EP2126134A1 - Procede et kit de test permettant la detection rapide de sequences d'acides nucleiques specifiques, notamment la detection de mutations ou de polymorphismes d'un seul nucleotide (snp) - Google Patents

Procede et kit de test permettant la detection rapide de sequences d'acides nucleiques specifiques, notamment la detection de mutations ou de polymorphismes d'un seul nucleotide (snp)

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Publication number
EP2126134A1
EP2126134A1 EP08735420A EP08735420A EP2126134A1 EP 2126134 A1 EP2126134 A1 EP 2126134A1 EP 08735420 A EP08735420 A EP 08735420A EP 08735420 A EP08735420 A EP 08735420A EP 2126134 A1 EP2126134 A1 EP 2126134A1
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EP
European Patent Office
Prior art keywords
fluorescence
detection
amplification
probes
reaction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP08735420A
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German (de)
English (en)
Inventor
Elmara Graser
Timo Hillebrand
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AJ Innuscreen GmbH
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AJ Innuscreen GmbH
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Filing date
Publication date
Application filed by AJ Innuscreen GmbH filed Critical AJ Innuscreen GmbH
Publication of EP2126134A1 publication Critical patent/EP2126134A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • SNP single nucleotide polymorphism
  • the target-specific amplification reaction is carried out, coupled with the probe hybridization reaction using fluorescence-labeled allele-specific amplification primers.
  • PCR Polymerase Chain Reaction
  • the fluorescence is detected by means of commercially available fluorescence readers.
  • a simple and inexpensive method for detecting specific nucleic acid sequences and mutative changes of the DNA sequence is the so-called RFLP analysis (Restriction Fragment Length Polymorphism).
  • RFLP analysis Restriction Fragment Length Polymorphism
  • a DNA segment containing the mutation site is amplified.
  • the PCR product is digested with an endonuclease which has its recognition sequence exactly at the mutation site.
  • the detection is carried out by means of electrophoresis.
  • the method can not be automated and does not allow parallel processing of large sample volumes.
  • a disadvantage of the method is that it is not universally applicable, since not every mutation site has a suitable nuclease recognition site. In addition, incomplete or inhibited nuclease digestion leads to false results.
  • SSCP analysis Single Strand Conformation Polymorphism
  • the method is based on the fact that the conformation of single-stranded DNA is essentially determined by the base sequence. After amplification of the target sequence to be assayed, the generated double-stranded PCR product is converted to single stranded conformation (e.g., by heating or treatment with a denaturing chemical) and then rapidly cooled on ice.
  • the single-stranded DNA is separated by native polyacrylamide gel electrophoresis (PAGE).
  • PAGE polyacrylamide gel electrophoresis
  • This method is also suitable in principle for the detection of mutations, but is labor intensive, highly error prone and does not have an automation potential.
  • DNA sequencing Another classic method for detecting genetic alterations is DNA sequencing.
  • This widespread method is a safe method for detecting changes in specific nucleic acid sequences.
  • the disadvantage is the time required for it and the very high costs in principle.
  • High-throughput device systems cost well over € 100,000.
  • a novel method for the rapid detection of altered nucleic acid sequences is pyrosequencing. Particularly in the field of detection of SNPs, this method can be used to process a large sample volume.
  • this method is also bound to a very expensive device system and also requires a highly complex reaction chemistry.
  • a large number of work steps are necessary before the actual detection reaction can be started. This concerns the amplification of the target sequence to be investigated, their purification and subsequently the sequencing reaction.
  • PCR ELISA Enzyme Linked Immunosorbent Assay
  • a widely used method for the detection of single base changes can be achieved by means of the Light Cycler technology (Roche).
  • the company Roche developed special hybridization probes, consisting of two different oligonucleotides, each labeled with only one fluorochrome. At the 3 "end of one probe is the acceptor, the other oligonucleotide is provided at the 5" end with a donor.
  • the probes are chosen so that they both bind to the same strand of DNA, wherein the distance between acceptor and donor may only be a maximum of 1 to 5 nucleotides, so that it can come to the so-called FRET effect (fluorescence resonance energy transfer).
  • Double Dye probes which are disclosed in the patent US 5,210,015 A and US 5,487,972 A (TaqMan probes). Double Dye Probes carry two fluorochromes on a probe.
  • the reporter dye is here at the 5 "end, the quencher dye at the 3" end.
  • the 3 'end of the probe there is still a phosphate group so that the probe can not function as a primer during elongation.As long as the probe is intact, the intensity of light released is low since almost all of the light energy following the reporter's excitation due to the proximity of the quencher, it is captured and reshaped, and the emitted light from the reporter dye is "quenched", ie deleted. This FRET effect is also retained after the probe has bound to the complementary DNA strand. During the elongation phase, the polymerase hits the probe and hydrolyzes it.
  • the ability of the polymerase to hydrolyze an oligonucleotide (or probe) during strand synthesis is referred to as 5 "-3" exonuclease activity. Not all polymerases have a 5 "-3" exonuclease activity (Taq and Tth polymerase).
  • Taq and Tth polymerase This principle has been described for the Taq polymerase. The principle is called the TaqMan principle. After probe hydrolysis, the reporter dye is no longer in close proximity to the quencher. The emitted fluorescence is no longer transformed, this increase in fluorescence is measured.
  • C ⁇ value is determined.
  • C T cycle threshold
  • the C ⁇ value thus indicates a number of cycles. It is directly related to the initial amount of DNA used. If the C ⁇ value is low, the amount of DNA used is large. If the C T value is high, the initial amount of DNA is small. The C T value is therefore the basis for the quantification of a reaction. With this evaluation method, genotyping is also possible by comparing the C ⁇ values of both probes.
  • Another way to detect single base differences using Real Time PCR technology is the use of intercalating dyes (ethidium bromide, Hoechst 33258, Yo-Pro-1 or SYBR Green TM, etc.). These dyes emit, after excitation by high-energy UV light, light in the visible lower-energy wavelength range (fluorescence). If the dye is present as a free dye in the reaction mixture, the emission is very low. Only by the intercalation of the dye, ie by the incorporation into the small groove of double-stranded DNA molecules, the light emission is greatly enhanced.
  • the dyes are inexpensive and can be used universally, since in principle any PCR reaction can be followed in real time. In addition, they have a high signal strength, since each DNA molecule binds several dye molecules.
  • intercalating dyes do not distinguish between correct product and amplification artefacts (such as primer dimers or defective products). Emerging primers Dimers and other artifacts naturally also bind intercalating dyes and thus lead to a nonspecific increase in fluorescence even in negative samples. A clear differentiation between specific Amplif ⁇ kationsereignis or artifact but is absolutely necessary. To achieve this, one uses a so-called melting point analysis at the end of the actual PCR reaction. The reaction mixture is heated in 1 degree increments from 50 0 C to 95 0 C. In this process, the fluorescence is measured continuously.
  • the point at which double-stranded DNA melts is characterized by a decrease (peak) in the fluorescence of the intercalating dye, as the intercalating dye dissociates from the single-stranded DNA. If the PCR is optimally adjusted, expect a peak of melting point to be tapered. This melting point represents the specific, expected product. Differently sized products and products of different sequence have different melting points.
  • Real Time PCR methods are also suitable for the detection of single base differences to answer different question positions (mutation analysis, SNP genotyping, etc.) as well as for the specific detection of nucleic acids in general (eg pathogenicity testing ).
  • the great advantage of the method is the complexity of the analysis, i. the process of target specific amplification and the detection of e.g. Single base differences take place in a reaction vessel.
  • the real-time methods which function on the basis of the use of the illustrated different probe systems in combination of reporter and quencher, are characterized by a very high degree of diagnostic specificity. For this reason, methods of real-time PCR have become established worldwide for the treatment of molecular diagnostic issues, including the detection of mutations or SNPs.
  • the invention was therefore based on the object of developing a simple, universally usable and inexpensive method which makes it possible to detect specific nucleic acid sequences, in particular to provide a method for the detection of mutations or for SNP genotyping.
  • the object has been solved according to the features of the claims.
  • the erfmdungssiee method uses the advantages of high diagnostic sensitivity and specificity of already established fluorescence probe technologies in the field of real-time PCR methods and the advantage of being able to work on standard laboratory and existing equipment systems.
  • the previously required process of experimental work by the molecular sample preparation until the final analysis of the analysis data is reduced to a minimum.
  • the method according to the invention for rapid detection of specific nucleic acid sequences comprises the following steps:
  • the real-time PCR methods known from the prior art are always based on the continuous measurement and preparation of fluorescence signals.
  • the preparation of the continuously measured fluorescence signals is a highly complex algorithm.
  • the diagnostic result is therefore based on the preparation of the continuously measured (in real time) fluorescence, not on a so-called endpoint fluorescence determination.
  • No. 6,154,707 B1 discloses a method which enables genotyping of multiple alleles also in the form of the end point measurement of fluorescence after a 5'-exonuclease assay. The method is based on an algorithm which is based on first determining a relative fluorescence by comparison with an internal control and then comparing the unknown and genotyping samples with a specific control used for one allele.
  • the present invention makes use of a novel and universal method for allelic discrimination, which is eminently suitable for the detection of specific nucleic acid sequences, in particular for the determination of SNPs or other nucleic acid sequence changes (for example mutations).
  • the inventive method does not require real-time device systems, but requires a known thermal cycler and a device for measuring fluorescence. The purchase price of such devices is thus significantly cheaper than the necessary price for a real-time device.
  • the method according to the invention requires no optimization steps and no DNA standard samples.
  • the primers and probes can be used in any concentration, which is not possible with the AD-TaqMan ® -PCR assay. Since the method according to the invention requires no controls, it can discriminate against the alleles which carry very few mutations and for which no homozygous mutant genotypes are present.
  • the comparison with the negative controls is used in the AD-TaqMan ® -PCR assay only the contamination control.
  • the allele discrimination is based on the measurements of the negative controls (as can be seen from embodiment 2).
  • Another principal advantage of the method according to the invention compared to the known method is the freedom of probe selection. In all writings on the TaqMan ® technology -PCR- is not recommended to provide dyes having a reporter molecule or guanine to place in its vicinity. The probes must not have a G / C content above 80%. In the method according to the invention, these limitations do not play a role.
  • the method can be used for a variety of functionally quite different commercially available probe systems used for PCR hybridization methods based on Fluorescence Resonance Energy Transfer (FRET) or other fluorescence detection methods. Possible is the use of so-called.
  • FRET Fluorescence Resonance Energy Transfer
  • Probe systems such as e.g. TaqMan, Uniprimer, TripleHyb, Scorpions, Molecular Beacons or e.g. Terbium chelated probes.
  • different detection technologies are also used and can be analyzed by the method, such as, e.g. the use of TaqMan probes in an exonuclease assay or the use of allele-specific double-labeled primers in a standard PCR reaction.
  • the detection reactions take place in two stages.
  • the target-specific amplification reaction is carried out, coupled with the probe hybridization reaction or using fluorescently labeled allele-specific amplification primers.
  • This reaction can be performed on all commercially available PCR devices and is not limited to real-time PCR devices.
  • the detection of fluorescence is then also on commercially available fluorescence readers.
  • the inventive method uses the combination of a so-called. SpeedCyclers (Analytik Jena AG) and a fluorescence reader (SpeedScan, Anlaytik Jena AG).
  • SpeedCyclers Analytik Jena AG
  • a fluorescence reader SpeedScan, Anlaytik Jena AG
  • the advantage of this device combination is based on the patented and extremely fast amplification technology (patent) by means of the SpeedCycler, which makes it possible to realize the amplification / hybridization reaction in about 30 minutes.
  • the reaction mixtures contain e.g. the required reagents, which are also used for real-time PCR's.
  • the determination of a successful Amplif ⁇ kation or verification of an existing contamination takes place in that the signal strengths of the samples are compared with the signal strengths of the negative controls. This is done in order to exclude the samples, in which no successful Amplif ⁇ kation has taken place, from the subsequent evaluation. Is the Signal strength of the negative controls as strong as the signal strength of the samples, then there is a contamination of the samples. This also takes into account the known problem of sample contamination in PCR-based methods.
  • Genotyping is carried out by determining the quotient (K) of the end-point fluorescence of probe 1 (eg labeling for allele 1 or for the wild-type sequence) and probe 2 (eg labeling for allele 2 or one of the wild-type differing nucleic acid sequence). This measurement is completely independent of the known potential fluctuations of the PCR conditions and efficiencies of probe binding. After determining the average of the quotients (K) for the negative controls, one obtains a statement as to whether a homozygous genotype with allele 1 or a homozygous wild type is present or not, that is, whether the samples are homozygous or heterozygous.
  • the inventive method thus allows simple, rapid and reproducible the intended characterization of specific nucleic acid sequences, in particular with regard to the detection of mutations or the genotyping of SNPs.
  • the evaluation of the endpoint fluorescence values can be mapped in the form of computer software. Statistical variables (such as the standard deviation) can be easily determined. The determination of standard deviations is known to the person skilled in the art.
  • the method according to the invention does not require any internal reference samples as positive controls or as controls for the calculation of the fluorescence signals to be evaluated.
  • the inventive method allows the precise determination of mutations or the genotyping of SNPs, only taking into account the measured fluorescence maxima and fluorescence minima.
  • the inventive method can be applied to a variety of assay formats used based on the generation of fluorescence signals. In particular, the so-called TaqMan probes are very suitable for the implementation of the evidence.
  • the inventive method circumvents an existing limitation of the use of TaqMan probes.
  • Essential in the selection and design of TaqMan probes is that the reporter dye must not be bound to a guanine, since even after Exonukleaseverdau the fluorescence of the probe is deleted.
  • the GC content of the probe to be selected according to the patent US 6,154,707 Bl was between 20% and 80% and the Schmelztempera- structure of the probe does not exceed 70 0 C.
  • the reporter dye can also be coupled to a guanine, without an evaluation of the measured signals is negatively influenced by the solution of the fluorescence. The sensitivity of the method according to the invention is thus significantly higher than the method disclosed in the above patent.
  • the goal of the method according to the invention to realize the detection of mutations or the genotyping of SNPs with minimal experimental effort can be achieved by surprisingly further simple methods or means. This takes into account the complex experimental process steps, which involve the modern methods of molecular diagnostic tests in a comprehensive overall process.
  • test kit according to the invention.
  • reaction mixtures are subsequently pipetted for the described molecular-biological detection methods necessary amplification / detection reactions.
  • the detection reaction can then be started.
  • the method according to the invention makes use of a very simple means to minimize the steps required to establish the amplification / detection reactions:
  • nucleic acids can be carried out with an elution buffer, the composition of salts; including Mg 2+ ions, and optionally further additives such as BSA (bovine serum albumin), and allows it to be used directly in subsequent amplification / detection reactions.
  • an elution buffer allows efficient elution of the bound DNA and thus inevitably ensures that the components required for the subsequent amplification / detection reactions (amplification buffer, Mg 2+ ions, further PCR additives) no longer have to be separately pipetted together.
  • the molar composition of the elution buffer according to the invention can be adjusted so that subsequent dilution with water can be carried out or such a dilution is also dispensed with, ie the eluent can then be used directly in the detection reactions.
  • the dual functionality of the elution buffer according to the invention thus enables a highly efficient elution and a time and labor savings in the necessary preparation of the reaction mixture for amplification / detection reaction.
  • reaction mixtures for example by lyophilization, can be converted into a storage-stable state and can reactivate such an approach by the addition of an aqueous phase.
  • stabilization do not always take place without problems and require considerable expenditure on equipment.
  • the biological activity of enzymes is no longer or not fully realized.
  • the invention makes it possible to extremely simplify the pipetting effort in the combination of a novel elution buffer in the process of DNA isolation and using the modified plastic produced according to the invention with the components required for the amplification / detection reactions.
  • a defined volume of the elution buffer is transferred only into a plastic which is storage-stable modified with the other specific reaction components and the amplification / detection reaction is started.
  • This simplification-in combination with the method according to the invention for allelic discrimination-thus makes it possible to carry out routinely feasible, simple and extremely rapid performance of molecular diagnostic tests for the detection of mutations or for SNP genotyping.
  • a particularly efficient embodiment is the use of a SpeedCycler (Analytik Jena AG) for the extremely fast amplification / detection reaction and the subsequent measurement on a commercially available fluorescence Reader (SpeedScan, Analytik Jena AG).
  • the use of this device combination allows the execution of the tests in an extremely short time due to the extremely fast amplification reactions. All the steps required, from DNA isolation using the elution buffer according to the invention, the use of the storage-stabilized reaction plastic for the amplification / detection reaction and the execution of these reactions on a SpeedCycler / SpeedScan and the integration of the evaluation algorithm into a computer-aided software solution, allow the determination of mutations or SNPs in less than an hour.
  • the present invention can make a valuable contribution to the routine establishment of molecular genetic tests, in particular to the so-called individualized medicine, which is increasingly becoming the focus of attention.
  • the coating solution was placed on the PCR microplate and dried for about 2 hours at physiological temperature conditions of 37 ° C. After drying, the plates were taped with a sealing film and stored at RT for 4 months and tested in a comparison reaction with freshly added reaction components.
  • the example illustrates that the use of the storage stable coated reaction plastic shows no loss of activity compared to fresh added reaction components.
  • the method according to the invention was subsequently used for SNP genotyping of human DNA samples.
  • the results obtained by the method according to the invention were then sequenced for verification.
  • the results of sequencing and genotyping by means of the method according to the invention are summarized.
  • a mutation in the promoter of the human MDM2 gene (SNP 309) [Bond GL et al. A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans. Cell. 2004 Nov 24; 119 (5): 591-602].
  • the amplification / detection reaction of the sample nucleic acids used were carried out by means of Analytik Jena AG SpeedCycler (amplification / hybridization) and SpeedScan (detection of end-point fluorescence).
  • Sense primer 5'CGC GGG AGT TCA GGG TAA-3 'Antisense primer: 5'-CCC AAT CCC GCC CAG ACT AC-3'
  • hybridization probes used were the following double-fluorescence-labeled oligonucleotides, wherein the GC content in both probes is> 80% and the melting temperature is greater than 70 ° C.: Wild type probe: 5'-FAM-GGG CCG CTT CGG CGC GGG -BHQ 1-3 'Probe Mutated: 5'-ROX-GGC CGC TGC GGC GCG-BHQ2-3'
  • the amplification / hybridization reaction took about 35 minutes at 40 performed
  • genotyping by means of the method according to the invention is listed below.
  • Step 1 Determination of a successful amplification or verification of an existing contamination.
  • sample 2 is homozygous wild type and further calculations for sample 2 need not be made since the difference between the mean of the negative controls and the quotient K of the sample 2 is larger as the difference between the maximum value and the quotient K of the sample 2 considering the standard deviation.
  • Sample 3 can in no case have a homozygous wild-type genotype and the affiliation to the other two genotype groups must be checked, since the difference between the maximum value and the quotient K of the sample 3 taking into account the standard deviation is greater than negative controls and the Quotient K of the sample 3.
  • sample 5 For sample 5:
  • Sample 5 can by no means have a homozygous wild-type genotype, and membership in the other two genotype groups must be checked, since the difference between the maximum value and the quotient K of sample 5, taking into account the standard deviation, is greater than negative controls and the quotient K of the sample 5.
  • sample 3 It follows from the comparison of the average of the negative controls with the minimum value that the sample 3 can by no means have a homozygous mutant genotype since it has already been calculated above and that it is not homozygous wild type but only can be heterozygous, since the difference between the average of the negative controls and the quotient K of the sample 3 is smaller than the difference between the minimum value and the quotient K of the sample 2 taking into account the standard deviation.
  • genotypes of the samples were defined.
  • the above calculation was performed in a logical sequence suitable for a computer algorithm.
  • genotyping was carried out on the basis of 48 different sample nucleic acids.
  • the results obtained by means of the method according to the invention were checked by means of sequencing. The comparison is shown in the following table.
  • Genotyping was carried out by the method according to the invention as follows:
  • the swabs were subsequently isolated by means of a commercially available DNA extraction kit (innuPREP DNA Mini Kit, aj Innuscreen GmbH).
  • the kit is based on the lysis of the Starting material, the subsequent binding of the DNA to the surface of a filter material (spin filter column), the washing of the bound DNA and finally the elution of the DNA by means of water or by means of a solution containing 10 mM Tris HCl.
  • the elution buffer according to the invention was used (33 mM tricine KOH, 14 mM KCl, 4.2 mM MgCl 2 , 3.5 ⁇ g / ml BSA, pH 9).
  • the DNA was eluted from the spin filter column after addition of 500 ⁇ l of the eluent.
  • the eluted DNA was then used directly for the amplification / detection reaction.
  • the reaction plastic was prepared as described under Example 1 and coated with the following components:
  • the coating solution was placed on the PCR microplate and dried for approximately 2 hours at physiological temperature conditions of 37 ° C.
  • Sense primer 5'-GCC TCT GGG CTA ATA GGA CTA CTT C-3 '
  • Antisense primer 5 '-TTT CTG AAA GGT TAC TTC AAG GAC AA-3'
  • hybridization probes used were the following double-fluorescence-labeled oligonucleotides: Probe wild-type: 5'-FAM-ACC TGT ATT CCT CGC CT-BHQ 1-3 '

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Abstract

La présente invention concerne un procédé et un kit de test permettant la détection rapide de séquences d'acides nucléiques spécifiques, notamment la détection de mutations ou de polymorphismes d'un seul nucléotide (SNP = Single nucleotid polymorphism), la réaction de détection étant réalisée en deux étapes. Dans une première étape, la réaction d'amplification spécifique à la cible est menée conjointement à une réaction d'hybridation de sonde, au moyen d'amorces d'amplification spécifiques aux allèles et marquées par fluorescence. Dans une seconde étape, la détection de la fluorescence est effectuée au moyen de lecteurs de fluorescence disponibles dans le commerce. Le génotypage est effectué à partir du quotient de la fluorescence finale des échantillons et des contrôles négatifs.
EP08735420A 2007-03-14 2008-03-14 Procede et kit de test permettant la detection rapide de sequences d'acides nucleiques specifiques, notamment la detection de mutations ou de polymorphismes d'un seul nucleotide (snp) Withdrawn EP2126134A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007013099A DE102007013099A1 (de) 2007-03-14 2007-03-14 Verfahren und Testkit zum schnellen Nachweis spezifischer Nukleinsäuresequenzen, insbesondere zum Nachweis von Mutationen oder SNP's
PCT/EP2008/053065 WO2008110622A1 (fr) 2007-03-14 2008-03-14 Procédé et kit de test permettant la détection rapide de séquences d'acides nucléiques spécifiques, notamment la détection de mutations ou de polymorphismes d'un seul nucléotide (snp)

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EP2126134A1 true EP2126134A1 (fr) 2009-12-02

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US (1) US20100099099A1 (fr)
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Publication number Priority date Publication date Assignee Title
DE102007029772B4 (de) 2007-06-22 2011-12-08 Aj Innuscreen Gmbh Verfahren und Schnelltest zum Nachweis spezifischer Nukleinsäuresequenzen
DE102010003781B4 (de) 2010-04-08 2012-08-16 Aj Innuscreen Gmbh Verfahren zum Nachweis spezifischer Nukleinsäuresequenzen
DE102010052524A1 (de) * 2010-11-22 2012-05-24 Aj Innuscreen Gmbh Verfahren zum qualitativen und quantitativen Nachweis von spezifischen Nukleinsäuresequenzen in Echtzeit
JP2014168442A (ja) * 2013-03-05 2014-09-18 Azbil Corp 微生物検出装置及び微生物検出方法
CN116724124A (zh) * 2020-09-30 2023-09-08 李峰 一种用于dna单核苷酸变异检测的方法、探针及试剂盒及其应用
CN116497098A (zh) * 2022-11-28 2023-07-28 广州奇辉生物科技有限公司 一种基于荧光标记引物的snp检测试剂盒及检测方法

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DE4139664A1 (de) 1991-12-02 1993-06-03 Diagen Inst Molekularbio Vorrichtung und verfahren zur isolierung und reinigung von nukleinsaeuren
DE4321904B4 (de) 1993-07-01 2013-05-16 Qiagen Gmbh Verfahren zur chromatographischen Reinigung und Trennung von Nucleinsäuregemischen
ATE184013T1 (de) 1994-06-14 1999-09-15 Invitek Gmbh Universelles verfahren zur isolierung und reinigung von nukleinsäuren aus extrem geringen mengen sowie sehr stark verunreinigten unterschiedlichsten ausgangsmaterialien
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DE102007013099A1 (de) 2008-09-18
US20100099099A1 (en) 2010-04-22

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