CN102643910A - Application of asymmetric multicolor fluorescence hairpin probe chain reaction in pathogenic bacterium detection - Google Patents
Application of asymmetric multicolor fluorescence hairpin probe chain reaction in pathogenic bacterium detection Download PDFInfo
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Abstract
The invention relates to application of an asymmetric multicolor fluorescence hairpin probe chain reaction in pathogenic bacterium DNA (deoxyribonucleic acid) detection, belonging to the field of biotechnological detection. In the invention, in combination with the asymmetric hairpin probe chain reaction technology and the FRET (fluorescence resonance energy transfer) technology, a pathogenic bacterium detection technology based on the asymmetric multicolor fluorescence hairpin probe chain reaction technology is established. If no target sequence exists in the system, the hairpin probe is in a sub-stable state, two groups form an FRET pair, and no fluorescence signal is generated; when the target sequence is added to the reaction system to trigger the chain polymerization reaction, the hairpin structure is opened, and a fluorescence signal is generated; and due to linear amplification, the strength of the fluorescence signal is directly proportional to the copy number of the target sequence. In the method provided by the invention, by introducing the high-sensitivity multicolor FRET technology into the chain reaction, a simple, quick and sensitive pathogenic bacterium DNA direct detection method is established, and the shortcoming of lower sensitivity of the agarose gel electrophoresis observation detection result in the chain reaction of the traditional asymmetric hairpin probe is overcome.
Description
Technical field
The present invention relates to a kind of bioreaction technology, particularly relate to based on asymmetric multicolor fluorescence hair clip probe chain reaction and in pathogenic bacteria detects, using.
Background technology
The molecular diagnostics method detects according to the bacterial nucleic acid characteristic, for clinical disease pathogenic microorganism rapid detection has been brought new opportunity.The priority of detection methods such as nucleic acid molecule solid-phase hybridization method, dna sequencing method, capillary electrophoresis analysis, biochip technology is set up; The bacteria molecule detection method is brought up to a brand-new stage; But these molecular diagnostics methods need special large-scale instrument mostly; Technical difficulty is big, is difficult to popularization and application in clinical.Therefore; To the problems referred to above; If can develop a kind ofly based on novel probe technology, quick, special, the easy molecular diagnostics method to multiple pathogenic bacteria genome carries out parallel detection obviously has crucial meaning to the practicability that advances molecular diagnosis to learn a skill.
The patent name of Applied Gene Technologies company is " hair clip forming core acid probe technology " (Nucleic Acid Hairpin Probes); The patent No. is 6380377 to relate to a kind of stem annular DNA probe technique; More stable with the single stranded nucleic acid probe structure compared of standard; Enzyme reaction is more efficient, and is responsive more to mispairing, produced non-specific target position still less and combined.These advantages have all improved the specificity of genetic analysis.With compared with techniques such as traditional P CR, solid-phase nucleic acid hybridization, this technology has more outstanding advantage: detection efficiency is high, and detection speed is fast, and this technology is carried out molecular recognition and cascade signal simultaneously and amplified, and can in 10 minutes, accomplish detection of nucleic acids; Reaction system is simple, need not enzyme and other complicated molecular biology reagent; The single tube reaction, all detections are carried out in single stopped pipe, and operation steps is simple, and has avoided the conventional art problem effectively, the problem includes: the laboratory pollution problem; This method also is fit to integrate with other detection methods.
FRET (Fluorescence resonance Energy Transfer; When FRET) technology is meant the excitation spectrum overlaid when the fluorescence spectrum of a fluorescence molecule (be called not only donor molecule) and another fluorescence molecule (but also being called acceptor molecule); And confession, (be generally 7～10nm) when the acceptor space length is close; Then the excitation energy of donor fluorescence molecule is brought out acceptor molecule and is sent fluorescence; The fluorescence intensity of donor fluorescence molecule self decay simultaneously, this phenomenon is called as FRET.Characteristics such as that the FRET technology has is highly sensitive, specificity strong, favorable reproducibility, and can avoid the influence of scattered light, have stronger immunity from interference than conventional fluorescent method and resonant light scattering method.The FRET technology has been widely used in aspects such as biology and medical research.The FRET probe is that the confession-acceptor of resonance energy transfer is right, mainly is divided into following several types: GFP, organic fluorescent dye; Group of the lanthanides dyestuff and quantum dot, wherein organic fluorescent dye is of a great variety, is widely used; Can select the dyestuff that is fit to as required, and can use with the pairing of other type probe.
Summary of the invention
The present invention has designed a kind of asymmetric hair clip probe chain reaction technology; The high efficiency that has combined the high specific and the chain polymerization of molecular hybridization dexterously; The free energy that discharges through the DNA polymerization drives molecule cascade amplification; And high-sensitive multicolor fluorescence resonance energy transfer techniques is incorporated in the Kettenreaktion, thereby set up a kind of simple, fast, sensitive pathogenic bacteria DNA detection method.
The application of asymmetric multicolor fluorescence hair clip probe chain reaction in the pathogenic bacteria DNA detection; Comprise solution hybridization chain reaction system; Adding simultaneously in the said reaction system has asymmetric hair clip probe 1 and asymmetric hair clip probe 2 and target sequence to be measured; Wherein asymmetric hair clip probe 1 comprises palindromic sequence, two stem arms and is connected in a sticky end on the stem arm that said two stem arms are connected in the two ends of palindromic sequence and have the complementary nucleotide sequence; Said palindromic sequence is and the irrelevant sequence of target sequence to be measured that the connected sticky end of said stem arm has the complementary nucleic acid sequence of target sequence; Wherein asymmetric hair clip probe 2; Comprise palindromic sequence, two stem arms and be connected in a sticky end on the stem arm; Said two stem arms are connected in the two ends of palindromic sequence and have the complementary nucleotide sequence; A coupled stem arm of said palindromic sequence has target sequence; The sticky end that links to each other on said another stem arm have with asymmetric hair clip probe 1 in the complementary nucleic acid sequence of palindromic sequence, 5 ' end of said asymmetric hair clip probe 1 and 3 ' the end 5 ' end and 3 ' of mark fluorescent acceptor molecule and fluorescence donor molecule or asymmetric hair clip probe 2 are respectively held difference mark fluorescent donor molecule and fluorescent receptor molecule.
Said fluorescence donor molecule and fluorescent receptor molecule mark respectively in the two ends of asymmetric hair clip probe 2.
Said fluorescence donor molecule is organic fluorescent dye FAM and HEX, and the fluorescent receptor molecule is BHQ1.
The sticky end of said asymmetric hair clip probe 1 is positioned at 5 ' end, and the sticky end of said asymmetric hair clip probe 2 is positioned at 3 ' end; The sticky end of perhaps said asymmetric hair clip probe 1 is positioned at 3 ' end, the sticky end 5 ' end of said asymmetric hair clip probe 2.
Preferably: the sticky end of said asymmetric hair clip probe 1 is positioned at 5 ' end, and the sticky end of said asymmetric hair clip probe 2 is positioned at 3 ' end.
Said sticky end is a 6-8 base.
The 16S rDNA sequence that said target sequence to be measured is a bacterium.
Said bacterium is a Pseudomonas aeruginosa, and said asymmetric hair clip probe 1, asymmetric hair clip probe 2 and target sequence to be measured are respectively:
PA-H1：CGTCCTGAGGGAGAAAGTGGGGGTCAAAGTACCCCCACTTTCTCCCTC，
PA-H2：ACCCCCACTTTCTCCCTCAGGACGGAGGGAGAAAGTGGGGGT，
Tpa：ACCCCCACTTTCTCCCTCAGGACG。
Said bacterium is a Klebsiella Pneumoniae, and said asymmetric hair clip probe 1, asymmetric hair clip probe 2 and target sequence to be measured are respectively:
KP-H1：
GGTGACGAGTGGCGGACGGGTGAGTTCAAAGTCTAACTCACCCGTCCGCCAC，
KP-H2：
AACTCACCCGTCCGCCACTCGTCACCGTGGCGGACGGGTGAGTTAGACTTTG，
Tkp：AACTCACCCGTCCGCCACTCGTCACC。
Its ultimate principle of asymmetric hair clip is: two specificity self-assembly probes are made up of a target nucleic acid distinguished sequence and a palindromic sequence; Under the situation that no target sequence exists; In a single day be in metastable hairpin structure, and add target sequence to be measured, promptly trigger need not the chain polymerization reaction that enzyme is participated in; The target nucleic acid distinguished sequence part of specificity self-assembly this moment probe combines with corresponding target nucleic acid site; All the other probes then in twos renaturation become two sections partially double stranded probes that are palindromic sequence, enhancing detection signal significantly in molecular recognition hybridization, thus realized the rapid detection of target sequence to be measured.
Asymmetric hair clip probe chain reaction technology and FRET technology are promptly united in this research, have set up a kind of pathogenic bacteria detection technique based on asymmetric multicolor fluorescence hair clip probe chain reaction technology.In this preferable methods, as the fluorescence donor, BHQ1 is marked on hair clip probe two ends respectively as fluorescent receptor with organic fluorescent dye (FAM and HEX); When no target sequence exists in the system; The hair clip probe is in metastable condition, and it is right that two groups constitute FRET, and no fluorescent signal produces; And adding reaction system when triggering the chain polymerization reaction when target sequence, hairpin structure is opened, and produces fluorescent signal, and owing to be linear amplification, fluorescence signal intensity be directly proportional with the target sequence copy number (as shown in Figure 1).In inventive method high-sensitive multicolor fluorescence resonance energy transfer techniques is incorporated in the Kettenreaktion; Thereby set up a kind of simple, fast, sensitive pathogenic bacteria dna direct detection method, agarose gel electrophoresis is observed the lower defective of detected result sensitivity when having overcome former asymmetric hair clip probe and carrying out Kettenreaktion.
In an embodiment of the present invention, the specific nucleic acid target sequence of directed toward bacteria, the asymmetric hair clip probe of design multiple specific in conjunction with the FRET technology, provides a kind of efficient, sensitive signal detecting method, can realize the purpose of fast parallel bacterial detection.
Description of drawings
Fig. 1. asymmetric hair clip probe chain reaction technical schematic diagram
H1, H2L, I among the figure: hair clip probe 1, hair clip probe 2, target sequence to be measured; A and a, B and b, C and c are the complementary nucleic acid sequence,
● be fluorescent receptor, ★ is a gleaming light donor,
Fig. 2: pcr amplification product,
M:DL2,000DNA maker, the K:KpnPCR product, the P:P.aerPCR product ,-: blank
Fig. 3: PCR expansion product enzyme is cut,
M:DL 2,000DNAmaker, and the K*:Kpn enzyme is cut product, and the P*:P.aer enzyme is cut product, K:Kpn PCR product, P:P.aer PCR product,
Fig. 4: PA group different concns target sequence fluorescence HCR,
Wherein, A: no target sequence exists; B: target sequence concentration is 1 μ M; C: target sequence concentration is 0.5 μ M; D: target sequence concentration is 0.1 μ M; E: target sequence concentration is 0.05 μ M; F: target sequence concentration is 0.01 μ M,
Fig. 5: KP group different concns target sequence fluorescence HCR,
Wherein, A: no target sequence exists; B: target sequence concentration is 1 μ M; C: target sequence concentration is 0.5 μ M; D: target sequence concentration is 0.1 μ M; E: target sequence concentration is 0.05 μ M; F: target sequence concentration is 0.01 μ M,
The fluorescence HCR of Fig. 6: PA group different concns asymmetric PCR product,
A:1 μ l single stranded DNA wherein; B:2 μ l single stranded DNA; C:3 μ l single stranded DNA; D:4 μ l single stranded DNA; E:5 μ l strand DN A; F:6 μ l strand DN A; G: no single stranded DNA exists; H: no probe exists
The fluorescence HCR of Fig. 7: KP group different concns asymmetric PCR product,
A:1 μ l single stranded DNA wherein; B:2 μ l single stranded DNA; C:3 μ l single stranded DNA; D:4 μ l single stranded DNA; E:5 μ l strand DN A; F:6 μ l strand DN A; G: no single stranded DNA exists; H: no probe exists.
Embodiment
Principle of the present invention is as shown in Figure 1.
(A) thus the strand replacement reaction that target sequence I to be measured begins to take place strict base pairing from the sticky end of probe H1 is opened the hairpin structure of probe, produce new sticky end;
(B) sticky end of the sticky end of H1 and H2L reacts and continues to open the hairpin structure of probe; Distance increases between quencher group and reporter group; Produce fluorescent signal, alternately hybridization takes place between probe H1 and the H2L then, form chain polymerization body amplifying signal significantly.
Specific embodiment of the present invention is primarily aimed in two kinds of pathogenic bacterias, and those skilled in the art can be applied to the detection of any little living body according to principle of the present invention, as long as find suitable target sequence specific to be measured.
1, the design of the asymmetric HCR hair clip of specificity probe
Adopt Array Designer 4.0 and the asymmetric hair clip formula of Primer Premier 5.0 software designs probe; With Omiga 2.0 softwares carry out between probe, the complementarity analysis of probe and target sequence; In the time of designing probe; Base corresponding on the position of sequence intermediate demand mark quencher group need be designed to T, so that the quencher group can be connected on the probe.The alternative probe sequence of design submits to the Genbank DB to carry out specificity analyses, and the probe that designs is submitted to
Http:// mfold.rna.albany.edu/The prediction of secondary structure is carried out in the website, has further verified the specificity and the validity of designing probe.
Table 1: probe and target sequence design
| Pseudomonas aeruginosa HCR probe | ? |
| ?PA-H1 | CGTCCTGAGGGAGAAAGTGGGGGTCAAAGTACCCCCACTTTCTCCCTC |
| ?PA-H2L | ACCCCCACTTTCTCCCTCAGGACGGAGGGAGAAAGTGGGGGTACTTTg |
| ?Tpa | ACCCCCACTTTCTCCCTCAGGACG |
| Klebsiella Pneumoniae HCR probe | ? |
| ?KP-H1 | GGTGACGAGTGGCGGACGGGTGAGTTCAAAGTCTAACTCACCCGTCCGCCAC |
| ?KP-H2L | AACTCACCCGTCCGCCACTCGTCACCGTGGCGGACGGGTGAGTTAGACTTTG |
| ?Tkp | AACTCACCCGTCCGCCACTCGTCACC |
2, the genomic dna that extracts is carried out the PCR experiment, preparation strand PCR product.
Primer 16S-a 5 ' end mark PO
4Group is beneficial to downstream and carries out endonuclease reaction.
Reaction system is following:
Table 2:PCR amplification reaction system
Reaction conditions is following:
Sex change is 94 ℃ in advance, 5min; 94 ℃ of sex change, 60s; Anneal 53 ℃ 45s; Extend 72 ℃, 45s; 30 circulations; Prolong 72 ℃, 10min, 4 ℃ of preservations.
The PCR reaction product is carried out 1.0% agarose gel electrophoresis, appearance on every hole 5.0 μ l.
Experimental result is as shown in Figure 2, uses the universal primer that is designed to carry out pcr amplification, the visible obviously purpose band of electrophoresis, and amplified production can be used for enzyme and tests conscientiously.
3, exonuclease digestion PCR product
The PCR product is digested preparation single stranded DNA (ssDNA) with exonuclease.
Reaction system is: get 45 μ l PCR reaction product, add 5 μ l, 10 * Reacton Buffer, add the 10U exonuclease again.
Reaction conditions: reaction system is placed 37 ℃ of water-bath 5min.Termination reaction in 80 ℃ of water-baths.
Its result is as shown in Figure 3, and the PCR product is after exonuclease digestion, and double-stranded DNA reduces in a large number, and it is higher that enzyme is cut efficient, and the gained single stranded DNA can be used for carrying out the downstream experiment.
4, different concns target sequence fluorescence HCR experiment:
3.0 μ l fluorescence HCR reaction system comprises: the probe H10.1 μ l of 40uM; The probe H2L 0.1 μ l of 40uM; 3 * HBVbuffer, 1 μ l; Sterilized water 0.8 μ l; The target sequence I 1.0 μ l of different concns.Reaction conditions is following: 99 ℃, and 8min; 50 ℃, 1h; 4 ℃ of preservations.The HCR reaction product is transferred in the kapillary, 3, the centrifugal 2min. of 000rpm.Kapillary is observed fluorescence and taken a picture as for causing under the fluorescent microscope.
Experimental result such as Fig. 4, shown in Figure 5; The HCR specific probe that is designed all can successfully start cross chain reaction; Need not the Kettenreaktion of enzyme effect, make the hair clip probe structure open, the fluorescence donor molecule sends fluorescence under the exciting light of the light of certain wavelength.When initial target sequence concentration during more than or equal to 0.1 μ M, can be observed obvious fluorescence, can detect fluorescence when promptly target sequence reaches 0.4pmol in the 12 μ l reaction systems, explain that institute's designed probe has specificity and susceptibility preferably.
5, the fluorescence HCR of strand PCR product experiment
Use the single stranded DNA of above asymmetric PCR method preparation to be template, carry out fluorescence HCR experiment.
Reaction system is following:
Table 3: the fluorescence HCR reaction system of strand PCR product
The HCR reaction product is transferred in the kapillary, 3, the centrifugal 2min. of 000rpm.Kapillary is observed fluorescence and photograph down as for inverted fluorescence microscope.
Experimental result is like Fig. 6, shown in 7, and PCR product enzyme is cut product can trigger cross chain reaction, produces fluorescence, and fluorescence intensity increases and raises with the asymmetric PCR product amount that adds.Our constructed fluorescent hybridization chain reaction method of this results suggest can be used for actual pathogenic micro-organism and detects.
SEQUENCE?LISTING
< 110>Hospital No.1 Attached to Military Medical Univ. No. 3
< 120>application of asymmetric multicolor fluorescence hair clip probe chain reaction in pathogenic bacteria detects
<130>P123105/DYF/CQ
<160>6
<170>PatentIn?version?3.3
<210>1
<211>48
<212>DNA
< 213>Pseudomonas aeruginosa HCR probe PA-H1
<400>1
cgtcctgagg?gagaaagtgg?gggtcaaagt?acccccactt?tctccctc 48
<210>2
<211>42
<212>DNA
< 213>Pseudomonas aeruginosa HCR probe PA-H2
<400>2
acccccactt?tctccctcag?gacggaggga?gaaagtgggg?gt 42
<210>3
<211>24
<212>DNA
< 213>Pseudomonas aeruginosa HCR target sequence Tpa
<400>3
acccccactt?tctccctcag?gacg 24
<210>4
<211>52
<212>DNA
< 213>Klebsiella Pneumoniae HCR probe KP-H1
<400>4
ggtgacgagt?ggcggacggg?tgagttcaaa?gtctaactca?cccgtccgcc?ac 52
<210>5
<211>52
<212>DNA
< 213>Klebsiella Pneumoniae HCR probe KP-H2
<400>5
aactcacccg?tccgccactc?gtcaccgtgg?cggacgggtg?agttagactt?tg 52
<210>6
<211>26
<212>DNA
< 213>Klebsiella Pneumoniae HCR target sequence Tkp
<400>6
aactcacccg?tccgccactc?gtcacc。
Claims (8)
1. the application of asymmetric multicolor fluorescence hair clip probe chain reaction in the pathogenic bacteria DNA detection; Comprise solution hybridization chain reaction system; Adding simultaneously in the said reaction system has asymmetric hair clip probe 1 and asymmetric hair clip probe 2 and target sequence to be measured; Wherein asymmetric hair clip probe 1 comprises palindromic sequence, two stem arms and is connected in a sticky end on the stem arm that said two stem arms are connected in the two ends of palindromic sequence and have the complementary nucleotide sequence; Said palindromic sequence is and the irrelevant sequence of target sequence to be measured that the connected sticky end of said stem arm has the complementary nucleic acid sequence of target sequence; Wherein asymmetric hair clip probe 2; Comprise palindromic sequence, two stem arms and be connected in a sticky end on the stem arm; Said two stem arms are connected in the two ends of palindromic sequence and have the complementary nucleotide sequence; A coupled stem arm of said palindromic sequence has target sequence; The sticky end that links to each other on said another stem arm have with asymmetric hair clip probe 1 in the complementary nucleic acid sequence of palindromic sequence, 5 ' end of said asymmetric hair clip probe 1 and 3 ' the end 5 ' end and 3 ' of mark fluorescent acceptor molecule and fluorescence donor molecule or asymmetric hair clip probe 2 are respectively held difference mark fluorescent donor molecule and fluorescent receptor molecule.
2. application according to claim 1, said fluorescence donor molecule and fluorescent receptor molecule mark respectively in the two ends of asymmetric hair clip probe 2.
3. application according to claim 2, said fluorescence donor molecule is organic fluorescent dye FAM and HEX, the fluorescent receptor molecule is BHQ1.
4. application according to claim 1, the sticky end of said asymmetric hair clip probe 1 are positioned at 5 ' end, and the sticky end of said asymmetric hair clip probe 2 is positioned at 3 ' end.
5. application according to claim 1, said sticky end are 6-8 base.
6. application according to claim 1, the 16S rDNA sequence that said target sequence to be measured is a bacterium.
7. application according to claim 1, said bacterium are Pseudomonas aeruginosa, and said asymmetric hair clip probe 1, asymmetric hair clip probe 2 and target sequence to be measured are respectively:
PA-H1：CGTCCTGAGGGAGAAAGTGGGGGTCAAAGTACCCCCACTTTCTCCCTC，
PA-H2：ACCCCCACTTTCTCCCTCAGGACGGAGGGAGAAAGTGGGGGT，
Tpa：ACCCCCACTTTCTCCCTCAGGACG。
8. application according to claim 1, said bacterium are Klebsiella Pneumoniae, and said asymmetric hair clip probe 1, asymmetric hair clip probe 2 and target sequence to be measured are respectively:
KP-H1：
GGTGACGAGTGGCGGACGGGTGAGTTCAAAGTCTAACTCACCCGTCCGCCAC，
KP-H2：
AACTCACCCGTCCGCCACTCGTCACCGTGGCGGACGGGTGAGTTAGACTTTG，
Tkp：AACTCACCCGTCCGCCACTCGTCACC。
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| CN109540856B (en) * | 2018-11-08 | 2022-01-28 | 南京师范大学 | Reagent for detecting different subtype breast cancer cells based on fluorescence resonance energy transfer |
| CN109613236A (en) * | 2018-12-12 | 2019-04-12 | 安邦（厦门）生物科技有限公司 | A kind of nucleic acid hybrid capture immunofluorescent detection method, immunofluorescence chromatography strip and kit |
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