or glass. Such solid phases can, for example, be porous or non-porous, in the form of, for example, particles, fibers, membranes, filters or simply appropriately modified vessel walls. Particulate materials are generally preferred because they have a greater binding capacity and kinetics, especially in larger sample volumes. Magnetizable particles are particularly preferred because their separation from a suspension is easy to automate. A According to the invention particularly suitable solid phase are MagPrep ^® silica particles (Merck KGaA, Darmstadt).

The method according to the invention for isolating viral nucleic acids from complex biological samples comprises the following steps:

The biological sample is brought to a pH less than or equal to 4 by adding a virus binding buffer, mixed with the solid phase and incubated for a short time. The sample is preferably brought to a pH between 1 and 3.

Any aqueous buffer which does not undesirably change the sample or which interferes with the binding of the virus particles is suitable as the virus binding buffer. For example, citrate or acetic acid buffers or a dilute acid such as 0.1 to 0.3 molar hydrochloric acid are suitable. Dilute hydrochloric acid is particularly preferably used as the virus binding buffer.

The mixing ratio between sample and virus binding buffer should be between 1: 0.25 and 1: 3, preferably between 1: 0.3 and 1: 2, particularly preferably between 1: 0.5 and 1: 1.5. Particularly good results can be achieved with a mixing ratio of 1: 1.

For particulate materials such as MagPrep ^® silica particles, the amount of solid phase added is typically 5 to 100 μl particle suspension (MagPrep ^® silica particles: c = 50 mg / ml) per ml sample.

It was found that only by selecting the suitable pH value and the suitable mixing ratio between the sample and the binding buffer is an optimal binding of the viruses to the solid phase achieved. This is illustrated by Examples 2 and 3. Depending on the type of sample, the binding buffer or the solid phase, the optimal extraction conditions can easily be within the specified range vary. The viruses are then bound to the solid phase, surprisingly, very selectively and effectively, despite the proteins, in particular glycoproteins, and other biological materials contained in the sample.

The solid phase can be added to the sample before or preferably after the addition of the virus binding buffer or at the same time as the binding buffer. The mixture or suspension of solid phase, sample and virus binding buffer is then incubated for a short time. Usually 10 minutes, preferably 1 to 5 minutes are sufficient.

b)

After the incubation, the solid phase with the viruses bound to it is separated from the mixture. This can e.g. Depending on the properties of the solid phase, centrifugation, filtration, application of a magnetic field or other physical methods are used. Magnetic particles are preferably used according to the invention, which are separated with the aid of a magnetic field.

c)

The viruses are then detected. This can be done by various methods, for example by means of immunological detection methods or by detection of the viral nucleic acids. Depending on the intended detection method, the solid phase A irus complex can either be directly mixed with a lysis buffer A which contains substances which allow the release of the viral nucleic acids and, if necessary, their subsequent purification, or the complex can be taken up in a suitable buffer which for example, immunological detection reactions allowed. The solid phase / irus complex can also first be washed as an intermediate step with a suitable buffer. The lysis of the viruses bound to the separated solid phase and the subsequent purification of the viral nucleic acids can be carried out according to known methods. Suitable methods are described, for example, in US Pat. No. 5,234,809 (binding of the nucleic acids to silica solid phases under chaotropic conditions) or WO 99/40098 (binding of the nucleic acids to solid phases under acidic conditions).

A possible procedure for lysing the viruses and purifying the viral nucleic acids is described below by way of example:

The separated solid phase with the viruses bound to it can either be fed directly to the nucleic acid isolation or can be taken up as an intermediate step in a resuspension buffer. In the simplest case, a suitable resuspension buffer can be pure water or consist of an aqueous buffer solution familiar to the person skilled in the art, e.g. Tris-HCI. The resuspension buffer can contain reagents that remove or inactivate interfering substances.

Subsequently, a virus-lysing and free nucleic acid-binding buffer is preferably added, which contains a chaotropic salt such as e.g. Contains guanidinium thiocyanate in high molar concentration (> 2M) and a non-ionic detergent. The binding buffer can also contain alcohol. The suspension of binding buffer and particles is mixed well and briefly incubated. Usually 1 to 5 minutes are sufficient. For the isolation and purification of the viral nucleic acids from the

The solid phase is removed from the mixture by suitable means and the supernatant is discarded.

The solid phase is washed (for example with 70% ethanol) and the nucleic acids are released with an elution buffer (for example with Tris-HCl pH 8.0). The released viral nucleic acids can then be fed directly, without further purification steps, for example to the known nucleic acid amplification techniques, such as polymerase chain reaction (PCR), branched DNA detection (bDNA) or nucleic acid sequence based amplification (NASBA).

Thus, for the first time, the method according to the invention opens up the possibility of isolating viruses from complex biological samples quickly and quantitatively using very simple techniques. In this way, the detection of lentiviruses, in particular HIV, in complex biological materials is greatly simplified and the sensitivity to detection is increased, particularly when enriched from large-volume samples. As shown in Figure 4, the five-fold diluted sample has the same signal strength as the undiluted sample of direct extraction. The viral nucleic acids can therefore be isolated quantitatively and representatively and used directly for detection in suitable methods.

The method according to the invention is very sensitive and can also be used by blood banks which form mini pools from several donor samples to detect viruses.

The present invention furthermore relates to a test kit for carrying out the method according to the invention. The test kit contains at least one solid phase, preferably a silica solid phase, and a virus binding buffer.

Other optional components are

- a reagent for resuspending the solid phase / virus particle complex

- a reagent for lysing the viruses - a reagent for binding the released viral nucleic acids to the solid phase a reagent for washing the nucleic acids bound to the silica solid phase

- A reagent for detaching the bound viral nucleic acids from the solid phase - Instructions for performing the method according to the invention

Even without further explanations, it is assumed that a person skilled in the art can use the above description in the broadest scope. The preferred embodiments and examples are therefore only to be regarded as descriptive, in no way as in any way limiting in any way.

The complete disclosure of all of the applications, patents and publications mentioned above and below, in particular the corresponding application DE 101 57624, filed on November 28, 2001, is incorporated by reference into this application.

Examples

1. Basic protocols for the isolation of viral nucleic acids

500μl HIV positive plasma (EDTA or citrate plasma) are mixed with 500μl

0.2N HCI adjusted to pH 2 and diluted 1: 1. After adding 30μ | Particle suspension (MagPrep ^® Silica, Merck KGaA), the mixture is mixed and incubated for 2-3 minutes at room temperature.

The silica particles are separated magnetically and the supernatant is discarded.

The adsorbed viruses can subsequently be lysed and viral RNA can be extracted in a suitable manner by, for example, the following various methods. A) The silica particles are resuspended in 400μl binding buffer X (5M guanidinium thiocyanate, 1% Triton X 100) and then 400μl ethanol is added. Binding buffer X also contains silica particles and an internal control RNA. After 10 minutes of incubation and subsequent magnetization, the supernatant is separated off and the

Silica particles washed twice with 70% ethanol. The viral RNA is eluted from the particles after incubation in 10 mM Tris / HCl pH 8.5 at 80 ° C. after 10 minutes.

0 B) The silica particles are resuspended in μOOμl binding buffer Y (10OmM acetate-Tris pH 4.0, 0.5% NP40, 200mM ammonium sulfate). Binding buffer Y also contains silica particles and an internal control RNA. After 10 minutes of incubation and subsequent magnetization, the supernatant is separated off and the particles are washed twice with washing buffer (10 mM Tris-HCl pH 5 6.6; 0.5 mg / ml proteinase K). The viral RNA is eluted from the particles after incubation in 10 mM Tris / HCl pH 8.5 at 80 ° C. after 10 minutes.

The RNA of the eluates obtained by means of variant A) or B) is amplified and detected by RT PCR and suitable gene probes (for example using a thermal cycler such as TaqMan ^® from Perkin Eimer or LightCycler ^® from Röche).

2. Effect of pH on the binding of HIV to the solid phase ⁵ 100μl HIV positive plasma (200 copies) is mixed with 400μl virus-free plasma and diluted 1: 1 in water. After adding the solid phase, the pH is adjusted to the desired value with concentrated HCl and the viral particles are adsorbed on the solid phase.

The silica particles are separated off as described in Example 1, the liquid supernatants are discarded, and the RNA is extracted. The result of this example is shown in Fig.1. The y-axis shows the percentage of viruses detected, the amplification signal achieved being set to 100% for a direct RNA extraction (without upstream enrichment of the virus particles) from the HIV-positive plasma sample (100 μl, 200 copies).

3. Effect of the mixing ratio with the binding buffer on the binding of HIV to the solid phase

100μl HIV positive plasma (200 copies) is mixed with 400μl virus-free plasma and diluted in the specified ratio with HCI acidic water, a pH of 2 being established. As described in Example 1, the viral particles adsorb to the solid phase, the silica particles are separated off, the liquid supernatants are discarded, and the RNA is extracted. The result is shown in FIG. 2. The y-axis shows the percentage

Proportion of the detected viruses, the amplification signal achieved being set to 100% for a direct RNA extraction (without upstream enrichment of the virus particles) from the HIV-positive plasma sample (100 μl, 200 copies). "N" denotes an undiluted sample on the x-axis.

4. Multiple analysis of virus enrichment from 1 ml plasma

Three batches of 100 .mu.l plasma (sample 2-4) with HIV particles (200 copies) are mixed with 400 .mu.l virus-free plasma and adsorbed onto the solid phase according to the basic protocol (example 1). The silica particles are separated off, the liquid supernatants are discarded, and the RNA is extracted. The result is shown in Fig. 3. The y-axis shows the percentage of viruses detected, the amplification signal achieved being set to 100% for a direct RNA extraction (without upstream enrichment of the virus particles) from the HIV-positive plasma sample (100 μl, 200 copies).

Claims

Expectations

1. A method for the detection of lentiviruses in complex biological samples, characterized by the following steps a) acidifying the sample with a virus binding buffer, the pH being adjusted to a value of less than or equal to 4 and the sample with the virus binding buffer in a volume ratio (sample: binding buffer) between 1: 0.3 and 1: 2 is mixed, addition of a solid phase and incubation of the mixture; b) separation of the solid phase with the bound viruses; c) Detection of the viruses.

2. The method according to claim 1, characterized in that in step a) the sample is acidified with the binding buffer to a pH value between 1 and 3.

3. The method according to claim 1 or 2, characterized in that in step a) the sample is mixed with the virus binding buffer in a volume ratio of sample to binding buffer of 1: 0.5 to 1: 1.5.

4. The method according to one or more of claims 1 to 3, characterized in that aqueous hydrochloric acid is used as the virus binding buffer in step a).

5. The method according to one or more of claims 1 to 4, characterized in that magnetic silica particles are used as solid phase in step a).

6. The method according to one or more of claims 1 to 5, characterized in that in step c) the detection of the viruses by d) lysis of the viruses c2) isolation of the viral nucleic acids from the lysate c3) Detection of the viral nucleic acids takes place.

7. The method according to claim 6, characterized in that the isolation of the viral nucleic acids in step c2) by binding to a

Solid phase takes place.

8. The method according to any one of claims 6 or 7, characterized in that the detection of the viral nucleic acids in step c3) is carried out by nucleic acid amplification techniques.

9. Test kit for performing the method according to one or more of claims 1 to 8 at least containing a solid phase and a virus binding buffer.