CN102534033A - High-sensitivity real-time fluorescence detection method - Google Patents
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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 complement and 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:
The Taqman probe (Taqman Probe) of the nineties invention in last century and molecular beacons technology (Molecular Beacon) 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 document CN101131347A just 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 document 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 Δ 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 Δ 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 deficiency of prior aries 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 primer wherein, probe.
Summary of the invention
The technical problem that the present invention will solve has been to provide new PCR in real time to detect the methods and applications of target nucleic acid.The present invention also provides and has been used for PCR in real time and detects 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 a 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 (like, 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 holds 3 ' end to be made up of first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence successively from 5 '.In an 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 possibly contain target nucleic acid, like food, blood, blood products, saliva, medical treatment product or medicine etc.The target that detection method of the present invention was directed against---target nucleic acid can be the nucleic acid that any suitable PCR in real time detects, and like gene or gene fragment, comprises 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 embodiment of the present invention, can detect the NS2 gene (RNA) of HCV (hepatitis C virus, it is a RNA viruses) and the S gene (DNA) of HBV (hepatitis B virus, it is a 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 target nucleic acid for HCV or HBV in the blood sample that utilizes detection method detection human or animal of the present invention; The existence that also can only directly draw target nucleic acid whether; Also need HCV or HBV that empirical doctor or sampling personnel judge that detected target nucleic acid pollutes when coming from HCV or HBV or the sampling in the blood accidentally, and can not directly obtain the diagnostic result or the healthy state of disease; Even the existence that target nucleic acid has characterized HCV and HBV whether; Because in 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 empirical doctor and just can judge whether to cause disease or influential, therefore can't judge directly whether its carrier suffers from third type or hepatitis B or ill risk is arranged, so directly purpose is not diagnosis according to the existence of this target nucleic acid to healthy state according to comprehensive conditions such as corresponding human or animal's physique, medical history, clinical symptom.So detection method of the present invention is not a 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 (like, 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, like commercial M-MLV ThermoScript II etc.In order to prevent RNA in the sample by fast degraded, the RT-PCR amplifing reagent can also comprise the RNase suppressor factor in addition, like commercial RNasin etc.There are many manufacturers can RT-PCR be provided directly 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 that be used to the to increase primer of HCVRNA or HBVDNA is right, and the primer that is more preferably be used to increase HBV S gene or HCVNS2 gene is right.In an 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 hold the order of 3 ' end to carry out according to 5 '; Each base on one of " complementary pairing " of nucleic acid expression or a part of single-chain nucleic acid from 5 ' hold 3 ' end successively with another or another part single-chain nucleic acid on each base hold 5 ' end complementary from 3 ', comprise that A and T are complementary, 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 an 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 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 an 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 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,, anneal, extend at about 72 ℃ at 50～65 ℃ carrying out sex change greater than 90 ℃.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 accomplish 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 holds 3 ' end to be made up of first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence successively from 5 '.In an 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.Further preferably in order to 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 suppressor factor.
Preferably in detection kit of the present invention, primer is to being that be used to the to increase primer of HCVRNA or HBVDNA is right, and the primer that is more preferably be used to increase HBV S gene or HCV NS2 gene is right.In an 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 an 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 an 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 embodiment of the present invention, extension sequence is AT.
In the third aspect, the test kit that the invention provides second aspect present invention is used for the application in the testing product of method of test sample target nucleic acid in preparation.Testing product comprises the test kit of second aspect present invention.The preferred detection product can further include other with test sample in the relevant goods of target nucleic acid.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 appearance, so the preferred detection product also comprises the fluorescent PCR appearance.
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.The preferably fluorescently-labeled probe of this probe can be used for the present invention first, second and the 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 an 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 an 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 embodiment of the present invention, extension sequence is AT.
Preferably hold 3 ' end to form by first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence successively from 5 ' at the nucleic acid moiety of probe of the present invention.In an 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 SNR, has improved the sensitivity and the specificity that detect; It is wide to detect the scope of using, can be to the detection of DNA and RNA; Shorten detection time, improve detection efficiency; Can use conventional fluorescent PCR appearance to carry out, practice thrift the detection cost; Detected result is reliably stablized, and is suitable for Industry Promotion and uses.
For the ease of understanding, below will describe in detail the present invention through concrete accompanying drawing, embodiment.What need particularly point out is 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 conspicuous 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 with template is: 1, and the probe of the present invention's optimization, 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 with template is: 1, the probe of the present invention's optimization, 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
Embodiment below in conjunction with concrete describes; Wherein synthetic, the reagent of primer, probe and sample all can obtain through the commercial channel; Experimental procedure is if any not using up part; All can be referring to " molecular cloning experiment guide " (third edition) teach book and laboratory manuals such as (Science Press, 2002), and can explain according to the manufacturer of business-like enzyme, reagent and equipment and carry out.
The detection of embodiment 1DNA sequence
We have designed primer and probe to HBV S gene order; Entrust six directions trade ltd of stimulating the menstrual flow in Beijing to synthesize following pcr amplification primer and probe (comprise and contrast probe); (hbv nucleic acid (HBV DNA) is qualitative and quantitatively national with reference to article to contain HBV; Numbering 300009 can be diluted 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 appearance (can available from Applied Biosystems company):
Forward primer: CGAGGCAGGTCCCCTAGAA
Reverse primer: CGGCGATTGAGACCTTCGT
The probe that traditional T aqman specificity method uses (contrast probe 1): FAM-AGAACTCCCTCGCCTCG-ECLIPSE, 5 ' end of its nucleic acid is marked with optical 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 optical dye FAM; 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 optical dye FAM; 3 ' end is marked with quenching group ECLIPSE; Wherein italics partly is the complementary pairing sequence, and boldface letter partly is an 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 the probe that above-mentioned contrast probe 1,2 or the present invention optimize respectively, and 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, and the fluorescence background values that 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, and detection sensitivity is high; Use contrast probe 2 to detect HBV (DNA) sample, though the fluorescence background values is similar with 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, reduced Δ Rn value because of 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.It is thus clear that; 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, low concentration sample can reach better reliability and stability when detecting; Through test, 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 to HCV NS2 gene; Entrust six directions trade ltd of stimulating the menstrual flow in Beijing to synthesize following pcr amplification primer and probe (comprise and contrast probe); (s-generation HCV RNA country is with reference to article to contain HCV; Numbering 300012 can be diluted 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, on ABI 7500 fluorescent PCR appearance (can available from Applied Biosystems company), carries out RT-PCR and detects:
Forward primer: TCGCCATATTACAAGCGCTACA
Reverse primer: GCGCTTCTACTCTGGTCAGAAAA
The specific probe that traditional T aqman method is used (contrast probe 1): FAM-CATGTGGTGGCTTCA-ECLIPSE, 5 ' end of its nucleic acid is marked with optical 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 optical dye FAM; 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 optical dye FAM; 3 ' end is marked with quenching group ECLIPSE; Wherein italics partly is the complementary pairing sequence, and boldface letter partly is an extension sequence
The PCR reaction system:
In the above reaction system; Template is got positive template, negative template or sensitivity 1,2 templates respectively, and probe is got the novel probe that above-mentioned contrast probe 1,2 or the present invention optimize respectively, and 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 height; Ct value (Ct value and the template number to be checked are inversely proportional to) minimum that real-time fluorescence detects, detection sensitivity is high; Use contrast probe 2) detect HCV (RNA) sample, though the fluorescence background values is similar with 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.It is thus clear that; 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, low concentration sample can reach better reliability and stability when detecting; Through test, 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 (16)
1. 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 pairing, the nucleic acid moiety of preferred probe holds 3 ' end to be made up of first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence successively from 5 '; With
(2) carry out PCR, and detect fluorescence in real time.
2. the described method of claim 1, wherein target nucleic acid is RNA or DNA, preferably HCV RNA or HBVDNA, for example HBV S gene or HCVNS2 gene.
3. the described method of claim 1, wherein pcr amplification reagent comprise can amplifying target nucleic acid sequence primer to, archaeal dna polymerase and dNTP.
4. claim 1 or 3 described methods, wherein PCR is RT-PCR; Preferably wherein pcr amplification reagent also comprises ThermoScript II, preferably also comprises the RNase suppressor factor.
5. the described method of claim 3, wherein primer is to being CGAGGCAGGTCCCCTAGAA and CGGCGATTGAGACCTTCGT, or TCGCCATATTACAAGCGCTACA and GCGCTTCTACTCTGGTCAGAAAA.
6. the described method of claim 1, wherein,
The target nucleic acid specific sequence is the length of 8～20 bases, is preferably the length of 9～15 bases, and more preferably the length of 10～12 bases most preferably is TCCCTCGCCTCG or GGTGGCTTCA;
First complementary sequence is respectively the length of 4～8 bases, is preferably the length of 5～7 bases, and more preferably the length of 5 bases most preferably is AGAAC or CATGT;
Second complementary sequence is respectively the length of 4～8 bases, is preferably the length of 5～7 bases, and more preferably the length of 5 bases most preferably is GTTCT or ACATG; And/or,
Extension sequence is the length of 2～5 bases, is preferably the length of 2～3 bases, and more preferably the length of 2 bases most preferably is AT.
7. the described method of claim 1, wherein PCR does not extend step, and the annealing temperature of preferred PCR is 55～65 ℃, more preferably 58～63 ℃, most preferably is 60 ℃; And/or the annealing time of preferred PCR is 20～60 seconds, more preferably 25～40 seconds, most preferably is 30 seconds.
8. 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 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 pairing, the nucleic acid moiety of preferred probe holds 3 ' end to be made up of first complementary sequence, target nucleic acid specific sequence, second complementary sequence and extension sequence successively from 5 '.
9. the described test kit of claim 8, wherein pcr amplification reagent comprise can amplifying target nucleic acid sequence primer to, archaeal dna polymerase and dNTP.
10. the described test kit of claim 8, 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, preferably also comprise the RNase suppressor factor.
11. claim 9 or 10 described test kits, wherein primer is to being CGAGGCAGGTCCCCTAGAA and CGGCGATTGAGACCTTCGT, or TCGCCATATTACAAGCGCTACA and GCGCTTCTACTCTGGTCAGAAAA.
12. the described test kit of claim 8, wherein
The target nucleic acid specific sequence is the length of 8～20 bases, is preferably the length of 9～15 bases, and more preferably the length of 10～12 bases most preferably is TCCCTCGCCTCG or GGTGGCTTCA;
First complementary sequence is respectively the length of 4～8 bases, is preferably the length of 5～7 bases, and more preferably the length of 5 bases most preferably is AGAAC or CATGT;
Second complementary sequence is respectively the length of 4～8 bases, is preferably the length of 5～7 bases, and more preferably the length of 5 bases most preferably is GTTCT or ACATG; And/or,
Extension sequence is the length of 2～5 bases, is preferably the length of 2～3 bases, and more preferably the length of 2 bases most preferably is AT.
13. the described test kit of claim 8～12 is used for the application in the testing product of method of test sample target nucleic acid in preparation.
14. the described application of claim 13, wherein the method for target nucleic acid is the described method of claim 1～7 in the test sample.
15. the described application of claim 13, testing product also comprise the specification sheets and/or the fluorescent PCR appearance of the described method of record claim 1～7.
16. probe or primer used in the claim 1～15 are right.
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| CN107746885A (en) * | 2016-09-26 | 2018-03-02 | 惠扬（上海）生物医药科技有限公司 | A kind of fluorescence probe that can be to the highly sensitive detection of nucleic acid amplification product and its application |
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2012
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| DAVID WHITCOMBE等: "《Detection of PCR products using self-probing amplicons and fluorescence》", 《NATURE BIOTECHNOLOGY》 * |
| DE-MING KONG等: "《A modified molecular beacon combining the properties of TaqMan probe》", 《CHEM. COMMUN.》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107746885A (en) * | 2016-09-26 | 2018-03-02 | 惠扬（上海）生物医药科技有限公司 | A kind of fluorescence probe that can be to the highly sensitive detection of nucleic acid amplification product and its application |
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