DE102006032610A1 - Method for the parallel isolation of viral nucleic acids - Google Patents

Abstract

The invention relates to a method for the parallel isolation of double- and single-stranded nucleic acids from samples containing these substances, without separating the double- and single-stranded nucleic acids, in which the samples are mixed with known lysis buffers (high salt concentrations or low salt concentrations or with proteolytic enzymes), wherein the nucleic acid-containing sample prior to its lysis or the already lysed or homogenized sample in the absence of alcohol with a binding buffer containing at least one nonionic detergent in high concentration is adjusted so that the total nucleic acid is adsorbed to a solid support.

Description

The The invention relates to a method for fast, simple and highly sensitive Isolation of viral nucleic acids.

The Examination of diagnostically relevant biological samples (serum, Plasma, blood, cerebrospinal fluid, swab samples, organ abrasions, etc.) for detection infectious Pathogen is becoming increasingly important. Viral infections like HIV, HCV or HBV are spreading worldwide. Furthermore requires the massive occurrence of novel viral diseases (e.g., avian influenza) a fast and flexible response for the sensitive diagnostic virus detection. New test methods based on use enable sensitive amplification techniques such as real-time PCR a highly efficient virus detection and are becoming stronger than used diagnostic tools. The step of the molecular Sample preparation in the form of isolation of the to be detected nucleic acids comes more and more important to.

The sensitivity of the test system is significantly influenced by the process of isolating the nucleic acid.

There are a number of extraction methods that allow the isolation of viral nucleic acids. The patent US 5,234,809 describes a process for isolating nucleic acids from nucleic acid-containing starting materials by incubating the starting material with a chaotropic buffer and a DNA-binding solid phase. The then binding solid phase consists of silicate mineral particles, which are> 50 nm. The chaotropic buffers realize both the lysis of the starting material and the binding of the nucleic acids to the solid phase. The method is well suited for isolating nucleic acids from small sample quantities and finds its practical application especially in the field of isolation of viral nucleic acids. However, in practice, the method described in the patent has a number of disadvantages. The use of particulate systems for the attachment of the nucleic acids to be isolated is in principle very cumbersome to handle. In addition, it has been shown that the use of buffers based on the use of chaotropic salts is often insufficient with respect to the need for highly sensitive virus detection.

An improvement of the method disclosed in the patent EP 9402056 , The method describes the chromatographic purification and separation of nucleic acid mixtures from a solution having a high salt concentration and a high alcohol concentration.

The solution will turn with a carrier material for binding the nucleic acids brought into contact, hereinafter the carrier material is known per se washed alcoholic washing buffers and the bound nucleic acid final removed again from the carrier material by means of water or a low salt buffer.

The method realizes an efficient isolation and possibly also separation of nucleic acids / nucleic acid mixtures and is simple and fast to carry out. The improvement over the patent cited above is achieved by the addition of an alcohol to the initial lysis buffer. This step allows an increase in the efficiency of the extraction process, which also allows to sensitively isolate nucleic acid targets from a clinical sample. Another method of separating and isolating single-stranded and double-stranded nucleic acids is disclosed in the patent EP 114604999 disclosed.

• 1. die einzelsträngige Nukleinsäure isoliert wird, indem man die Behandlungsbedingungen durch ein wässriges Gemisch von chaotropen Salzen und niederen aliphatischen Alkoholen einstellt, derart, dass nachfolgend vorrangig die einzelsträngige Nukleinsäure an einen mineralischen Träger adsorbiert wird, die doppelsträngige Nukleinsäure dagegen nicht adsorbiert. Die gebundene einzelsträngige Nukleinsäure wird nachfolgend gewaschen und final mittels eines Niedrigsalzpuffers vom Trägermaterial eluiert.
• 2. die doppelsträngige Nukleinsäure isoliert wird, indem man die Behandlungsbedingungen durch ein Gemisch von Erdalkali-Ionen komplexierenden Substanzen ohne niedere aliphatische Alkohole einstellt, derart, dass nachfolgend vorrangig die doppelsträngige Nukleinsäure an einen mineralischen Träger adsorbiert wird, die einzelsträngige Nukleinsäure dagegen nicht adsorbiert. Die gebundene doppelsträngige Nukleinsäure wird nachfolgend gewaschen und final mittels eines Niedrigsalzpuffers vom Trägermaterial eluiert.
• 3. die doppelsträngige Nukleinsäure isoliert wird, in dem man die Behandlungsbedingungen durch die Anwesenheit eines Sarkosinats aber ohne niedere aliphatische Alkohole einstellt, derart, dass nachfolgend vorrangig die doppelsträngige Nukleinsäure an einen mineralischen Träger adsorbiert wird, die einzelsträngige Nukleinsäure dagegen nicht adsorbiert. Die gebundene doppelsträngige Nukleinsäure wird nachfolgend gewaschen und final mittels eines Niedrigsalzpuffers vom Trägermaterial eluiert.
• 4. die doppelsträngige oder einzelsträngige Nukleinsäuren isoliert wird, in dem man die Behandlungsbedingungen durch ein wässriges Gemisch von chaotropen Salzen und niederen aliphatischen Alkoholen einstellt, derart, dass beide Nukleinsäurefraktionen an einen mineralischen Träger adsorbieren. Die Trennung der Nukleinsäuren erfolgt durch eine selektive Flution. Dabei wird die doppelsträngige Nukleinsäure mittels einer Lösung verminderter Innenstärke und niederen aliphatischen Alkoholen vom Trägermaterial abgelöst. Die verbleibende einzelsträngige Nukleinsäure wird nachfolgend gewaschen und final mittels eines Niedrigsalzpuffers vom Trägermaterial abgelöst.

The method is based on the treatment of a source containing nucleic acids with at least one mineral carrier material in the form:

• 1. the single-stranded nucleic acid is isolated by adjusting the treatment conditions by an aqueous mixture of chaotropic salts and lower aliphatic alcohols, such that subsequently predominantly the single-stranded nucleic acid is adsorbed to a mineral carrier, the double-stranded nucleic acid, however, not adsorbed. The bound single-stranded nucleic acid is subsequently washed and finally eluted from the support by means of a low salt buffer.
• 2. the double-stranded nucleic acid is isolated by adjusting the treatment conditions by a mixture of alkaline earth ion-complexing substances without lower aliphatic alcohols, such that subsequently predominantly the double-stranded nucleic acid is adsorbed to a mineral carrier, however, does not adsorb the single-stranded nucleic acid. The bound double-stranded nucleic acid is subsequently washed and finally eluted from the support by means of a low salt buffer.
• 3. the double-stranded nucleic acid is isolated by changing the treatment conditions through the An but does not adjust the presence of a sarcosinate without lower aliphatic alcohols, such that subsequently the double-stranded nucleic acid is predominantly adsorbed to a mineral carrier, whereas the single-stranded nucleic acid is not adsorbed. The bound double-stranded nucleic acid is subsequently washed and finally eluted from the support by means of a low salt buffer.
• 4. the double-stranded or single-stranded nucleic acids is isolated by adjusting the treatment conditions by an aqueous mixture of chaotropic salts and lower aliphatic alcohols such that both nucleic acid fractions adsorb to a mineral carrier. The separation of the nucleic acids is carried out by a selective elution. In this case, the double-stranded nucleic acid is detached from the carrier material by means of a solution of reduced internal strength and lower aliphatic alcohols. The remaining single-stranded nucleic acid is subsequently washed and finally detached from the carrier material by means of a low-salt buffer.

alternative this can be the single-stranded nucleic acid by means of a solution, which alkaline earth metal ions complexing substances and / or sarcosinates contains selectively from the carrier material be eluted. The now remaining double-stranded nucleic acid is subsequently washed again and finally by means of a low salt buffer from the carrier material replaced.

Again, the process is simple to carry out and can isolate DNA or RNA from biological samples. Even if the patent refers to the separation of nucleic acid mixtures, the skilled person will read the dependent claim 4, which in turn, as in the patent EP 9402056 described, the combination of a saline solution with an alcohol, the attachment of nucleic acids to a mineral material is realized. Decisive for the step of attaching nucleic acids to mineral materials is again the existence of an alcohol in the buffers used for the extraction process. Thus, the prior art suggests that isolation of nucleic acids from biological samples using chaotropic salts alone works, but the process becomes truly efficient only by the addition of an alcohol (ie, a combination of a saline solution with an aliphatic alcohol).

Of the Invention was based on the object, the disadvantages of standing the technique called to eliminate.

The inventive task could in surprising Simply solved become. The method is characterized by claim 1 with his Characterized method alternatives. The dependent claims 2 to 8 relate to specific embodiments the method according to the invention.

According to the invention was provided an alternative method which allows to quickly, simple and inexpensive nucleic acids from a nucleic acid to isolate the sample containing for the selective binding of the nucleic acids necessary solutions contain an alcohol. The method allows simultaneous and thus causing no separation of nucleic acid mixtures, DNA (double Nucleic acid) and RNA (single-stranded Nucleic acid) from an initial sample. In particular, the method able to detect highly sensitive viral nucleic acids from diagnostically relevant isolate biological samples.

surprisingly Way can be a highly efficient isolation of nucleic acids a biological sample also in a very different way, than in the State of the art, can be achieved, where entirely the one for that previously necessary essential component alcohol is dispensed with.

The inventive method is based on the lysis of a biological sample with lysis buffers known per se. The lysis buffers contain e.g. known chaotropic salts of high internal strengths or Combinations of chaotropic salts of high internal strengths with a sarcosinate and possibly other additives. These buffer systems allow in their known function the digestion and denaturation a biological sample and also cause inactivation of endogenous RNases. This is especially true with regard to the isolation of RNA essential. It can also lysis buffer can be used, which is a combination of chaotropic salts and other salts and which in support of the Lysing process may still contain detergents and possibly other additives, wherein in the chaotropic salts no known high internal strengths are needed. Such a combination then allows for efficient inclusion proteolytic enzymes (e.g., proteinase K) for efficient digestion of the starting material.

To Lysis of the starting material then becomes the binding conditions for Connection of the nucleic acids contained in the sample to a mineral carrier material set.

This takes place in the process according to the invention not by the addition of an alcohol, but surprisingly by the Addition of a nonionic detergent, e.g. Tween-20, Tween-80 or Triton X-100 in a high concentration. The addition of the detergent is the essential step. The detergent completely replaced according to the invention the function of adding a previously necessary alcoholic Component. The lysis mixture which is mixed with the detergent, is subsequently with a carrier, preferably a mineral carrier or surface functionalized Magnetic particles or iron oxide particles, brought into contact the nucleic acids to be isolated to the mineral carrier adsorb. The mineral carrier is subsequently washed with known washing buffers and final again by means of water or a low salt buffer of mineral Material detached. The for the binding of the nucleic acids needed Detergent component can about it also as a mixture of detergent with other salts and if necessary further additives available. With that you can always optimal binding conditions in the combination of Salt / detergent can be achieved without this on the initial use an efficient lysis buffer has an impact. Thus, e.g. one Lysis buffers are used for the initial sample digestion, which has only a low salt concentration (which would not be sufficient to in the combination with a detergent efficient connection of the to be isolated nucleic acids convey). After sample digestion, the reaction mixture is then a binding buffer added, which is a nonionic detergent high concentration as well as additional has a high salt concentration. This then becomes the binding conditions adjusted, the quantitative isolation of viral nucleic acids efficiently convey. Decisive is always that about the binding buffer on the basis of a high concentration of nonionic detergents Also, the other components are provided which are efficient Enable connection of the nucleic acids to be isolated.

The Method is thus completely alternative to the described methods, which for an efficient attachment of nucleic acids to mineral substrates need an alcohol. It also allows parallel isolation of double-stranded and single-stranded nucleic acids a biological sample without the double-stranded and single-stranded nucleic acids separate. About that In addition, there is a decisive advantage in that in relation on the selection of the lysis buffer a high flexibility is achieved. With that you can also known, efficiently acting lysis buffer including proteolytic Enzymes are used. The setting of the binding conditions for nucleic acids for their Adsorption to a mineral carrier material always takes place after the lysis of the starting material by the addition of the binding buffer according to the invention, containing a high concentration of a nonionic detergent and optionally containing other additives.

The Invention allows For the first time, the parallel isolation of double-stranded and single-stranded nucleic acids a nucleic acid-containing Sample, wherein the nucleic acid containing Sample with a buffer made either from a saline solution chaotropic salts or a salt solution of chaotropic salts low ionic strength (less than 100 mM) and other non-chaotropic salts, as well if necessary further additives such as detergents (SDS, sarcosinate, LDS), proteolytic enzymes, complexing compounds (EDTA, EGTA), is incubated under these conditions, a digestion of the biological sample takes place, so that the nucleic acids be released into the buffer,

• a) einem nichtionischen Detergenz oder
• b) einem Gemisch eines nichtionischen Detergenzes mit Wasser oder
• c) einem Gemisch eines nichtionischen Detergenzes mit einer Salzlösung

und ggf. weiteren Zusätzen, aber ohne Alkohol, Bedingungen eingestellt, die es ermöglichen, sowohl doppelsträngige als auch einzelsträngige Nukleinsäuren an ein mineralisches Material (Träger) zu adsorbieren. Die Konzentration an Detergenz zur Anbindung der Nukleinsäuren beträgt 5–50 %; vorzugsweise 10–30 % im finalen Gemisch von Lysepuffer/Bindungspuffer.According to the invention, by adding a binding buffer consisting of:

• a) a nonionic detergent or
• b) a mixture of a nonionic detergent with water or
• c) a mixture of a nonionic detergent with a saline solution

and optionally further additives, but without alcohol, set conditions that make it possible to adsorb both double-stranded and single-stranded nucleic acids to a mineral material (carrier). The concentration of detergent for binding the nucleic acids is 5-50%; preferably 10-30% in the final mixture of lysis buffer / binding buffer.

The subsequently scrubbing mineral material with washing buffer and the nucleic acids using water or low salt buffer of mineral material again replaced.

Also The invention relates to a kit for carrying out the method according to the invention, in the claims 9 to 11 will be described.

The use according to the invention is that nonionic detergents in a concentration of 5-50%; preferably 10-30%, in the absence of alcohol, for the parallel isolation of single and double-stranded nucleic acids from samples containing these substances, without the double- and single-stranded ones Nucleic acids can be used by binding the total nucleic acids to a solid support. The invention is particularly advantageous in the isolation of viral nucleic acids.

The Method will be described below by way of example, without being limited to this example to become.

embodiment

Example: Isolation of viral nucleic acids blood plasma

Two Blood plasma samples were spiked with HIV viruses and HBV viruses and for isolation the viral nucleic acid used. 150 μl of the sample were transferred to a 1.5 ml reaction vessel. The sample was subsequently with 450 μl a lysis buffer (4 M guanidine thiocyanate, 80 mM trisodium citrate dihydrate) and 10 seconds ahead. Subsequently The sample was incubated at room temperature for 10 min. After the lysis and denaturation of the starting sample, 600 μl of a binding buffer (30% Tween-20, 5mM EDTA, 20mM trisodium citrate dihydrate) and the sample completely mixed. The approach was followed by a filter column, which a commercially available one Glass fiber filter paper (Whatman) contained, centrifuged. The filter column was subsequently washed with alcoholic wash buffers. After that, shortly centrifuged to dry the filter material. The flow of bound viral nucleic acid was carried out by the addition of 50 ul RNAse free water.

The isolated nucleic acid was then analyzed for specific virus detection of HIV or HBV by means of real-time PCR. The detection of viral nucleic acids was successful. Results: Plasma sample virus detection CT value Sample 1 HIV 1000 IU / ml 34.33 Sample 1 HBV 500 IU / ml 30.52 Sample 2 HIV 1000 IU / ml 34.86 Sample 2 HBV 500 IU / ml 30.78

Claims (13)

Method for parallel isolation double and single nucleic acids from samples containing these substances, without the double- and single-stranded nucleic acids too separating the samples with known lysis buffers (high salt concentrations or lower salt concentrations or with proteolytic enzymes) be offset, characterized by the following steps: a) the nucleic acid-containing Sample (before lysis) or the already lysed or homogenized Sample is incubated in the absence of alcohol with a binding buffer, the at least one nonionic detergent in high concentration contains adjusted so that the total nucleic acid adsorbs to a solid support becomes b) Removing the carrier with the adsorbed total nucleic acid c) washing and elution the adsorbed total nucleic acid according to known methods. Method according to claim 1, characterized in that that the double- and single-stranded nucleic acids are um viral nucleic acids is. Method according to claim 1, characterized in that that in known lysis buffers low salt concentrations (smaller than 100mM) the nonionic detergent additionally a salt solution higher Concentration (greater than 1 M) contains. Method according to claim 1 or 2, characterized that the detergent concentration for binding the nucleic acids 5-50%; preferably 10-30 %, in the final mixture of lysis buffer / binding buffer. Method according to claim 4, characterized in that that the detergent used is Tween-20, Tween-80 or Triton X-100. Method according to one of claims 1 to 5, characterized that the binding buffer in addition a) SDS, sarcosinate, LDS and / or b) complexing compounds, in particular EDTA or EGTA, contains. Method according to claim 1, characterized in that that as a solid carrier a mineral carrier or magnetic iron oxide particles, preferably with modified surfaces used become. Method according to one of Claims 4, characterized in that that the solid carrier Part of a mini-centrifugation column is. Kit to carry out of the method according to any one of claims 1 to 5, comprising • known lysis buffer • one Detergent solution for binding the nucleic acids • at least a solid carrier • known Wash and elution buffer. Kit according to claim 9, characterized in that it in addition a saline solution greater than 1M. Kit according to claim 9 or 10, characterized that in addition a) SDS, sarcosinate, LDS and / or b) complexing compounds, especially EDTA or EGTA. Use of nonionic detergents in one Concentration of 5-50 %; preferably 10-30%, in the absence of alcohol, for parallel isolation single and double nucleic acids from samples containing these substances, without the double- and single-stranded nucleic acids too by binding the total nucleic acids to a solid support. Use according to claim 12 for the isolation of viral Nucleic acids.