CN102534033B - High-sensitivity real-time fluorescence detection kit and application thereof - Google Patents
High-sensitivity real-time fluorescence detection kit and application thereof Download PDFInfo
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Abstract
The invention discloses a high-sensitivity real-time fluorescence detection method. The invention provides a method for detecting a target nucleic acid in a detected sample, which comprises: mixing the sample with a polymerase chain reaction(PCR) amplification agent and a fluorescence labeling probe, wherein the nucleic acid part of the fluorescence labeling probe comprises a first complementary sequence, a target nucleic acid specific sequence, a second complementary sequence, and an extension sequence, and the first complementary sequence and the second complementary sequence can complementand match with each other; and performing PCR and real-time fluorescence detection. The invention also provides a detection kit, use of the detection kit in preparation of a detection product, and primers, probes and other reagents used in the detection kit.
Description
Technical field:
The invention belongs to the detection of nucleic acids field, particularly, the present invention relates to highly sensitive real-time fluorescence detection method and reach wherein used detection kit and reagent etc.
Background technology:
Taqman probe (Taqman Probe) and the molecular beacons technology (Molecular Beacon) of the nineties invention in last century have promoted fluorescence application and the development of (Real-Time) polymerase chain reaction (PCR) technology aspect detection of nucleic acids in real time greatly.In PCR in real time detected, the specific nucleic acid that common applying marking has fluorophor and quenching group was implemented real-time detection as probe in the process of pcr amplification.For example, Chinese patent literature CN101131347A discloses the PCR kit for fluorescence quantitative of rapid detection Schistosoma japonicum, comprises forward primer, reverse primer and fluorescent probe, is used for the DNA of fluorescence quantitative PCR detection Schistosoma japonicum; And for example, Chinese patent literature CN101591716A discloses real-time fluorescent RT-PCR detection reagent for banana bract mosaic virus (BBrMV), comprises primer and fluorescently-labeled probe, is used for the RNA that RT-PCR detects banana bract mosaic virus (BBrMV).
Yet the inventor finds that in the practical application of PCR in real time, the Taqman probe can make the fluorescence background values of whole detection raise, and causes the reduction of fluorescence △ R value, thereby reduces the sensitivity that detects; And the use molecular beacon probe though can effectively reduce the fluorescence background values, can reduce detected value simultaneously, thereby also can dwindle △ R value, and the amplitude of dwindling even can surpass use the Taqman probe, might further reduce the sensitivity that detects.
For this reason, the inventor gropes through long-term practice, has invented a kind of new highly sensitive real time PCR detection method unexpectedly, and it can improve sensitivity, overcomes the deficiencies in the prior art such as Taqman probe and molecular beacons technology.In addition, this method high specificity can effectively reduce the too high and phenomenon of the non-specific fluorescence signal take-off that is easy to generate of fluorescence background, and the detected result of this method is reliable and stablize, is suitable for the Industry Promotion application.In addition, the inventor has also invented the test kit that can be used for this real time PCR detection method, and reagent such as wherein primer, probe.
Summary of the invention
The technical problem to be solved in the present invention has been to provide new PCR in real time to detect the methods and applications of target nucleic acid.The present invention also provides for PCR in real time and has detected the test kit of target nucleic acid and reagent wherein, comprises primer and/or probe.
Particularly, in first aspect, the invention provides the method for target nucleic acid in the test sample, it comprises:
(1) sample is mixed with pcr amplification reagent and fluorescently-labeled probe, the nucleic acid moiety of its middle probe comprises first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence, and first complementary sequence and second complementary sequence can complementary pairings; With
(2) carry out PCR, and detect fluorescence in real time.
Fluorescent mark is technology commonly used in the PCR in real time, usually the end (preferably 5 ' end) of the nucleic acid moiety of probe connect fluorophor (as, commercial FAM), and at the other end (preferably 3 ' end) connect quenching group (as, commercial ECLIPSE).Therefore, in this article, fluorophor and quenching group that probe comprises nucleic acid moiety and is connected to the nucleic acid moiety two ends preferably are made up of nucleic acid moiety and the fluorophor and the quenching group that are connected to the nucleic acid moiety two ends.Preferably in detection method of the present invention, the nucleic acid moiety of probe is made up of first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence to 3 ' end successively from 5 ' end.In a specific embodiment of the present invention, probe is FAM-AGAACTCCCTCGCCTCGGTTCTAT-ECLIPSE, and wherein nucleic acid moiety is AGAACTCCCTCGCCTCGGTTCTAT.In another embodiment of the present invention, probe is FAM-CATGTGGTGGCTTCAACATGAT-ECLIPSE, and wherein nucleic acid moiety is CATGTGGTGGCTTCAACATGAT.
In this article, sample is the potential vitro samples that may contain target nucleic acid, as food, blood, blood products, saliva, medical treatment product or medicine etc.Detection method of the present invention at target---target nucleic acid, can be the nucleic acid that any suitable PCR in real time detects, as gene or gene fragment, comprise DNA and RNA, can be single-chain nucleic acid, also can be double-strandednucleic acid.Those skilled in the art can judge used nucleic acid species according to technical spirit.Discover through the inventor, adopt detection method of the present invention, not only can detect RNA, also can detect DNA.For example, in the specific embodiment of the present invention, can detect the NS2 gene (RNA) of HCV (hepatitis C virus, it is RNA viruses) and the S gene (DNA) of HBV (hepatitis B virus, it is dna virus).These nucleic acid are the existence of characterization of HCV and HBV to a certain extent.Therefore, target nucleic acid is RNA or DNA in the detection method of the present invention, preferably HCV RNA or HBVDNA.For example HBV S gene or HCV NS2 gene.
It is pointed out that detection method of the present invention is used for the detection to vitro samples, the direct result of detection be target nucleic acid existence whether, and be not diagnostic result.Even the target nucleic acid for HCV or HBV in the blood sample that utilizes detection method detection human or animal of the present invention, whether also can only directly draw the existence of target nucleic acid, also need experienced doctor or sampling personnel to judge HCV or HBV that detected target nucleic acid pollutes when coming from HCV in the blood or HBV or sampling accidentally, and can not directly obtain diagnostic result or the healthy state of disease; Even the existence that target nucleic acid has characterized HCV and HBV whether, because in the normal HCV of liver function or HBV carrier, some carrier's viral detected result can be turned out cloudy naturally, some can not fallen ill all the life, also need experienced doctor just can judge whether to cause disease or influential to healthy state according to comprehensive conditions such as corresponding human or animal's physique, medical history, clinical symptom, therefore can't judge directly whether its carrier suffers from third type or hepatitis B or ill risk is arranged according to the existence of this target nucleic acid, so directly purpose is not diagnosis.So detection method of the present invention is not diagnostic method.
In detection method of the present invention, pcr amplification reagent is that those skilled in the art are familiar with.Usually, pcr amplification reagent comprise can amplifying target nucleic acid sequence primer to, archaeal dna polymerase and dNTP, can also comprise damping fluid in addition.Wherein, a large amount of reagent commercializations, for example archaeal dna polymerase (as, Taq polysaccharase), dNTP (dATP, dTTP, dCTP and dGTP) and damping fluid are usually included in the pcr amplification test kit of manufacturer.
In detection method of the present invention, PCR is RT-PCR in addition.Like this, detection method of the present invention can directly detect RNA.RT-PCR and amplifing reagent thereof also are that those skilled in the art are familiar with.Usually, the RT-PCR amplifing reagent also comprises ThermoScript II on the basis of common pcr amplification reagent, as commercial M-MLV ThermoScript II etc.In order to prevent that the RNA in the sample from being degraded fast, the RT-PCR amplifing reagent can also comprise the RNase inhibitor in addition, as commercial RNasin etc.There are many manufacturers directly to provide RT-PCR amplification kit at present, comprise the required reagent of above all RT-PCR.
In detection method of the present invention, primer is to being used for amplifying target nucleic acid, and preferred primer is to being right for the primer of amplification HCV RNA or HBVDNA, and it is right to be more preferably for the primer of amplification HBV S gene or HCVNS2 gene.In a specific embodiment of the present invention, primer is to being CGAGGCAGGTCCCCTAGAA and CGGCGATTGAGACCTTCGT; In another embodiment of the present invention, primer is to being TCGCCATATTACAAGCGCTACA and GCGCTTCTACTCTGGTCAGAAAA.Through inventor's research, these concrete primers are to being particularly suitable for the PCR step in the specific embodiment of the invention.
In this article, if do not add special instruction, the expression of nucleotide sequence all is to carry out according to the order of 5 ' end to 3 ' end; Each base on one of " complementary pairing " of nucleic acid expression or a part of single-chain nucleic acid from 5 ' end to 3 ' end successively with another or another part single-chain nucleic acid on each base complementary to 5 ' end from 3 ' end, comprise A and T complementation, C and G complementation.These all are terms well-known to those skilled in the art.For example, a part of specific fragment of target nucleic acid specific sequence and target nucleic acid can complementary pairing; And " AGAAC " among the AGAACTCCCTCGCCTCGGTTCTAT and " GTTCT " can complementary pairings.
The target nucleic acid specific sequence can design according to target nucleic acid.Preferably in detection method of the present invention, the length of target nucleic acid specific sequence is 8～20 bases, is preferably 9～15 bases, more preferably 10～12 bases.In a specific embodiment of the present invention, the target nucleic acid specific sequence is TCCCTCGCCTCG.In another embodiment of the present invention, the target nucleic acid specific sequence is GGTGGCTTCA.
First complementary sequence and second complementary sequence are on same the single-chain nucleic acid, lay respectively at the target nucleic acid specific sequence, and these two complementary sequences can complementary pairing.Wherein, " first ", " second " only are used for distinguishing sequence, sequential structure are not constituted to limit.For example, first complementary sequence and/or second complementary sequence also can be respectively and the partial sequence complementary pairing of target nucleic acid, and the partial sequence complementary pairing of a sequence and target nucleic acid is arranged in common first complementary sequence and second complementary sequence.In the specific embodiment of the present invention, the partial sequence complementary pairing of first complementary sequence and target nucleic acid, the integral body of first complementary sequence and target nucleic acid specific sequence all is that target nucleic acid is had specificity, and second complementary sequence and the first complementary sequence complementary pairing.Preferably in detection method of the present invention, the equal in length of first complementary sequence and second complementary sequence.For example, first complementary sequence is respectively the length of 4～8 bases, is preferably the length of 5～7 bases, more preferably the length of 5 bases; And second complementary sequence is respectively the length of 4～8 bases, is preferably the length of 5～7 bases, more preferably the length of 5 bases.In a specific embodiment of the present invention, first complementary sequence is AGAAC, and second complementary sequence is GTTCT.In another embodiment of the present invention, first complementary sequence is CATGT, and second complementary sequence is ACATG.
Extension sequence can be connected 5 ' end of first complementary sequence, also can connect 3 ' end, the preferably latter of second complementary sequence.Preferably in detection method of the present invention, the length of extension sequence is 2～5 bases, is preferably 2～3 bases, more preferably 2 bases.Extension sequence is the sequence of high AT content, and for example AT content is preferably greater than 80% greater than 60%, and most preferably all Nucleotide all are selected from A and T.In the specific embodiment of the present invention, extension sequence is AT.
PCR generally includes these three steps of sex change, annealing and extension, and RT-PCR also further comprises reverse transcription step.For example, about 42 ℃, carry out reverse transcription, carrying out sex change greater than 90 ℃, anneal at 50～65 ℃, extend at about 72 ℃.Preferably in detection method of the present invention, the annealing temperature of PCR is 55～65 ℃, more preferably 58～63 ℃, most preferably is 60 ℃.Also preferably in detection method of the present invention, the annealing time of PCR is 20～60 seconds, more preferably 25～40 seconds, most preferably is 30 seconds.The inventor discovers that detection method of the present invention can not comprise the extension step, still can finish detection, can shorten detection time like this.Therefore preferably in detection method of the present invention, PCR does not extend step.
In second aspect, the invention provides the detection kit of target nucleic acid in the sample, it comprises pcr amplification reagent and fluorescently-labeled probe, and the nucleic acid moiety of its middle probe comprises first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence.
Preferably in detection kit of the present invention, the nucleic acid moiety of probe is made up of first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence to 3 ' end successively from 5 ' end.In a specific embodiment of the present invention, probe is FAM-AGAACTCCCTCGCCTCGGTTCTAT-ECLIPSE, and wherein nucleic acid moiety is AGAACTCCCTCGCCTCGGTTCTAT.In another embodiment of the present invention, probe is FAM-CATGTGGTGGCTTCAACATGAT-ECLIPSE, and wherein nucleic acid moiety is CATGTGGTGGCTTCAACATGAT.
In detection kit of the present invention, pcr amplification reagent comprise can amplifying target nucleic acid sequence primer to, archaeal dna polymerase and dNTP, can also comprise damping fluid in addition.Can carry out conventional PCR like this.More preferably carry out RT-PCR, pcr amplification reagent is the RT-PCR amplifing reagent.The RT-PCR amplifing reagent comprise can amplifying target nucleic acid sequence primer to, archaeal dna polymerase, dNTP and ThermoScript II, preferably also comprise the RNase inhibitor.
Preferably in detection kit of the present invention, primer is to being right for the primer of amplification HCVRNA or HBVDNA, and it is right to be more preferably for the primer of amplification HBV S gene or HCV NS2 gene.In a specific embodiment of the present invention, primer is to being CGAGGCAGGTCCCCTAGAA and CGGCGATTGAGACCTTCGT; In another embodiment of the present invention, primer is to being TCGCCATATTACAAGCGCTACA and GCGCTTCTACTCTGGTCAGAAAA.Through inventor's research, these concrete primers are to being particularly suitable for the PCR step in the specific embodiment of the invention.
Preferably in detection kit of the present invention, the length of target nucleic acid specific sequence is 8～20 bases, is preferably 9～15 bases, more preferably 10～12 bases.In a specific embodiment of the present invention, the target nucleic acid specific sequence is TCCCTCGCCTCG.In another embodiment of the present invention, the target nucleic acid specific sequence is GGTGGCTTCA.
Preferably in detection kit of the present invention, the equal in length of first complementary sequence and second complementary sequence.For example, first complementary sequence is respectively the length of 4～8 bases, is preferably the length of 5～7 bases, more preferably the length of 5 bases; And second complementary sequence is respectively the length of 4～8 bases, is preferably the length of 5～7 bases, more preferably the length of 5 bases.In a specific embodiment of the present invention, first complementary sequence is AGAAC, and second complementary sequence is GTTCT.In another embodiment of the present invention, first complementary sequence is CATGT, and second complementary sequence is ACATG.
Preferably in detection kit of the present invention, the length of extension sequence is 2～5 bases, is preferably 2～3 bases, more preferably 2 bases.Extension sequence is the sequence of high AT content, and for example AT content is preferably greater than 80% greater than 60%, and most preferably all Nucleotide all are selected from A and T.In the specific embodiment of the present invention, extension sequence is AT.
In the third aspect, the invention provides the application in the testing product of test kit method of target nucleic acid in for the preparation of test sample of second aspect present invention.Testing product comprises the test kit of second aspect present invention.The preferred detection product can further include other goods relevant with target nucleic acid in the test sample.For example, the method for target nucleic acid can be the method for first aspect present invention in the test sample, and therefore preferred, testing product also comprises the specification sheets of the method for putting down in writing first aspect present invention.And for example, the test kit of second aspect present invention can with the tie-in sale of fluorescent PCR instrument, so the preferred detection product also comprises the fluorescent PCR instrument.
In fourth aspect, the invention provides probe, its nucleic acid moiety comprises first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence.This probe is fluorescently-labeled probe preferably, can be used for the present invention first, second, and third aspect.
Preferably in probe of the present invention, the length of target nucleic acid specific sequence is 8～20 bases, is preferably 9～15 bases, more preferably 10～12 bases.In a specific embodiment of the present invention, the target nucleic acid specific sequence is TCCCTCGCCTCG.In another embodiment of the present invention, the target nucleic acid specific sequence is GGTGGCTTCA.
Preferably in probe of the present invention, the equal in length of first complementary sequence and second complementary sequence.For example, first complementary sequence is respectively the length of 4～8 bases, is preferably the length of 5～7 bases, more preferably the length of 5 bases; And second complementary sequence is respectively the length of 4～8 bases, is preferably the length of 5～7 bases, more preferably the length of 5 bases.In a specific embodiment of the present invention, first complementary sequence is AGAAC, and second complementary sequence is GTTCT.In another embodiment of the present invention, first complementary sequence is CATGT, and second complementary sequence is ACATG.
Preferably in probe of the present invention, the length of extension sequence is 2～5 bases, is preferably 2～3 bases, more preferably 2 bases.Extension sequence is the sequence of high AT content, and for example AT content is preferably greater than 80% greater than 60%, and most preferably all Nucleotide all are selected from A and T.In the specific embodiment of the present invention, extension sequence is AT.
Preferably formed by first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence successively to 3 ' end from 5 ' end at the nucleic acid moiety of probe of the present invention.In a specific embodiment of the present invention, probe is FAM-AGAACTCCCTCGCCTCGGTTCTAT-ECLIPSE, and wherein nucleic acid moiety is AGAACTCCCTCGCCTCGGTTCTAT.In another embodiment of the present invention, probe is FAM-CATGTGGTGGCTTCAACATGAT-ECLIPSE, and wherein nucleic acid moiety is CATGTGGTGGCTTCAACATGAT.
Aspect the 5th, it is right to the invention provides primer, its primer that is selected from amplification HBV S gene is to right with the primer of amplification HCV NS2 gene, the primer of HBV S gene of wherein increasing is CGAGGCAGGTCCCCTAGAA and CGGCGATTGAGACCTTCGT, and the primer of amplification HCV NS2 gene is to being TCGCCATATTACAAGCGCTACA and GCGCTTCTACTCTGGTCAGAAAA.
Beneficial effect of the present invention is, reduces the fluorescence background and improves the fluoroscopic examination signal, has very high signal to noise ratio, has improved the sensitivity and the specificity that detect; It is wide to detect the scope of using, can be at the detection of DNA and RNA; Shorten detection time, improve detection efficiency; Can use conventional fluorescent PCR instrument to carry out, save the detection cost; Detected result is reliably stablized, and is suitable for Industry Promotion and uses.
For the ease of understanding, below will the present invention describes in detail by concrete accompanying drawing, embodiment.It needs to be noted that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this specification sheets, many variations of the present invention, change all are apparent concerning one of ordinary skill in the art.In addition, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, just looks like that repeated description is the same excessively in this article for their full text.
Description of drawings
Fig. 1 is for detecting the comparison diagram as a result of nucleic acid (DNA), and wherein each curve is numbered employed probe and template is: 1, and the probe that the present invention optimizes, positive template; 2, contrast probe 1, positive template; 3, contrast probe 1, negative template; 4, contrast probe 2, positive template; 5, the probe that the present invention optimizes: negative template; 6, contrast probe 2, negative template.
Fig. 2 is for detecting nucleic acid (RNA) figure as a result, comparison diagram as a result, wherein each curve is numbered employed probe and template is: 1, the probe that the present invention optimizes, positive template; 2, contrast probe 1, positive template; 3, contrast probe 1, negative template; 4, contrast probe 2, positive template; 5, the probe that the present invention optimizes: negative template; 6, contrast probe 2, negative template.
Embodiment
Describe below in conjunction with specific embodiment, wherein synthetic, the reagent of primer, probe and sample all can obtain by the commercial channel, experimental procedure is if any not using up part, all can be referring to " molecular cloning experiment guide " (third edition) (Science Press, teach book and laboratory manual such as 2002), and can illustrate according to the manufacturer of business-like enzyme, reagent and equipment to carry out.
The detection of embodiment 1DNA sequence
We have designed primer and probe at HBV S gene order, entrust six directions trade company limited of stimulating the menstrual flow in Beijing to synthesize following pcr amplification primer and probe (comprising the contrast probe), to contain HBV (the qualitative and quantitative national reference material of hbv nucleic acid (HBV DNA), numbering 300009, can dilute respectively with purified water according to the rules available from National Institute for Food and Drugs Control) 1 * 10
2The aqueous solution of IU/ml (positive), 20IU/ml (sensitivity 1) and 10IU/ml (sensitivity 2) or pure water (feminine gender) be respectively as template, detects at the enterprising performing PCR of ABI 7500 fluorescent PCR instrument (can available from Applied Biosystems company):
Forward primer: CGAGGCAGGTCCCCTAGAA
Reverse primer: CGGCGATTGAGACCTTCGT
The probe that traditional Taqman specificity method uses (contrast probe 1): FAM-AGAACTCCCTCGCCTCG-ECLIPSE, 5 ' end of its nucleic acid is marked with fluorescence dye FAM, and 3 ' end is marked with quenching group ECLIPSE
The probe that traditional molecular beacon method is used (contrast probe 2): FAM-
AGAACTCCCTCGCCTCG
-ECLIPSE, 5 ' end of its nucleic acid is marked with fluorescence dye FAM, and 3 ' end is marked with quenching group ECLIPSE, and wherein italics partly is the complementary pairing sequence
The probe that the present invention optimizes: FAM-
TCCCTCGCCTCG
-ECLIPSE, 5 ' end of its nucleic acid is marked with fluorescence dye FAM, and 3 ' end is marked with quenching group ECLIPSE, and wherein italics partly is the complementary pairing sequence, and boldface letter partly is extension sequence
The PCR reaction system:
In the above reaction system, template is got positive template, negative template or sensitivity 1,2 templates respectively, probe is got above-mentioned contrast probe 1,2 or the probe optimized of the present invention respectively, the step of PCR reaction is: 94 ℃ 10 minutes, 45 circulations (94 ℃ 10 seconds, 60 ℃ 30 seconds), and detect fluorescent signals at 60 ℃.
Detected result is shown in Fig. 1 and table 1, under the same detection condition, the new amplification system (containing novel probe) that uses the present invention to optimize detects HBV (DNA) sample, the fluorescence background values that the feminine gender that obtains and positive detect is all lower, and the fluorescent signal value that positive obtains when detecting is big, its Δ Rn (difference of fluorescent signal value and fluorescence background) is worth high, Ct value (Ct value and the template number to be checked are inversely proportional to) minimum that real-time fluorescence detects, detection sensitivity height; Use contrast probe 2 to detect HBV (DNA) sample, though the fluorescence background values is similar to the present invention, the fluorescent signal value is less, and its Δ Rn value is very low, therefore when detecting low concentration sample, crosses low can't detecting because of Δ Rn value, has reduced detection sensitivity; Use contrast probe 1 to detect HBV (DNA) sample, the fluorescence background values is bigger, though the fluorescent signal value of its positive sample is higher, but reduced Δ Rn value because the fluorescence background values is too high, make low concentration sample be difficult for stable detecting, and too high fluorescence background is easy to generate non-specific fluorescent signal take-off, thereby has reduced detection specificity.As seen, the amplification system of optimization of the present invention has reached high specific and highly sensitive DNA detection effect when realizing high s/n ratio, particularly when detecting, low concentration sample can reach better reliability and stability, after tested, with respect to traditional method, the method for optimization of the present invention detects the DNA sample can improve sensitivity 5-10 doubly approximately.
Table 1 detects the comparison sheet as a result of HBV nucleic acid
The detection of embodiment 2RNA sequence
We have designed primer and probe at HCV NS2 gene, entrust six directions trade company limited of stimulating the menstrual flow in Beijing to synthesize following pcr amplification primer and probe (comprising the contrast probe), to contain HCV (s-generation HCV RNA country reference material, numbering 300012, can dilute with purified water according to the rules available from National Institute for Food and Drugs Control) 5 * 10
2IU/ml (positive), 2.5 * 10
2IU/ml (sensitivity 1) and 1 * 10
2The aqueous solution (positive) of IU/ml (sensitivity 2) or water (feminine gender) is respectively as template, carries out RT-PCR at ABI 7500 fluorescent PCR instrument (can available from Applied Biosystems company) and detects:
Forward primer: TCGCCATATTACAAGCGCTACA
Reverse primer: GCGCTTCTACTCTGGTCAGAAAA
The specific probe that traditional Taqman method is used (contrast probe 1): FAM-CATGTGGTGGCTTCA-ECLIPSE, 5 ' end of its nucleic acid is marked with fluorescence dye FAM, and 3 ' end is marked with quenching group ECLIPSE
The probe that traditional molecular beacon method is used (contrast probe 2): FAM-
CATGTGGTGGCTTCA
-ECLIPSE, 5 ' end of its nucleic acid is marked with fluorescence dye FAM, and 3 ' end is marked with quenching group ECLIPSE, and wherein italics partly is the complementary pairing sequence.
The probe that the present invention optimizes: FAM-
GGTGGCTTCA
-ECLIPSE, 5 ' end of its nucleic acid is marked with fluorescence dye FAM, and 3 ' end is marked with quenching group ECLIPSE, and wherein italics partly is the complementary pairing sequence, and boldface letter partly is extension sequence
The PCR reaction system:
In the above reaction system, template is got positive template, negative template or sensitivity 1,2 templates respectively, probe is got above-mentioned contrast probe 1,2 or the novel probe optimized of the present invention respectively, the step of PCR reaction is: 42 ℃ 30 minutes, 94 ℃ 10 minutes, 45 circulations (94 ℃ 10 seconds, 60 ℃ 30 seconds), and in the time of 60 ℃, detect fluorescent signal.
Detected result is shown in Fig. 2 and table 2, although passed through reverse transcription step, the DNA detection of itself and embodiment 1 is basically identical as a result.Under same testing conditions, the new amplification system (containing novel probe) that uses the present invention to optimize detects HCV (RNA) sample, the fluorescence background values that feminine gender and positive detect is all lower, and the fluorescent signal value that positive obtains when detecting is big, its Δ Rn value is high, Ct value (Ct value and the template number to be checked are inversely proportional to) minimum that real-time fluorescence detects, the detection sensitivity height; Use contrast probe 2) detect HCV (RNA) sample, though the fluorescence background values is similar to the present invention, the fluorescent signal value is less, its Δ Rn value is very low; Use contrast probe 1 to detect HCV (RNA) sample, the fluorescence background values is bigger.As seen, the amplification system of optimization of the present invention has reached high specific and highly sensitive DNA detection effect when realizing high s/n ratio, particularly when detecting, low concentration sample can reach better reliability and stability, after tested, with respect to traditional method, the method for optimization of the present invention detects the DNA sample can improve 2～5 times of sensitivity approximately.
Table 2 detects the comparison sheet as a result of HCV nucleic acid
Claims (43)
1. the detection kit of target nucleic acid in the sample, it comprises pcr amplification reagent and fluorescently-labeled probe, the nucleic acid moiety of its middle probe is made up of first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence to 3 ' end successively from 5 ' end, and first complementary sequence and second complementary sequence can complementary pairing, wherein the target nucleic acid specific sequence is the length of 8～20 bases, first complementary sequence is the length of 4～8 bases, second complementary sequence is that length and the extension sequence of 4～8 bases is AT.
2. the described test kit of claim 1, wherein pcr amplification reagent comprise can amplifying target nucleic acid sequence primer to, archaeal dna polymerase and dNTP.
3. the described test kit of claim 1, wherein pcr amplification reagent is the RT-PCR amplifing reagent, it comprise can amplifying target nucleic acid sequence primer to, archaeal dna polymerase, dNTP and ThermoScript II.
4. the described test kit of claim 3, wherein the RT-PCR amplifing reagent also comprises the RNase inhibitor.
5. claim 2 or 3 described test kits, wherein primer is to being CGAGGCAGGTCCCCTAGAA and CGGCGATTGAGACCTTCGT, or TCGCCATATTACAAGCGCTACA and GCGCTTCTACTCTGGTCAGAAAA.
6. the described test kit of claim 1, wherein the target nucleic acid specific sequence is the length of 9～15 bases.
7. the described test kit of claim 6, wherein the target nucleic acid specific sequence is the length of 10～12 bases.
8. the described test kit of claim 7, wherein the target nucleic acid specific sequence is TCCCTCGCCTCG or GGTGGCTTCA.
9. the described test kit of claim 1, wherein first complementary sequence is the length of 5～7 bases.
10. the described test kit of claim 9, wherein first complementary sequence is the length of 5 bases.
11. the described test kit of claim 10, wherein first complementary sequence is AGAAC or CATGT.
12. the described test kit of claim 1, wherein second complementary sequence is the length of 5～7 bases.
13. the described test kit of claim 12, wherein second complementary sequence is the length of 5 bases.
14. the described test kit of claim 13, wherein second complementary sequence is GTTCT or ACATG.
15. the application in the testing product of arbitrary described test kit method of target nucleic acid in for the preparation of test sample of claim 1～14.
16. the described application of claim 15, wherein the method for target nucleic acid comprises in the test sample:
(1) sample is mixed with pcr amplification reagent and fluorescently-labeled probe, the nucleic acid moiety of its middle probe is made up of first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence to 3 ' end successively from 5 ' end, and first complementary sequence and second complementary sequence can complementary pairing, wherein the target nucleic acid specific sequence is the length of 8～20 bases, first complementary sequence is the length of 4～8 bases, second complementary sequence is that length and the extension sequence of 4～8 bases is AT; With
(2) carry out PCR, and detect fluorescence in real time.
17. the described application of claim 16, wherein target nucleic acid is RNA or DNA.
18. the described application of claim 17, wherein target nucleic acid is HCV RNA or HBV DNA.
19. the described application of claim 18, wherein target nucleic acid is HBV S gene or HCV NS2 gene.
20. the described application of claim 16, wherein pcr amplification reagent comprise can amplifying target nucleic acid sequence primer to, archaeal dna polymerase and dNTP.
21. claim 16 or 20 described application, wherein PCR is RT-PCR.
22. the described application of claim 20, wherein pcr amplification reagent also comprises ThermoScript II.
23. the described application of claim 22, wherein pcr amplification reagent also comprises the RNase inhibitor.
24. the described application of claim 20, wherein primer is to being CGAGGCAGGTCCCCTAGAA and CGGCGATTGAGACCTTCGT, or TCGCCATATTACAAGCGCTACA and GCGCTTCTACTCTGGTCAGAAAA.
25. the described application of claim 16, wherein the target nucleic acid specific sequence is the length of 9～15 bases.
26. the described application of claim 25, wherein the target nucleic acid specific sequence is the length of 10～12 bases.
27. the described application of claim 26, wherein the target nucleic acid specific sequence is TCCCTCGCCTCG or GGTGGCTTCA.
28. the described application of claim 16, wherein first complementary sequence is the length of 5～7 bases.
29. the described application of claim 28, wherein first complementary sequence is the length of 5 bases.
30. the described application of claim 29, wherein first complementary sequence is AGAAC or CATGT.
31. the described application of claim 16, wherein second complementary sequence is the length of 5～7 bases.
32. the described application of claim 31, wherein second complementary sequence is the length of 5 bases.
33. the described application of claim 32, wherein second complementary sequence is GTTCT or ACATG.
34. the described application of claim 16, wherein PCR does not extend step.
35. the described application of claim 34, wherein the annealing temperature of PCR is 55～65 ℃.
36. the described application of claim 35, wherein the annealing temperature of PCR is 58～63 ℃.
37. the described application of claim 36, wherein the annealing temperature of PCR is 60 ℃.
38. the described application of claim 34, wherein the annealing time of PCR is 20～60 seconds.
39. the described application of claim 38, wherein the annealing time of PCR is 25～40 seconds.
40. the described application of claim 39, wherein the annealing time of PCR is 30 seconds.
41. the described application of claim 16, wherein testing product also comprises specification sheets and/or the fluorescent PCR instrument of the method for target nucleic acid in the described test sample of record.
42. probe, it is FAM-AGAACTCCCTCGCCTCGGTTCTAT-ECLIPSE, or FAM-CATGTGGTGGCTTCAACATGAT-ECLIPSE.
43. primer is right, it is CGAGGCAGGTCCCCTAGAA and CGGCGATTGAGACCTTCGT, or TCGCCATATTACAAGCGCTACA and GCGCTTCTACTCTGGTCAGAAAA.
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| 《A modified molecular beacon combining the properties of TaqMan probe》;De-Ming Kong等;《CHEM. COMMUN.》;20020320;第854－855页 * |
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