CN105301237A - Method for detecting nucleic acid by colloidal gold chromatography technology and reagent kit - Google Patents
Method for detecting nucleic acid by colloidal gold chromatography technology and reagent kit Download PDFInfo
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Abstract
The invention discloses a method for detecting nucleic acid by a colloidal gold chromatography technology and a reagent kit, and belongs to the technical field of medical biology. A universal test paper strip for nucleic acid detection is provided; colloidal gold particles are marked on a universal probe 1, and then, the universal probe is fixed on a glass cellulose membrane; the probe sequence is designed into a universal sequence; the test paper strip is fixed on a PVC (polyvinyl chloride) bottom plate; a sample pad, glass fiber, an NC membrane and water absorption paper are in sequential arrangement from the left side to the right side, wherein a T line (detection line) and a C line (quality control line) are arranged on the NC membrane; antibodies to labels b cover the detection line; antibodies to labels a cover the quality control line; the specificity probe A series and the specificity B series successfully combine a gold mark probe and nucleic acid amplification fragments in series; the specificity detection of nucleic acid amplification fragments is realized. The method has the advantages that the technical requirement on the experiment personnel is low; the required detection time is short; special instrument equipment is not needed; the popularization to basic levels and remote rural area medical institutions is easy.
Description
Technical field
The present invention relates to medical biotechnology, particularly a kind of method of colloidal gold chromatographic technology for detection nucleic acid and kit.
Background technology
Colloidal gold technique is the solid phase labelling immunoassay grown up after three large labelling techniques (fluorescein, radioactive isotope and enzyme) eighties in last century.Only for immunoelectronmicroscopy, but along with the development of biotechnology, there are immunodotting diafiltration and immunochromatography technique etc. now in initial the method.Colloidal gold immunochromatographimethod technology is widely used in clinical diagnosis, as the detection of early pregnancy, hepatitis B, parasite, venereal disease and virus and bacteria.The maximum feature of the method is cheap, simple and quick, special sensitivity, without any need for instrument and equipment and reagent, a few minutes just can observe with the naked eye the experimental result of color clear.
The detection kit overwhelming majority that the technological development of application colloidal gold immunochromatographimethod goes out on the market is now all detect certain antigen or antibody, and namely their detected object is all protein, applies almost not having of this technology for detection pathogen nucleic acid.When adopting gold label test strip detectable antigens or antibody, most employing be two sandwich method immunoassay principle, first colloid gold particle is marked on corresponding pathogen antigen or antigen, if have during detection corresponding antigens or antibody will with the protein combination of standard gold, bond moves forward along nitrocellulose membrane, the bag run on film will be formed golden labeling antibody-antigen-antibody complex and is fixed on this detection line during line, form macroscopic enrichment line.These exist a lot of not enough to detect the colloidal gold strip that albumen is target merely, a large amount of monoclonal antibody as required is expensive and manufacturing cycle is long, the specificity detected neither be very high, in shortening detection window phase, raising pathogen recall rate, also do not have advantage.
Nucleic acid detection technique has the features such as highly sensitive, high specificity compared with the Serologic detection of routine, and in the shortening detection window phase, improving in pathogen recall rate and have suitable advantage, is the important supplement to immunological detection method.
Nucleic acid detection technique conventional is at present polymerase chain reaction (PolymeraseChainReaction, PCR).In clinical practice, the amplified production obtained by round pcr is judge yin and yang attribute by the method for agarose gel electrophoresis according to the size of special band at first, such operation not only time-consuming, loaded down with trivial details, easy generation is polluted, and only judges that yin and yang attribute specificity is not high yet by amplified production size.Along with the develop rapidly of Protocols in Molecular Biology, round pcr is also with rapid changepl. never-ending changes and improvements, the particularly invention of quantitative fluorescent PCR (RealtimefluorescencequantitativePCR, RTFQPCR), is filled with new vitality to whole molecular diagnosis industry.Fluorescent quantitative PCR technique has the features such as highly sensitive, high specificity in clinical practice, but this technology proposes very high requirement to experimenter and instrument and equipment.A lot of fluorescent quantificationally PCR detecting kit all specifies special instrument and equipment on the market now, and these equipment prices are expensive, apply single; Meanwhile, kit operating personnel require experimental skill and the knowledge background of specialty, and like this, a lot of kit for detecting nucleic acid that application fluorescent quantitative PCR technique is derived just are difficult to spread to basic hospital, for the health of the people seeks the well-being of.
PCR is alternating temperature nucleic acid amplification technologies, with PCR unlike isothermal amplification technology in the last few years also gradually development and application get up.Isothermal amplification technology required temperature when increasing is constant, doing so avoids significantly floating up and down as temperature during pcr amplification.So isothermal amplification technology also reduces the requirement of instrument, general water-bath just can meet experiment condition.These nucleic acid constant-temperature amplification technology as: rely on the amplification technique (NucleicAcidSequence-basedAmplification of nucleotide sequence, NASBA), rely on unwindase amplification technique (Helicase-dependentAmplification, HDA), rolling circle amplification (RollingCircleAmplification, RCA), loop-mediated isothermal amplification technique (Loop-mediatedIsothermalAmplification, LAMP), the amplification technique (TranscriptionMediatedAmplification, TMA) etc. of transcriptive intermediate.These technology realize the amplification of specific nucleic acid DNA or RNA according to different principle (as 42 DEG C) under a certain specific temperature conditions.No matter be adopt which kind of isothermal amplification technology, amplified production all needs to realize qualitative detection by certain technological means.As, someone introduces the detection that molecular beacon (molecularbeacon) probe realizes constant-temperature amplification product.But molecular beacon extremely unstable, high especially to solion environmental requirement, and the detection of molecular beacons signals also needs high end instrument, is unfavorable for popularizing.
How the advantage quick, simple to operate of colloidal gold chromatographic technology is applied to detection of nucleic acids? 1996, Mirkin seminar of Northwestern Univ USA utilizes the character that can form stable Au-S key between nm of gold and sulfydryl to prepare nm of gold-DNA composite Nano probe, can be used for detecting DNA, the Au-S key that nm of gold and sulfydryl are formed is firmly covalent bond.The people such as BrittanyA.Rohrman successfully have detected HIV viral nucleic acid amplified production by colloidal gold immunochromatographimethod technology.They are when testing, devise three kinds of probes altogether, a kind of probe mark colloid gold particle, HIV specific amplified product is handed over as specific probe impurity elimination, other two kinds of nucleic acid probes are coated on detection line and nature controlling line respectively, be used for catching HIV nucleic acid amplification product---gold mark nucleic acid probe compound and free gold mark nucleic acid probe, thus form T line and C line.This is the once successful example that collaurum detects nucleic acid.But nucleic acid probe being coated on film and going to catch HIV nucleic acid amplification product as single capture probe---gold mark nucleic acid probe compound efficiency is not high, liquid (goldenhancementsolution) must be strengthened by gold colour developing and increase colour developing, improve sensitivity.Collaurum has relevant report in the detection of DNA chip, normally by upper for target molecule mark sulfydryl, surface because of colloid gold particle surrounds the more weak charged part of one deck adhesion as citric acid, the mercapto groups that these ligand moleculars are easy to combined power strong replaced, thus collaurum can be connected upper target nucleic acid, then the Probe Hybridization on the target molecule of mark and chip be realized the detection to it.But this method complex operation, and each hybridization all needs to mark special target molecules, does not possess versatility.
Summary of the invention
For solving problems of the prior art, the invention provides a kind of collaurum mark detection kit detecting nucleic acid, comprising:
(1), general colloidal gold strip: test strips used is fixed on PVC base plate, is sample pad, glass fibre, NC film, thieving paper from left to right successively; NC film has T line (detection line) and C line (nature controlling line), described detection line wraps by the material of energy specific binding label b, on described nature controlling line, bag is by the molecule of energy specific binding label a, and described label b is different from label a; General probe 1 be marked with colloid gold particle or be marked with label a at the other end simultaneously, and being fixed on the glass fibre element film of test strips;
(2) probe tube of specific probe A series, specific probe B series, is filled respectively: one end of specific probe A series and determined nucleic acid hybridization, and be marked with label a, the other end and general probe 1 are hybridized, or when general probe 1 is not only marked with colloid gold particle but also is marked with label a, specific probe A series is not with any mark, and specific probe A series can have multiple, as A1, A2 ... hybridize with the zones of different complementary pairing of determined nucleic acid respectively, be contained in different probe tubes; Specific probe B series one end and determined nucleic acid hybridization, the other end is marked with label b, or when also comprising the general probe 2 with label b, its simultaneously and general probe 2 complementary pairing hybridize, now specific probe B series is not with any mark, and specific probe B series can have multiple, as B1, B2 ... hybridize with the zones of different complementary pairing of determined nucleic acid respectively, be contained in respectively in different probe tubes.
Described label a, b can be antigen or haptens, as being biotin, digoxin or fluorescent dye (as Cy3, Cy5, Fam, Fit etc.), then the T line in test strips or C line are Streptavidin, anti digoxin antibody, corresponding anti-fluorescent dye antibody to the molecule of their specific bindings.
Described general probe 1 may be used for the detection of all pathogen nucleic acid fragments, must focus on the ratio of GC%, avoiding some non-specific bindings with gold grain as far as possible when designing; General probe 1 and general probe 2 also must be noted that the Tm value with other probe joint sub-sequences, improve the validity of hybridizing under lower temperature conditions as far as possible, and through the comparison of experiment effect, general probe 1 sequence announced is optimal design sequence.Described general probe 1 sequence is:
5-SH-CATCTTCCAGCGGCCTTATGCAGTTGCTCTCCATTTTTAGAAGGCGTCCGT CTTTGAGGC-3(SeqNo.1), wherein SH is sulfydryl, and 5 ' end is sulfydryl modification, also can carry out other chemical group modifications, as-NH
2deng.
The invention provides a kind of nucleic acid gold-labeled rapid detection method, by simple to operate, quick, the detection that lower-price characteristic is applied to nucleic acid of colloidal gold immunochromatographimethod technology.Comprise the steps:
(1), designing probe: general probe 1, specific probe A series, specific probe B series, wherein, 5 ' end sulfhydrylation of general probe 1 is modified and (also can be carried out other chemical group modifications, as-NH
2deng) mark colloid gold particle afterwards, or be marked with label a at the other end simultaneously; Specific probe A series, its one end and determined nucleic acid hybridization, the other end and general probe 1 are hybridized, be marked with label a, or when general probe 1 is not only marked with colloid gold particle but also is marked with label a, specific probe A series is not with any mark, and specific probe A series can have multiple, as A1, A2 ..., hybridize with the zones of different complementary pairing of determined nucleic acid respectively; Specific probe B series one end and determined nucleic acid hybridization, the other end is marked with label b, or when kit also comprises the general probe 2 with label b, its simultaneously and general probe 2 complementary pairing hybridize, now specific probe B series is not with any mark, specific probe B series can have multiple, as B1, B2 ..., hybridize with the zones of different complementary pairing of determined nucleic acid respectively;
(2), preparing universal test paper bar: described test strips is fixed on PVC base plate, is sample pad, glass fibre, NC film, thieving paper from left to right successively; NC film there are T line (detection line) and C line (nature controlling line); Described detection line wraps by the antibody of anti-label b, specificity can catch the general probe 2 of binding label b or the specific probe B series of binding label b; Described nature controlling line wraps by anti-label a, specificity can catch the specific probe A of binding label a or be not only marked with colloid gold particle but also be marked with the general probe 1 of label a; General probe 1 marked upper colloid gold particle or be fixed on glass fibre element film with after tense marker subscript note thing a; chromatography in sample pad is dropped in by specific probe A, specific probe B or after adding general probe 2 and determined nucleic acid hybridization simultaneously; detection line colour developing indicates that determined nucleic acid exists, and nature controlling line colour developing represents that detection effectively.
Described label b and label a can be digoxin, biotin and other antigen or haptens, as fluorescent dye (Cy3, Cy5, Fam, Fit), be then corresponding anti digoxin antibody, Streptavidin or anti-fluorescent dye antibody to the molecule of their specific bindings on test strips T line or C line.
Described specific probe A series or general probe 1 mark digoxin, and specific probe B series or general probe 2 mark biotin, then wrap by Streptavidin at test strips T line place, and C line place bag is by anti digoxin antibody.
Described general probe 1 may be used for the detection of all pathogen nucleic acid fragments, must focus on the ratio of GC%, avoiding some non-specific bindings with gold grain as far as possible when designing; General probe 1 and general probe 2 also must be noted that the Tm value of dividing with other probe joint, improve the validity of hybridizing under lower temperature conditions as far as possible, and through the comparison of experiment effect, general probe 1 sequence announced is optimal design sequence.Described general probe 1 sequence is: 5-SH-CATCTTCCAGCGGCCTTATGCAGTTGCTCTCCATTTTTAGAAGGCGTCCGT CTTTGAGGC-3(SeqNo.1), and wherein SH is sulfydryl, and 5 ' end is sulfydryl modification, also can carry out other chemical group modifications, as-NH
2(amino) etc.
In conjunction with principle of work, the course of work of said method of the present invention is described below:
1, design four kinds of probes: general probe 1, general probe 2, specific probe A(can have multiple, as A1, A2......), specific probe B(can have multiple, as B1, B2......).Wherein, general probe 1 sulfhydrylation marks colloid gold particle after modifying, and can hybridize with specific probe A complementary pairing; General probe 2 marks biotin (Biotin also can be other materials, as digoxin or fluorescent dye, as Cy3, Cy5, Fam, Fitc etc.), and can hybridize with specific probe B complementary pairing; Specific probe A series of markings digoxin (also can be other materials, as biotin or fluorescent dye, as Cy3, Cy5, Fam, Fitc etc.), its one end can be hybridized with determined nucleic acid fragment, and the other end also can be hybridized with general probe 1; Specific probe B series one end can be hybridized with determined nucleic acid fragment, and the other end also can be hybridized with general probe 2.
2, in test strips, " T line " (detection line) place bag, by Streptavidin (this place can also wrap by other materials, but this material can combine with the label on general probe 2), can catch the biotin on general probe 2, form T line; " C line " (nature controlling line) place bag (also can be other materials by DigiTAb, as antibody, but this material must combine with the material of specific probe A series of markings) be used for catching the free general probe 1--specific probe A series compound indicating collaurum, form C line.
3, after general probe 1 marks colloid gold particle, can with specific probe A serial hybridization, form gold grain general probe 1--specific probe A digoxin compound.
4, indicate biotin general probe 2 can with specific probe B serial hybridization, form biotin general probe 2--specific probe B compound.
5, when there being nucleic acid fragment to be measured, determined nucleic acid fragment can with 4 in complex hybridization combine, formed nucleic acid fragment--specific probe B--biotin general probe 2 compound.
6, by formed in 5 determined nucleic acid fragment--the gold grain general probe 1--specific probe A digoxin compound formed in specific probe B--biotin general probe 2 compound and 3 carries out hybridization and combines, formation gold grain general probe 1--specific probe A digoxin--determined nucleic acid fragment--specific probe B--biotin general probe 2 compound.
7, the colloidal gold composite obtained in step 6 on NC film by capillarity along tunica fibrosa dialysis forward, when arrival T line place, be combined with the Streptavidin of T line place bag quilt, thus the compound obtained in 6 is trapped on T line, form macroscopic coloured band, this is positive (as Fig. 1).
8, when determined nucleic acid fragment does not exist, 5 ~ 6 steps would not be there are, just can not form gold grain general probe 1--specific probe A digoxin--determined nucleic acid fragment--specific probe B--biotin general probe 2 compound, colloid gold particle just can not be assembled at T line place, would not form macroscopic band, this is negative (as Fig. 2).
9, no matter with or without determined nucleic acid fragment, the gold grain general probe 1--specific probe A digoxin compound formed in step 3 all can have more than needed, then unnecessary gold grain general probe 1--specific probe A digoxin compound can be crossed T line and continue along tunica fibrosa flow forward, when arriving C line, be combined with the DigiTAb of C line place bag quilt, thus be trapped in C line place, form macroscopic coloured band, this is that experimental result is effective.(as shown in Figure 1)
Based on the thinking that said method is similar, the present invention also provides the detection nuclei aoid methods of another single general probe, and its principle of work is as follows:
1, design three kinds of probes: general probe 1, specific probe A(can have multiple, as A1, A2......), specific probe B(can have multiple, as B1, B2......).Wherein, general probe 1 marks colloid gold particle, and can hybridize with the pairing of specific probe A complementary series; Specific probe A series of markings digoxin (also can be other materials, as biotin or fluorescent dye, as Cy3, Cy5, Fam, Fitc etc.), its one end can be hybridized with determined nucleic acid fragment, and the other end also can be hybridized with general probe 1; Specific probe B series of markings biotin(also can be other materials, as digoxin or fluorescent dye, as Cy3, Cy5, Fam, Fitc etc.), it can combine with the hybridization of determined nucleic acid fragment.
2, on NC film, " T line " (detection line) place bag, by Streptavidin (this place can also wrap by other materials, but this material can combine with the label in specific probe B series), can catch the biotin in specific probe B series, form T line; " C line " (nature controlling line) place bag (also can be other materials by DigiTAb, as antibody, but this material must combine with the material of specific probe A series of markings) be used for catching the free general probe indicating collaurum--specific probe A series compound, forms C line.
3, after general probe 1 marks colloid gold particle, can with specific probe A serial hybridization, form gold grain general probe 1--specific probe A digoxin compound.
4, when there being nucleic acid fragment to be measured, determined nucleic acid fragment can combine with the specific probe B serial hybridization indicating biotin, forms determined nucleic acid fragment--specific probe B--biotin compound.
5, by formed in 4 determined nucleic acid fragment--the gold grain general probe 1--specific probe A digoxin compound formed in specific probe B--biotin compound and 3 carries out hybridization and combines, formation gold grain general probe 1--specific probe A digoxin--determined nucleic acid fragment--specific probe B--biotin compound.
6, the colloidal gold composite obtained in step 5 in the solution by capillarity along tunica fibrosa flow forward, when flowing to T line place, be combined with the Streptavidin of T line place bag quilt, thus the compound obtained in 5 is trapped on T line, form macroscopic coloured band, this is positive (as Fig. 3).Or,
7, when determined nucleic acid does not exist, 4 ~ 6 steps would not be there are, just can not form gold grain general probe 1--specific probe A digoxin--determined nucleic acid--specific probe B--biotin compound, colloid gold particle just can not be assembled at T line place again, would not form macroscopic band, this is negative (as Fig. 4).
8, no matter with or without determined nucleic acid, in step 3 formed gold grain general probe--specific probe A digoxin compound all can have more than needed, then unnecessary gold grain general probe 1--specific probe A digoxin compound can be crossed T line and continue along tunica fibrosa flow forward, when arriving C line, be combined with the DigiTAb of C line place bag quilt, thus be trapped in C line place, form macroscopic coloured band, this is that experimental result is effective.(as shown in Figure 3)
Based on the thinking that said method is similar, the present invention also provides the detection nuclei aoid methods of another single general probe, and its principle of work is as follows:
1, design three kinds of probes: general probe 1, specific probe A(can have multiple, as A1, A2......), specific probe B(can have multiple, as B1, B2......).Wherein, general probe 1 marks colloid gold particle after marking digoxin (also can be other materials, as biotin or fluorescent dye, as Cy3, Cy5, Fam, Fitc etc.) again, and can hybridize with the pairing of specific probe A complementary series; Its one end of specific probe A series can be hybridized with determined nucleic acid, and the other end also can be hybridized with general probe 1; Specific probe B series of markings biotin(also can be other materials, as digoxin or fluorescent dye, as Cy3, Cy5, Fam, Fitc etc.), can hybridize with determined nucleic acid.
2, on NC film, " T line " (detection line) place bag, by Streptavidin (this place can also wrap by other materials, but this material can combine with the label in specific probe B series), can catch the biotin in specific probe B series, form T line; " C line " (nature controlling line) place bag is used for catching the free general probe 1 indicating collaurum by DigiTAb (can be also other materials, as antibody, but this material must combine with the material of general probe mark), forms C line.
3, general probe mark colloid gold particle after, can with specific probe A serial hybridization, formed digoxin--gold grain general probe 1--specific probe A compound.
4, when there being determined nucleic acid, determined nucleic acid can combine with the specific probe B serial hybridization indicating biotin, forms determined nucleic acid--specific probe B--biotin compound.
5, determined nucleic acid--digoxin formed in specific probe B--biotin compound and 3--the gold grain general probe 1--specific probe A compound formed in 4 is carried out hybridization to combine, form digoxin--gold grain general probe 1--specific probe A--determined nucleic acid--specific probe B--biotin compound.
6, the colloidal gold composite obtained in step 5 in the solution by capillarity along tunica fibrosa flow forward, when flowing to T line place, the Streptavidin of biotin and the T line place bag quilt in specific probe B series combines, thus the compound obtained in 5 is trapped on T line, form macroscopic coloured band, this is positive (as Fig. 5).Or,
7, when determined nucleic acid does not exist, 4 ~ 5 steps would not be there are,--gold grain general probe 1--specific probe A--determined nucleic acid--the specific probe B--biotin compound that just can not form digoxin, colloid gold particle just can not be assembled at T line place again, would not form macroscopic band, this is negative.(as Fig. 6)
8, no matter with or without determined nucleic acid, digoxin--gold grain general probe 1 all can have more than needed, then unnecessary digoxin--gold grain general probe 1 can be crossed T line and continue along tunica fibrosa flow forward, when arriving C line, be combined with the DigiTAb of C line place bag quilt, thus be trapped in C line place, form macroscopic coloured band, this is that experimental result is effective.(as shown in Figure 5)
Nucleic acid method for quick of the present invention, combines the advantage such as quick, easy that colloidal gold technique detects cleverly.The maximum feature of the inventive method is colloid gold particle to be directly marked on nucleic acid probe, and the nucleic acid probe sequence of mark is designed to universal sequence, and this universal sequence directly and the combination of specific probe sequence.The introducing of general probe can directly apply to the detection of different pathogens nucleic acid, avoiding problems the trouble all probe will being carried out standard gold when detecting different pathogens nucleic acid for often kind of pathogen, after having had the general probe of standard gold, only need design different specific probes when detecting different pathogens.The general probe 2 of mark biotin also has the phase same-action of the general probe 1 of above-mentioned mark collaurum.The label such as biotin, digoxin (being referred to as antigen or haptens) is also introduced in the present invention.Biotin can combine with Streptavidin, and digoxin can combine with anti digoxin antibody, both specific binding, and adhesion is strong.At detection line and nature controlling line place bag by the antibody of anti-label, form the universal test paper bar detecting nucleic acid together with the general probe 1 of mark collaurum, thus raise the efficiency and reduce costs.In addition, the introducing of these antigens or hapten-marked thing, successfully changed into by nucleic acid hybridization compound (specific probe-pathogen nucleic acid-specific probe compound) and have immunogenic molecular complex, this has immunogenic molecular complex and just can be detected by universal test paper bar delicately.
The present invention is when detecting a kind of pathogen nucleic acid, and specific probe, i.e. specific probe A and specific probe B are overlapped in design two.The use of two cover probes, wherein any a set of probe can not be hybridized with pathogen nucleic acid coupling, all can not form the compound detected by test strips, also just not occur positive test symbol, ensure that the specificity of detection.Wherein often overlap probe and can design more than two, such design is conducive to again the sensitivity improving test strips.The specific probe A introduced during design in the present invention and specific probe B has the effect of bridging molecule composition, and gold is successfully marked probe and nucleic acid fragment series combination to be checked to one piece by two kinds of probes, realizes the specific detection of nucleic acid fragment.The introducing of bridging molecule avoids gold mark probe direct impurity elimination knot and closes nucleic acid fragment to be checked, like this can according to the zones of different of nucleic acid fragment to be checked, some specific probes of many designs A, realizes a nucleic acid fragment to be checked in conjunction with many gold mark probes, greatly improves detection sensitivity.
The present invention can detect the current techique of nucleic acid specificity amplified matter as one, apply the detection that detection method of the present invention and detection kit can be widely used in various pathogen nucleic acid.Common pathogen has parasite, fungi, bacterium, virus, mycoplasma and Chlamydia etc., as long as design different specific detection probes for these pathogen, can detect by this method.Because this method detects the versatility of nucleic acid, naturally also have and apply very widely.The inventive method can be applied to the detection of Clinic infectivity disease pathogen, as caused pathogen (as influenza virus, syncytial virus, mycoplasma pneumoniae, Chlamydia pneumoniae etc.), the sexual reverse (as AIDS virus, gonococcus etc.) and hand-foot-and-mouth disease pathogen (as enterovirus) etc. of breathing problem.The inventive method and test strips can be applied to the every aspects such as agricultural, animal husbandry, import and export inspection and quarantine, food industry, antenatal exaination and forensic identification.
Direct for general nucleic acid standard gold technology is applied to the detection of nucleic acid specificity amplified matter by the present invention first, provides universal test paper bar, has the advantage of its uniqueness:
Detection specificity is strong: the inventive method introduces two cover specific detection probes, can accurately detect nucleic acid amplification thing;
Highly sensitive: the specific detection probe that the inventive method is introduced has two covers, often overlap specific probe and can design more than two, nucleic acid detection test strip sensitivity is improved greatly;
Simple to operate, technical requirement is low: only need drip on detection of nucleic acids test paper by detection thing specific nucleic acid amplified matter, low to experimenter's technical requirement, can be widely used in basic unit and remote countryside medical institutions;
Result interpretation is simple, without the need to specific apparatus: by ELISA test strip nucleic acid, can direct visual results, result interpretation intuitively, without the need to by specific apparatus;
Testing result is quick, consuming time short: by ELISA test strip nucleic acid, only needs about 10min to carry out result interpretation.
Low price, testing cost is low: though the method is for detecting nucleic acid, expense is low more than the expense of existing fluorescent quantitation and genetic chip.
Accompanying drawing explanation
Fig. 1 is for detecting the principle schematic of determined nucleic acid positive findings by colloidal gold chromatographic technology (general probe 1,2, specific probe A, B)
Fig. 2 is for detecting the principle schematic of determined nucleic acid negative findings by colloidal gold chromatographic technology (general probe 1,2, specific probe A, B)
Fig. 3 is for detecting the principle schematic of determined nucleic acid positive findings by colloidal gold chromatographic technology (only marking the general probe 1 of gold grain, specific probe A, B)
Fig. 4 is for detecting the principle schematic of determined nucleic acid negative findings by colloidal gold chromatographic technology (only marking the general probe 1 of gold grain, specific probe A, B)
Fig. 5 is for detecting the principle schematic of determined nucleic acid positive findings by colloidal gold chromatographic technology (not only marked digoxin but also marked general probe 1, specific probe A, B of gold grain)
Fig. 6 is for detecting the principle schematic of determined nucleic acid negative findings by colloidal gold chromatographic technology (not only marked digoxin but also marked general probe 1, specific probe A, B of gold grain)
Fig. 7 is the assembling assumption diagram of detection of nucleic acids universal test paper bar
Fig. 8 is, negative findings figure positive with colloidal gold chromatographic technology for detection Respiratory Syncytial Virus(RSV) nucleic acid
Fig. 9 is, negative findings figure positive with colloidal gold chromatographic technology for detection parainfluenza virus III type nucleic acid
Figure 10 is, negative findings figure positive with colloidal gold chromatographic technology for detection Chlamydia pneumoniae nucleic acid.
Embodiment
The features and advantages of the invention can be understood further by reference to the accompanying drawings by following detailed description.The embodiment provided is only the explanation to the inventive method, and does not limit the present invention in any way all the other contents of announcement.
The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, the condition as described in " molecular cloning: lab guide " the 3rd edition (NewYork:ColdSpringHarborlaboratoryPress, 2005) is carried out.
[embodiment 1] general labeled nucleic acid probe colloid gold particle
1, the general probe sequence of sulfhydrylation is designed:
5’-SH-CATCTTCCAGCGGCCTTATGCAGTTGCTCTCCATTTTTAGAAGGCGTCCGTCTTTGAGGC-3’，（SeqNo.1）
2, the general probe designed (final concentration 0.1mM) is joined TCEP-HCl(final concentration 100mM) in, reduction sulfhydrylation DNA general probe;
3, joined in the collaurum liquid of 30nm diameter particle by the general probe after process, ambient temperature overnight is incubated
Educate.
4, adding 2%SDS liquid makes its final concentration be 0.01%, incubated at room 30min.
5, in solution, dropwise add the NaCl of 2M, be 0.15M to final concentration.
6, the centrifugal 15min of centrifugal purification gold mark nucleic acid probe: 15000rpm, precipitation cleansing solution (0.15M
NaCl, 0.01%SDS) wash four times, collaurum precipitation be resuspended in re-suspension liquid (0.15MNaCl, 5%BSA,
0.25%Tween, 10% sucrose) in, the general labeled nucleic acid probe collaurum that both must mark
Grain.
The preparation of [embodiment 2] nucleic acid detection test strip
The main raw material(s) needed when preparing nucleic acid detection test strip: glass fibre membrane, nitrocellulose filter (NC film), sample pad, PVC base plate etc.
1, the preparation of colloidal gold pad: glass fibre membrane is cut into the square little module of 0.5 × 1cm, each module evenly drips the standard gold nucleic acid probe liquid of 10 μ l with rifle, room temperature makes it dry, and Seal and preservation is for subsequent use.
2, film is sprayed:
Detection line (T line): Avidin (about 0.5 ~ 1.0mg/ml), spray film amount: 1.5 ~ 3 μ l/cm;
Nature controlling line (C line): anti digoxin antibody (about 0.5 ~ 1.0mg/ml), spray film amount: 1.5 ~ 3 μ l/cm;
After spray film, film bar is put in 37 DEG C of clean constant temperature oven inner dryings 5 ~ 6 hours, is stored in dry environment for subsequent use.
3, test strips assembling
Cut the long thieving paper of 2cm respectively, wrap and be fixed on successively from top to bottom on PVC base plate by the glass fibre membrane of good NC film, ADSORPTION OF GOLD mark probe, sample pad, test strip.The assembling assumption diagram of test strips is as Fig. 7.
[embodiment 3] general purpose core acid probe direct standard gold method (two general probe), detects Respiratory Syncytial Virus(RSV) (RSV) T7 linear amplification product
The primer sequence of amplification RSV nucleic acid:
R primer: 5 '-TAATACGACTCACTATAGGGAGAATGCAGGTGTAACTACACCTGTAAG-3 '
F primer: 5 '-GTGGTAATTGTACTACATATGCTAAG-3 '
The specific probe A sequence detecting RSV is:
5’-Dig-ATCATTGATTAATGATTTTTGCCTCAAAGACGGACGCCTTCT-3’
Detect specific probe B1, B2(two of RSV) be:
5’-TTAACATATAAGTGCTTTTTGGCCTCTAAGTCGTAGCCCA-3’
5’-TACAGGTGTAGTTACATTTTGGCCTCTAAGTCGTAGCCCA-3’
General probe 2 sequence indicating biotin is:
5’-Biotin-TGGGCTACGACTTAGAGGCC-3’
A, T7 linear amplification Respiratory Syncytial Virus(RSV) nucleic acid:
| Component | Volume (μ l) |
| Respiratory Syncytial Virus(RSV) nucleic acid (or lysate) | 2 |
| Amplification reaction solution: containing dNTPs, NTPs, primer R&F and various salt ion | 17 |
| Add up to | 19 |
95 DEG C, 2min, adds amplification enzyme mixation (AMV & T7 polymerase & RNaseH) 1 μ l by 42 DEG C after 2min, after 42 DEG C of reaction 45min, to be checked.
B, the Respiratory Syncytial Virus(RSV) specific amplified product detected in a:
Amplified production: 10 μ l
Indicate the general probe 2(1 μM of biotin): 1 μ l
Indicate the specific probe A(5 μM of Dig): 1 μ l
Specific probe B1(5 μM): 1 μ l
Specific probe B2(5 μM): 1 μ l
Chromatographic solution (4 × SSC containing 5% formamide) is to cumulative volume 100 μ l
Observations (as Fig. 8) after 5 ~ 10min is put in test strips after 42 DEG C of 10min.
[embodiment 4] general purpose core acid probe direct standard gold method (single general probe), detects human parainfluenza viruses (PIV3) T7 linear amplification product
The primer sequence of amplification PIV3 nucleic acid:
R primer: 5'-TAATACGACTCACTATAGGGAGAACAACAATGCATACAATATTGTC-3 '
F primer: 5 '-GATTAATTCTGTGATTGTCTCT-3 '
The specific probe A sequence detecting PIV3 is:
5’-Dig-GTCTCTTTGATTGTTTTTTTGCCTCAAAGACGGACGCCTTCT-3’
Detect specific probe B1, B2(two that PIV3 indicates biotin) be:
5’-Biotin-TGGGCTACGACTTAGAGGCCTTTTGGAGGATACTGTCATG-3’
5’-Biotin-TGGGCTACGACTTAGAGGCCTTTTATATTCGTTTTCATAC-3’
A, T7 linear amplification human parainfluenza viruses (PIV3) nucleic acid:
| Component | Volume (μ l) |
| PIV3 nucleic acid (or lysate): | 2 |
| Amplification reaction solution: containing dNTPs, NTPs, primer R&F and various salt ion | 17 |
| Add up to | 19 |
95 DEG C, 2min, adds amplification enzyme mixation (AMV & T7 polymerase & RNaseH) 1 μ l by 42 DEG C after 2min, after 42 DEG C of reaction 45min, to be checked.
B, the people PIV3 specific amplified product detected in a
Amplified production: 10 μ l
Indicate the specific probe A(5 μM of Dig): 1 μ l
Indicate the specific probe B1(1 μM of biotin): 1 μ l
Indicate the specific probe B2(1 μM of biotin): 1 μ l
Chromatographic solution (4 × SSC containing 5% formamide) is to cumulative volume 100 μ l
Observations (as Fig. 9) after 5 ~ 10min is put in test strips after 42 DEG C of 10min.
The general probe direct standard gold method (single general probe) of digoxin on [embodiment 5] mark, detects Chlamydia pneumoniae (Cpn) T7 linear amplification product
The primer sequence of amplification Cpn nucleic acid:
R primer: 5 '-TAATACGACTCACTATAGGGAGAACACTACATTCGGTATTAGCGATC-3 '
F primer: 5 '-CTGAGAATTTGATCTTGGTTCAGAT-3 '
The gold mark general probe of digoxin on mark:
5’-SH-CATCTTCCAGCGGCCTTATGCAGTTGCTCTCCATTTTTAGAAGGCGTCCGTCTTTGAGGC-Dig-3’
The specific probe A sequence detecting Cpn is:
5’-CTGAGAATTTGATCTTTTTTGCCTCAAAGACGGACGCCTTCT-3’
Detect specific probe B1, B2(two that Cpn indicates biotin) be:
5’-Biotin-TGGGCTACGACTTAGAGGCCTTTTGAATAATGACTTCGGT-3’
5’-Biotin-TGGGCTACGACTTAGAGGCCTTTTAAGGGTTAGTAATACA-3’
A, T7 linear amplification Chlamydia pneumoniae nucleic acid:
| Component | Volume (μ l) |
| Chlamydia pneumoniae nucleic acid (or lysate) | 2 |
| Amplification reaction solution: containing dNTPs, NTPs, primer R&F and various salt ion | 17 |
| Add up to | 19 |
95 DEG C, 2min, does not add amplification enzyme mixation (AMV & T7 polymerase & RNaseH) 1 μ l by 42 DEG C after 2min, respectively after 42 DEG C of reaction 45min, to be checked.
B, detect the Chlamydia pneumoniae specific amplified product obtained in a by the direct standard gold method of general purpose core acid probe:
Amplified production: 10 μ l
Specific probe A(5 μM): 1 μ l
Indicate the specific probe B1(1 μM of biotin): 1 μ l
Indicate the specific probe B2(1 μM of biotin): 1 μ l
Chromatographic solution (4 × SSC containing 5% formamide) is to cumulative volume 100 μ l
Observations (as Figure 10) after 5 ~ 10min is put in test strips after 42 DEG C of 10min.
SEQUENCELISTING
Zhong Zhi biotech inc, <110> Wuhan
<120> mono-kind method of colloidal gold chromatographic technology for detection nucleic acid and kit
<130>1
<160>17
<170>PatentInversion3.3
<210>1
<211>60
<212>DNA
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<220>
<223> general probe 1
<220>
<221>misc_feature
<222>(1)..(1)
<223>5' end is sulfydryl modification or amido modified
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catcttccagcggccttatgcagttgctctccatttttagaaggcgtccgtctttgaggc60
<210>2
<211>48
<212>DNA
<213>Artificial
<220>
The R primer of <223> amplification RSV nucleic acid
<400>2
taatacgactcactatagggagaatgcaggtgtaactacacctgtaag48
<210>3
<211>26
<212>DNA
<213>Artificial
<220>
The F primer of <223> amplification RSV nucleic acid
<400>3
gtggtaattgtactacatatgctaag26
<210>4
<211>42
<212>DNA
<213>Artificial
<220>
<223> detects the specific probe A of RSV
<220>
<221>misc_feature
<222>(1)..(1)
<223>5' holds Dig to modify
<400>4
atcattgattaatgatttttgcctcaaagacggacgccttct42
<210>5
<211>40
<212>DNA
<213>Artificial
<220>
<223> detects the specific probe B1 of RSV
<400>5
ttaacatataagtgctttttggcctctaagtcgtagccca40
<210>6
<211>40
<212>DNA
<213>Artificial
<220>
<223> detects the specific probe B2 of RSV
<400>6
tacaggtgtagttacattttggcctctaagtcgtagccca40
<210>7
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<212>DNA
<213>Artificial
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<223> indicates the general probe 2 of biotin
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tgggctacgacttagaggcc20
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The R primer of <223> amplification PIV3 nucleic acid
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taatacgactcactatagggagaacaacaatgcatacaatattgtc46
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The F primer of <223> amplification PIV3 nucleic acid
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gattaattctgtgattgtctct22
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<223> detects the specific probe A of PIV3
<220>
<221>misc_feature
<222>(1)..(1)
<223>5 ' holds Dig to modify
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gtctctttgattgtttttttgcctcaaagacggacgccttct42
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<212>DNA
<213>Artificial
<220>
<223> detects the specific probe B1 of PIV3
<220>
<221>misc_feature
<222>(1)..(1)
<223>5' end mark Bio
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tgggctacgacttagaggccttttggaggatactgtcatg40
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tgggctacgacttagaggccttttatattcgttttcatac40
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taatacgactcactatagggagaacactacattcggtattagcgatc47
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ctgagaatttgatcttggttcagat25
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tgggctacgacttagaggccttttaagggttagtaataca40
Claims (8)
1. detect a collaurum mark detection kit for nucleic acid, comprising:
(1), general colloidal gold strip: test strips used is fixed on PVC base plate, is sample pad, glass fibre, NC film, thieving paper from left to right successively; NC film has T line (detection line) and C line (nature controlling line), described detection line wraps by the material of energy specific binding label b, on described nature controlling line, bag is by the molecule of energy specific binding label a, and described label b is different from label a; General probe 1 be marked with colloid gold particle or be marked with label a at the other end simultaneously, and being fixed on the glass fibre element film of test strips;
(2) probe tube of specific probe A series, specific probe B series, is filled respectively: one end of specific probe A series and determined nucleic acid hybridization, and be marked with label a, the other end and general probe 1 are hybridized, or when general probe 1 is not only marked with colloid gold particle but also is marked with label a, specific probe A series is not with any mark, and specific probe A series can have multiple, as A1, A2 ... hybridize with the zones of different complementary pairing of determined nucleic acid respectively, be contained in different probe tubes; Specific probe B series one end and determined nucleic acid hybridization, the other end is marked with label b, or when also comprising the general probe 2 with label b in kit, its simultaneously and general probe 2 complementary pairing hybridize, now specific probe B series is not with any mark, and specific probe B series can have multiple, as B1, B2 ... hybridize with the zones of different complementary pairing of determined nucleic acid respectively, be contained in respectively in different probe tubes.
2. the collaurum mark detection kit of detection nucleic acid according to claim 1, it is characterized in that: described label a, b can be antigen or haptens, as biotin, digoxin or fluorescent dye, wherein fluorescent dye can be Cy3, Cy5, Fam, Fit etc., then the T line in test strips or C line are Streptavidin, anti digoxin antibody, corresponding anti-fluorescent dye antibody to the molecule of their specific bindings.
3. the collaurum mark detection kit of detection nucleic acid according to claim 3, it is characterized in that: described specific probe A series of markings digoxin, general probe 2 marks biotin, then wrap by Streptavidin at test strips T line place, and C line place bag is by anti digoxin antibody.
4. the application of collaurum mark detection kit in pathogen nucleic acid detects of the detection nucleic acid described in any one of claim 1-3, is characterized in that: described pathogen is parasite, fungi, bacterium, virus, mycoplasma and Chlamydia.
5. the collaurum mark detection kit of the detection nucleic acid according to any one of claim 1-3, it is characterized in that: described general probe 1 sequence is: 5 '-SH-CATCTTCCAGCGGCCTTATGCAGTTGCTCTCCATTTTTAGAAGGCGTCCGTCT TTGAGGC-3 ' (SeqNo.1), wherein SH is sulfydryl, 5 ' end is sulfydryl modification, also other chemical group modifications can be carried out, as-NH
2.
6. a nucleic acid gold-labeled rapid detection method, is characterized in that: comprise the steps:
(1), designing probe: general probe 1, specific probe A series, specific probe B series, wherein, 5 ' end sulfhydrylation of general probe 1 is modified, and also can carry out other chemical group modifications, as-NH
2, then mark colloid gold particle, or be marked with label a at the other end simultaneously; Specific probe A series, its one end and determined nucleic acid hybridization, the other end and general probe 1 are hybridized, be marked with label a, or when general probe 1 is not only marked with colloid gold particle but also is marked with label a, specific probe A series is not with any mark, and specific probe A series can have multiple, as A1, A2 ..., hybridize with the zones of different complementary pairing of determined nucleic acid respectively; Specific probe B series one end and determined nucleic acid hybridization, the other end is marked with label b, or when kit also comprises the general probe 2 with label b, its simultaneously and general probe 2 complementary pairing hybridize, now specific probe B series is not with any mark, specific probe B series can have multiple, as B1, B2 ..., hybridize with the zones of different complementary pairing of determined nucleic acid respectively;
(2), preparing universal test paper bar: described test strips is fixed on PVC base plate, is sample pad, glass fibre, NC film, thieving paper from left to right successively; NC film there are T line (detection line) and C line (nature controlling line); Described detection line wraps by the antibody of anti-label b, specificity can catch the general probe 2 of binding label b or the specific probe B series of binding label b; Described nature controlling line wraps by anti-label a, specificity can catch the specific probe A of binding label a or be not only marked with colloid gold particle but also be marked with the general probe 1 of label a; General probe 1 marked upper colloid gold particle or be fixed on glass fibre element film with after tense marker subscript note thing a; chromatography in sample pad is dropped in by specific probe A, specific probe B or after adding general probe 2 and determined nucleic acid hybridization simultaneously; detection line colour developing indicates that determined nucleic acid exists, and nature controlling line colour developing represents that detection effectively.
7. nucleic acid gold-labeled rapid detection method according to claim 6, it is characterized in that: described label b and label a can be digoxin, biotin and other antigen or haptens, as fluorescent dye (Cy3, Cy5, Fam, Fit), be then corresponding anti digoxin antibody, Streptavidin or anti-fluorescent dye antibody to the molecule of their specific bindings on test strips T line or C line.
8. the nucleic acid gold-labeled rapid detection method according to claim 6-7, is characterized in that: described specific probe A series of markings digoxin, and general probe 2 marks biotin, then wrap by Streptavidin at test strips T line place, and C line place bag is by anti digoxin antibody.
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