CN105018601B - Encephalic bacterial infection common causative bacterium MLPA detection probes and its application after a kind of neurosurgery - Google Patents

Encephalic bacterial infection common causative bacterium MLPA detection probes and its application after a kind of neurosurgery Download PDF

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CN105018601B
CN105018601B CN201510361873.XA CN201510361873A CN105018601B CN 105018601 B CN105018601 B CN 105018601B CN 201510361873 A CN201510361873 A CN 201510361873A CN 105018601 B CN105018601 B CN 105018601B
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probe
seq
rpo
side probe
group
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CN105018601A (en
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王明席
李云振
王庆波
姜涛
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Xiamen Baijia Biotechnology Co Ltd
Huaqiao University
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Xiamen Baijia Biotechnology Co Ltd
Huaqiao University
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses encephalic bacterial infection common causative bacterium MLPA detection probes and its applications after a kind of neurosurgery, the probe includes first group of probe to the 17th group of probe, it is sequentially detection Neisseria meningitidis, Pseudomonas aeruginosa, Salmonella, germ oligotrophy unit cell, staphylococcus aureus, staphylococcus epidermis, Klebsiella pneumoniae, serratia marcescens, Acinebobacter lwoffi, corynebacterium diphtheriae, streptococcus mitis, Acinetobacter baumannii, streptococcus pneumonia, propionibacterium acnes, listeria spp, the probe of enterobacter cloacae and haemophilus influenzae.The present invention is according to MLPA technical researches, and once, after quick diagnosis neurosurgery 17 kinds of common causative bacteriums of encephalic bacterial infection, the quick diagnosis for such patient pathogenic bacteria provide possibility, help to reduce this para-infectious lethality and disability rate energy.

Description

Encephalic bacterial infection common causative bacterium MLPA detections after a kind of neurosurgery Probe and its application
Technical field
The invention belongs to the pathogenic microorganism examination technical fields, and in particular to encephalic is bacillary after a kind of neurosurgery Infect common causative bacterium MLPA detection probes and its application.
Background technology
Encephalic bacterial infection most often shows as meningitis, subdural empyema disease or brain abscess after neurosurgery.State The outer report intracranial infection death rate is 27.4%~35% [42,43].Domestic scholars report, encephalic after severe neurosurgery The bacterial infection death rate may be up to 57% [44].
Encephalic bacterial infection has tens kinds of pathogenic bacterias after neurosurgery, common are nearly 20 kinds, comes from nearly 10 A different bacterium category.Most of is gram-positive bacteria, and one of the most common germ is staphylococcus aureus and epidermis grape Coccus (can account for 31% and 37%) respectively, secondly for streptococcus and Propionibacterium (respectively account for nearly 10%), it is mostly Cutaneous Flora；And in Gram-negative bacteria, Enterobacter and acinetobacter (can account for nearly 10%) respectively for the main pathogenic fungi.In addition, Pseudomonas aeruginosa and Klebsiella pneumoniae are more typical gram negative pathogenic bacterias, such germ is essentially from respiratory tract With enteron aisle [45].And it is ground both at home and abroad in relation to the epidemiology of encephalic bacterial infection and microbial characteristic after neurosurgery Study carefully result substantially with etc [46], be shown in Table 1.
Have been applied to the pathogenic microorganisms diagnosis of infection for many years using round pcr as the nucleic acid detection technique of representative.With Traditional detection method is compared, and bacterial infection has many advantages after nucleic acid detection technique application diagnosis neurosurgery.It is first First, nucleic acid detection technique is not necessarily to the culture of isolated of bacterium, only need to extract DNA, or even can directly be detected, be greatly improved inspection Degree of testing the speed；Secondly, nucleic acid detection technique has very high specificity and sensitivity (theoretically can detect a cell), is protecting Under the premise of demonstrate,proving detection accuracy, the positive rate of encephalic bacterial infection is substantially increased；Furthermore nucleic acid detection technique is not It is not only influenced by antibiotic usage, also detectable pathogenic bacteria antibiotic resistance genes instruct doctor's medication.Common nucleic acid inspection Survey technology includes mainly fluorescence in situ hybridization technique (Fluorescence in situ hybridization, FISH), polymerization Enzyme chain reaction (Polymerase chain reaction, PCR), real-time quantitative PCR (Real-time PCR), wide spectrum PCR (Broad range PCR), multiple Real time PCR, micro-array chip (Microarray) and sequencing (Sequencing). Wherein, it is said in terms of detection specificity, sensibility and detection speed etc. are several, real-time quantitative PCR is still currently for brain ridge Pathogenic bacteria detect ideal technology in liquid sample.However, this technology detection flux is relatively low, 8 kinds can only be at most detected simultaneously Pathogenic bacteria, rate of missed diagnosis are high.Theoretically, although micro-array chip and sequencing technologies may be implemented to examine while most of pathogenic bacteria It surveys, but these technologies are directed to the complicated analysis of mass data that it is necessary to have the personnel of professional skill.In short, currently, clinical 8 or more the neurosurgery Craniotomy encephalic bacterial infection of upper disposable diagnostic not yet plants the kit of pathogenic bacteria.
Invention content
It is an object of the invention to overcome prior art defect, encephalic bacterial infection after a kind of neurosurgery is provided Common causative bacterium MLPA detection probes and its application.
The specific technical solution of the present invention is as follows：
Encephalic bacterial infection common causative bacterium MLPA detection probes after a kind of neurosurgery, including first group of spy Needle is sequentially detection Neisseria meningitidis, Pseudomonas aeruginosa, Salmonella, thermophilic malt oligotrophy to the 17th group of probe Monad, staphylococcus aureus, staphylococcus epidermis, Klebsiella pneumoniae, serratia marcescens, Acinebobacter lwoffi, Corynebacterium diphtheriae, streptococcus mitis, Acinetobacter baumannii, streptococcus pneumonia, propionibacterium acnes, listeria spp, cloaca intestines bar The probe of bacterium and haemophilus influenzae contains left side probe LPO and right side probe RPO in every group of probe respectively：
The left side probe LPO of first group of probe contains sequence shown in SEQ ID NO 1, and right side probe RPO contains SEQ ID Sequence shown in NO 2；
The left side probe LPO of second group of probe contains sequence shown in SEQ ID NO 3, and right side probe RPO contains SEQ ID Sequence shown in NO 4；
The left side probe LPO of third group probe contains sequence shown in SEQ ID NO 5, and right side probe RPO contains SEQ ID Sequence shown in NO 6；
The left side probe LPO of 4th group of probe contains sequence shown in SEQ ID NO 7, and right side probe RPO contains SEQ ID Sequence shown in NO 8；
The left side probe LPO of 5th group of probe contains sequence shown in SEQ ID NO 9, and right side probe RPO contains SEQ ID Sequence shown in NO 10；
The left side probe LPO of 6th group of probe contains sequence shown in SEQ ID NO 11, and right side probe RPO contains SEQ ID Sequence shown in NO 12；
The left side probe LPO of 7th group of probe contains sequence shown in SEQ ID NO 13, and right side probe RPO contains SEQ ID Sequence shown in NO 14；
The left side probe LPO of 8th group of probe contains sequence shown in SEQ ID NO 15, and right side probe RPO contains SEQ ID Sequence shown in NO 16；
The left side probe LPO of 9th group of probe contains sequence shown in SEQ ID NO 17, and right side probe RPO contains SEQ ID Sequence shown in NO 18；
The left side probe LPO of tenth group of probe contains sequence shown in SEQ ID NO 19, and right side probe RPO contains SEQ ID Sequence shown in NO 20；
The left side probe LPO of 11st group of probe contains sequence shown in SEQ ID NO 21, and right side probe RPO contains SEQ Sequence shown in ID NO 22；
The left side probe LPO of 12nd group of probe contains sequence shown in SEQ ID NO 23, and right side probe RPO contains SEQ Sequence shown in ID NO 24；
The left side probe LPO of 13rd group of probe contains sequence shown in SEQ ID NO 25, and right side probe RPO contains SEQ Sequence shown in ID NO 26；
The left side probe LPO of 14th group of probe contains sequence shown in SEQ ID NO 27, and right side probe RPO contains SEQ Sequence shown in ID NO 28；
The left side probe LPO of 15th group of probe contains sequence shown in SEQ ID NO 29, and right side probe RPO contains SEQ Sequence shown in ID NO 30；
The left side probe LPO of 16th group of probe contains sequence shown in SEQ ID NO 31, and right side probe RPO contains SEQ Sequence shown in ID NO 32；
The left side probe LPO of 17th group of probe contains sequence shown in SEQ ID NO 33, and right side probe RPO contains SEQ Sequence shown in ID NO 34.
In a preferred embodiment of the invention, first group of probe to each right side of the 17th group of probe is visited 5 ' end connections of needle are just like BsmI restriction enzyme site sequences shown in SEQ ID NO 35, and 3 ' end connections are just like 36 institutes of SEQ ID NO The Ecor V restriction enzyme site sequences shown.
Encephalic bacterial infection common causative bacterium MLPA detection methods after a kind of neurosurgery, using above-mentioned probe Hybridized as template using the DNA of measuring samples.
In a preferred embodiment of the invention, further include using a pair of of universal primer to being connected after hybridization Left side probe LPO and right side probe RPO carries out PCR amplification, and the sense primer and downstream primer of the universal primer contain respectively Sequence shown in SEQ ID NO 37 and SEQ ID NO 38.
Encephalic bacterial infection common causative bacterium MLPA detection kits after a kind of neurosurgery, including above-mentioned spy Needle.
In a preferred embodiment of the invention, further include a pair of of universal primer, the upstream of the universal primer is drawn Object and downstream primer contain sequence shown in SEQ ID NO 37 and SEQ ID NO 38 respectively.
It is further preferred that further include Ligase-65, Ligase buffer, Probe Mix and PCR Primer Mix, Wherein,
The formula of Ligase-65 is：1U/ μ L, storing liquid be 20mM Tris, pH 7.5,50mM KCl, 1mM DTT, 0.1mM EDTA and 50% (v/v) glycerine；
The formula of Ligase buffer is：10×Ligation Buffer：400mM Tris, pH 7.8,100mM MgCl2, 100mM DTT, 5mM ATP；
The formula of Probe Mix is：A concentration of 4fM of each probe, working solution are 10mM Tris-Cl, pH 8.0,1mM EDTA；
The formula of PCR Primer Mix is：Sense primer containing the universal primer and downstream primer, each primer A concentration of 10pM, sense primer be Cy 5.0 mark, dNTPs 2.5mM, working solution be 10mM Tris-Cl, pH 8.0,1mM EDTA。
The beneficial effects of the invention are as follows：
The present invention, can encephalic bacterial infection 17 after primary, quick diagnosis neurosurgery according to MLPA technical researches Kind of common causative bacterium, the quick diagnosis for such patient pathogenic bacteria provide possibility, help to reduce this para-infectious lethal Rate and disability rate.
Description of the drawings
Fig. 1 is pcr amplification reaction product electrophoretogram before the clone of PrbX in the embodiment of the present invention 5 (to detect No. 1 micro- life For the right side probe RPO Prb1 of object Neisseria meningitidis, PCR amplification length is 165bp)；
Fig. 2 is the electrophoretogram of 5 pnagus medius particle suspension PCR verifications of the embodiment of the present invention (to detect No. 1 microorganism meninx For the right side probe RPO Prb1 of scorching Neisseria, PCR amplification length is 278bp)；
Fig. 3 is 5 pnagus medius particle suspension PCR product sequence verification result of the embodiment of the present invention (to detect No. 1 microorganism For the right side probe RPO Prb1 of Neisseria meningitidis)；
Fig. 4 is the preparation process of right side long probe RPO in the embodiment of the present invention 6；
Fig. 5 is PrbX-M13ssDNA electrophoresis results figure in the embodiment of the present invention 6 (to detect No. 1 microorganism meningitis Neisser For the right side probe RPO Prb1 of Salmonella)；
Fig. 6 is PrbX-M13ssDNA solution digestion products electrophoretic analysis figure in the embodiment of the present invention 6 (to detect No. 1 micro- life For the right side probe RPO Prb1 of object Neisseria meningitidis)；
Fig. 7 is that list target spot PrbX MLPA verify electrophoretogram (how to detect No. 1 microorganism meningitis in the embodiment of the present invention 7 For the right side probe RPO Prb1 of plucked instrument Salmonella, the length that MLPA is detected is 144bp)；
Fig. 8 is GeneScan in the embodiment of the present invention 7TM- 600 LIZ Size Standard Capillary Electrophoresis figures；
The MLPA that Fig. 9 is PrbX in the embodiment of the present invention 7 reacts Capillary Electrophoresis result (to detect No. 1 microorganism meninx For the right side probe RPO Prb1 of scorching Neisseria, the length that MLPA is detected is 144bp)；
Figure 10 is that the MLPA of 17 target spots in the embodiment of the present invention 8 reacts Capillary Electrophoresis result figure；
Figure 11 is using PrbX in the embodiment of the present invention 9 (with No. 17 microorganism haemophilus influenzae positive criteria product plasmids For) MLPA detection electrophoretograms (M, the DL of the plasmid of the target spot of haemophilus influenzae containing the Prb17 DNA of the different diluted concentrations of detection 1000DNA Marker；Detection template is diluted 1,10 by 1-10 respectively1, 102, 103, 104, 105, 106, 107, 108, 109, 1010 Times, the length that MLPA is detected is 512bp)；
Figure 12 be the embodiment of the present invention 9 in (by taking No. 17 microorganism haemophilus influenzae positive criteria product plasmids as an example) template Detection limit repeated experiment result (A, template 108Dilute MLPA electrophoretograms；B 107Dilution MLPA reacts electrophoresis detection figure again； 1-20,20 repetition parallel laboratory tests, the length that MLPA is detected are 512bp)；
Figure 13 is that PrbX probes in the embodiment of the present invention 9 (are with probe Prb17 on the right side of No. 17 microorganism haemophilus influenzaes Example) MLPA reaction gels electrophoretogram (M, DL1000DNA Marker after various concentration gradient dilution；1-8, probe do 1,101, 102, 103, 104, 105, 106, 107, 108Concentration gradient dilutes, and the length detected is 512bp)；
Figure 14 is PrbX in the embodiment of the present invention 9 (by taking probe Prb17 on the right side of No. 17 microorganism haemophilus influenzaes as an example) 103With 10420 parallel repetition reaction result of concentration gradient dilution (A, 104Dilution repeats；B, 103Dilution repeats；M, DL 1000DNA Marker；1-20,20 repetition parallel laboratory tests, the length that MLPA is detected are 512bp)；
Figure 15 is in the embodiment of the present invention 10 with probe Prb1 (No. 1 microorganism Neisseria meningitidis, the length detected Respectively 144bp), (No. 8 microorganism serratia marcescens, the length detected are respectively that 271) (No. 17 micro- with Prb17 to Prb8 Probe Prb17 on the right side of biological haemophilus influenzae, the length detected is respectively 512bp) probe of short, medium and long three length For, Repeatability detects gel electrophoresis figure.
Specific implementation mode
Technical scheme of the present invention is further detailed and is described below by way of specific implementation mode combination attached drawing.
The detection shot design of 1 intracranial infection pathogenic bacteria of embodiment
Encephalic bacterial infection common pathogen after neurosurgery is summarized first, is shown in Table 1：
Encephalic bacterial infection common pathogen summarizes after table 1. shows neurosurgery
In principle, the detection target spot of intracranial infection pathogenic bacteria constant current journey really：It is searched into PubMed to each pathogenic bacteria The conserved sequence of specificity, such as the sequence of house-keeping gene 16S rRNA, rpoB, gyrB or ITS, as shown in table 2：
The detection target spot of encephalic bacterial infection common pathogen after 2. neurosurgery of table
2 MLPA probes of embodiment hybridize the reference sequences of target spot
After the conservative sequence for comparing neurosurgery 17 kinds of pathogenic bacteria of intracranial infection by Blast tool analysis, determine Reference sequences are to design hybridization probe, such as table 3：
Table 3. shows the reference sequences of MLPA probes hybridization target spot
3 left side probe LPO of embodiment
17 kinds of pathogenic bacteria of intracranial infection carry out the left side probe of MLPA analyses after neurosurgery designed by the present invention The sequence of LPO, is shown in Table 4：
4. left side probe LPO sequence designs result of table (for upstream universal primer+hybridization sequences)
4 right side probe RPO of embodiment
17 kinds of pathogenic bacteria of neurosurgery intracranial infection designed by the present invention carry out the sequence of the right side probe RPO of MLPA analyses Row, are shown in Table 5：
5. right side probe RPO sequence designs result (including 4 parts) of table
Probe RPO cloning procedures on the right side of 5 MLPA of embodiment
Design synthesis RPO PrbX segments are (to detect the right side probe RPO of No. 1 microorganism Neisseria meningitidis first Prb1 (No. 1 Bsm I restriction enzyme sites sequence+hybridization sequences+EcoR V restriction enzyme sites sequence+downstream universal primers in above-mentioned table 5 Sequence) for preparation process), carry out PCR (sense primer Primer F：5'- GCTATGACCATGATTACG-3'(such as SEQ ID Shown in NO 57)；Downstream primer Primer R：5'-GGCCAGTGCCAAGCTT-3'(is as shown in SEQ ID NO 58)) amplification, Product purification, connection are cloned into double-strand M13mp18RF I DNA (NEB companies, N4018S) and are transformed into JM109 competent cells (Promega companies, L2005) carries out PCR amplification and sequencing (sense primer Clo_veri_ by blue hickie screening picking locus coeruleus pimer_F：5'-TCCGGCTCGTATGTTGTG-3'(is as shown in SEQ ID NO 59)；Downstream primer Clo_veri_pimer_ R：5'-CTGCAAGGCGATTAAGTT-3'(is as shown in SEQ ID NO 60)) confirm.It is as follows：
The pcr amplification reaction system of 1.RPO PrbX, is shown in Table 6：
Pcr amplification reaction system before probe RPO clones on the right side of table 6.MLPA
The pcr amplification reaction condition of 2.RPO PrbX, is shown in Table 7：
PCR amplification program before probe RPO clones on the right side of table 7.MLPA
The PCR product electrophoresis of 3.PrbX is shown in Fig. 1 (to detect the right side probe RPO of No. 1 microorganism Neisseria meningitidis For Prb1 preparation process, PCR product 165bp).
4.PCR product purifications
Use PCR product purification kit MiniElute PCR Purification KIT (Qiagen companies, 28004) The PCR product of rite-directed mutagenesis segment is purified, 10 μ L water is finally taken to be eluted (specific steps reference reagent box explanation Book).
5. coupled reaction system is shown in Table 8：
The DNA coupled reaction systems of 8. right side probe RPO of table
6. reaction condition：16 DEG C,>8h
7. transformation experiment
Transformation experiment is carried out using Single-Use JM109 Competent Cells (Promega companies, L2005) (specific steps reference explanation book).
8.PrbX-M13 phage vectors are verified
1) the single blue plaque that the connection product of picking PrbX DNA obtains after converting successfully, prepares phage particle Suspension appoints and a phage particle suspension PCR that carries out is taken to react verification.
2) phage particle suspension PCR reacts, and is shown in Table 9,10：
9. phage particle suspension PCR reaction systems of table
10. phage particle suspension PCR reaction conditions of table
3) electrophoresis is verified, and sees Fig. 2.
4) sequence verification：Phage particle suspension PCR product is sequenced, sequencing result is with standard sequence ((with detection For the right side probe RPO Prb1 preparation process of No. 1 microorganism Neisseria meningitidis, PCR amplification length is 278bp) into Row comparison, as a result such as Fig. 3.
According to the verification of the electrophoresis of phage particle suspension PCR product and sequence verification as a result, Prb1 synthesising probing needles DNA has become It is inserted into work(in M13 phage vectors.In other words, Prb1-M13 phage vectors are verified has built success.The phagocytosis Body carrier can be used for the preparation of probe Prb1 on the right side of MLPA.The PrbX-M13 that other 17 probes are cloned using identical method is bitten Bacteriophage vectors.
The preparation of 6 right side probe RPO PrbX of embodiment
Flow includes：Extraction purification PrbX-M13 phage vectors, probe prepare the design and Bsm I and EcoR of primer The contents such as V double digestions, see Fig. 4 (with detect No. 1 microorganism Neisseria meningitidis right side probe RPO Prb1 preparation process be Example).
1. the preparation of right side probe RPO PrbX
1) extraction purification of PrbX-M13 single stranded DNAs
(1) PrbX-M13 bacteriophages liquid 1mL cultivates 5h, volume of culture 1mL.
(2) PrbX-M13 bacteriophage liquid cultures are centrifuged, it is single-stranded that obtained supernatant is used to prepare PrbX-M13 DNA, bacterial precipitation object are then used for the preparation of PrbX-M13 double-stranded DNAs.
(3) it is carried out according to kit specification extraction using QIAprep Spin M13Kit (Qiagen companies, 27704) PrbX-M13 single stranded DNAs are purified, 100 μ L EB elutions are finally taken, obtain PrbX-M13 single stranded DNA solution；It uses simultaneously QIAprep Spin Miniprep Kit (Qiagen companies, 27104) kit prepares PrbX-M13 double-stranded DNAs, using as ginseng According to.
(4) the PrbX-M13 single stranded DNAs and double stranded DNA solutions obtained extraction purification carries out electrophoretic analysis, electrophoresis result Such as Fig. 5 (by taking probe RPO Prb1 on the right side of No. 1 microorganism Neisseria meningitidis of detection as an example)：
2) probe prepares the design of primer
It includes two that probe, which prepares primer, respectively on PrbX-M13ssDNA at EcoR V restriction enzyme sites and Bsm I digestions Annealed combination forms double-stranded DNA at site, then passes through the digestion of EcoR V and Bsm I restriction enzymes and can obtain Purpose long-chain probe.The design that probe prepares primer mainly considers that several factors include the length of primer, primer sequence bound site It sets, the Tm values of primer, G/C content etc., specific design result is as shown in table 11：
Table 11. prepares the primer of right side single-stranded probe RPO
3) probe prepares the annealing experiment of primer
This step focuses on the molecular weight of PrbX-M13ssDNA and the calculating of concentration.Probe prepares the molal quantity of primer It should be generally equalized with the M13ssDNA molal quantitys containing other right sides probe RPO PrbX.How to detect No. 1 microorganism meningitis On the right side of plucked instrument Salmonella for probe RPO Prb1, by calculate it is found that the relative molecular weight of Prb1-M13ssDNA be about 4.5 × 106.Therefore, the molal quantity of 500ng Prb1-M13ssDNA is about 100pmol.Using 50 μ L as final volume, PrbX-M13ssDNA Final concentration then be 2 μM.It is specific experiment process below：
(1) probe prepares primer annealing reaction system, is shown in Table 12：
Table 12. prepares the primer annealing reaction system of probe
(2) probe prepares primer annealing reaction condition, is shown in Table 13：
Table 13 prepares the primer annealing reaction condition of probe
4) EcoR V and Bsm I digestions experiment
(1) after annealed reaction, EcoR V restriction enzymes are firstly added into PrbX-M13ssDNA solution (43 μ L) Enzyme is digested, and the endonuclease reaction system of EcoRV is shown in Table 14：
Table 14.PrbX-M13ssDNA solution E coR V endonuclease reaction systems
(2) PrbX-M13ssDNA solution Es coR V endonuclease reaction conditions：37 DEG C of warm bath 2h.
(3) after EcoR V restriction enzymes are digested, the Bsm directly into PrbX-M13ssDNA solution (49 μ L)
I restriction enzymes are digested, and the endonuclease reaction system of Bsm I is shown in Table 15：
Table 15.PrbX-M13ssDNA solution B sm I endonuclease reaction systems
(4) PrbX-M13ssDNA solution Bs sm I endonuclease reaction conditions：65 DEG C of warm bath 2h.
(5) PrbX-M13ssDNA solution digestion products electrophoretic analysis is shown in Fig. 6 (to detect No. 1 microorganism meningitis Neisser On the right side of Salmonella for probe RPO Prb1).
According to electrophoresis result, Prb1-M13ssDNA solution is respectively after EcoR V and Bsm I digestion with restriction enzyme Product obtains two electrophoretic bands after electrophoretic separation, thus can tentatively judge, Prb1-M13ssDNA solution digestions success, reason By above saying, the band of the position of 100bp is long probe product, and concentration should be 2 μM.The long probe solution (PrbX- M13ssDNA digestions remainder need not remove) probe Prb1 on the right side of first MLPA of this project will be used as to be used for downstream needle to list The MLPA of target spot detects confirmatory experiment.In addition, according to above-mentioned set experiment flow, the phage vector of other 16 probes Same structure is completed, and is used for downstream needle together and is detected confirmatory experiment to the MLPA of multiple target point.
The single target spot MLPA analyses of embodiment 7
After the completion of on the right of MLPA prepared by probe RPO, it is most important that can verify it with probe LPO on the left of MLPA together It completes to detect the MLPA for detecting target spot.Steps are as follows：
1. the design and preparation of the single target spot positive criteria product of detection
1) it is directed to each detection target spot, chooses the DNA sequence dna for including MLPA probes (LPO+RPO) hybridization sequences respectively (length controls between 200-300bp as possible), is shown in Table 16：
The DNA sequence dna of the positive criteria product detection target spot of 16. 17 kinds of pathogenic bacterias of table
2) each above-mentioned DNA sequence dna is synthesized by method for synthesizing gene, and synthetic sequence is cloned into PUC57 matter It (is completed by Jin Sirui companies) in grain carrier.
3) amplification cultivation is carried out in plasmid conversion e. coli host bacteria.
4) extraction of plasmid DNA purifies, which is to detect target spot positive criteria product.
The preparation of 2.MLPA probe reaction liquid
1) prepared by MLPA LPO and RPO standard solution：TE (10mM Tris-HCl, pH 8.0,1mM EDTA) points are used respectively The final concentration of MLPA LPO (100 μM) and RPO stostes (2 μM, 50 μ L) 1 μM is not diluted to.
2) prepared by MLPA probe reactions liquid：
1 μ L mixings are taken per portion LPO and RPO standard solution, then add TE buffer solutions to 600 μ L, mixing, this solution is For MLPA probe reactions liquid (1.5 μ L is taken to carry out MLPA reactions every time).
3. single target spot MLPA confirmatory experiments
1) single detection target spot PrbX positive criteria product solution prepares：
It is suspended with TE and detects target spot positive criteria product (10 μ g), and be diluted to final concentration of 100ng/ μ L, as storing liquid. Then it takes 10 μ L storing liquids to dilute 10 times and obtains single detection target spot positive criteria product solution (a concentration of 10ng/ μ L).
2) hybridization reaction premixed liquid is prepared and (uses preceding 1h), is shown in Table 17：
Table 17.MLPA hybridization reactions premix liquid making method
3) connection reaction premixed liquid is prepared and (uses preceding 1h), is shown in Table 18：
Table 18.MLPA connection reaction premixed liquid preparation methods
4) PCR reaction premixed liquids are prepared and (use preceding 1h), are shown in Table 19：
Table 19.PCR reaction premixed liquid preparation methods
5) PCR response procedures are arranged, and are shown in Table 20：
Table 20.MLPA hybridizes PCR response procedures
6) DNA reactions of degeneration (RD)：5 μ L detection target spot positive criteria product solution are added into 200 μ L PCR reaction tubes, are placed in PCR instrument, operation PCR react Step1 and Step2.Reaction tube can be removed from PCR instrument after reaction, is started in next step.
7) hybridization reaction：3 μ L hybridization reaction premixed liquids are added to each reaction tube.Blow and beat mixing, operation PCR reactions Step3 and Step4.Hybridization time>8h.
8) connection reaction：It runs PCR and reacts Step5.When sample temperature is 54 DEG C, it is anti-that 32 μ L connections are added in each sample Premixed liquid is answered, mixing is blown and beaten.Continue to run with PCR reactions Step6-8.
9) pcr amplification reaction：At room temperature, 10 μ L PCR reaction premixed liquids are added into each reaction tube, gently blow and beat Mixing.Then operation PCR reacts Step9-11.
10) electrophoretic analysis, see Fig. 7 (by taking probe RPO Prb1 on the right side of No. 1 microorganism Neisseria meningitidis of detection as an example, The length detected is 144bp).
11) detection of operation MLPA reaction products is analyzed with specificity
(1) Preparatory work of experiment：Detect sample：MLPA reaction products；The relevant reagent of instrument and consumptive material is sequenced in ABI3500；Liz Standard and fornamide protection liquid as dissolving on ice：Genmapper analysis softwares.
(2) loading Injection mixture：1 μ L of MLPA reaction products in taking the PCR reaction tubes of 200 μ L to be added 1) With the TE buffer solutions of 99 μ L, the PCR reaction tubes of a 200 μ L are then taken again, and it is fully mixed then to sequentially add following table reagent Of short duration centrifugation is done after even.
Injection mixture systems：
(3) it is denaturalized：Injection mixture in reaction tube are denaturalized 2min as 80 DEG C of constant temperature of PCR instrument, after Reaction tube is taken out immediately as several minutes on ice.
(4) model on a clean ABI3500 is taken, is then added to the Injection mixture being denaturalized In the hole slot of model, pay attention to when sample-adding trying not that sample is made to stay in tube wall, doing rotating centrifugal after sample-adding makes sample Originally it is sunken to slot bottom and ensures that no bubble generates.
(5) upper model is closed using mating silicagel pad, then upper model is put into loading slot and is closed.
(6) ABI3500 sub computers is opened, ABI3500 hosts is opened after then waiting for machine self-test, waits for master (display lamp becomes green) opens instrument software after machine self-test.
(7) ABI3500 software kits are opened, check various mating reagent consumptive materials whether have run out with it is expired, it is ensured that Machine is in when can be with detection performance, creates a template, and carrying out detection parameters, (present invention carries out MLPA using Pop7 running gels Reaction product is analyzed), the sample slot of operation is so clicked after name and the settings such as save location, select the sample position to be run and The position in loading hole.
(8) ABI3500 hatch doors are opened, then loading slot are put into the automatic loading arm of machine, closing chamber is clicked behind the door Open the pre- hot function of instrument so that the temperature at instrument position reaches service requirement, then clicks operation instrument.
(9) instrument automatic running, after the data that generate can be automatically saved to specific position, the data of generation use Genmapper analysis softwares are analyzed.
Capillary electrophoresis technique is a kind of very high DNA detection techniques of resolution ratio, and it is 4bp that can clearly tell difference Segment, be most widely used to MLPA reaction products detection.This research is using ABI3500 sequenators to MLPA reaction results Capillary Electrophoresis is carried out, wherein 1 electrocapillary phoresis pipe is to GeneScanTM- 600LIZ Size Standard Marker carry out pipe Electrophoresis, discovery can highly precisely navigate to each each segment, and (20bp to 600bp sizes, orange absorption peak are such as schemed 8), and Capillary Electrophoresis, such as Fig. 9 are carried out to single target spot MLPA reaction products of 17 diagnostic probes one by one simultaneously.It confirms to use The MLPA product sheet segment lengths of this 17 pairs of probes pair, 17 positive plasmid standard items comply fully with the detection length of design, single Peak also demonstrates the preferable specificity of each pair of probe.
The multiple MLPA analyses of 8 pathogenic bacteria positive criteria product of embodiment, 17 detection target spots
1. the positive criteria product solution of 17 detection target spots：17 detection target spot positive criteria product solution mix totally 5 μL。
2. multiple MLPA probe reactions liquid：Each 1 μ L of standard list target spot MLPA probe reaction liquid are taken to be added to the PCR of 200 μ L In reaction tube, piping and druming mixing is prepared into multiple reaction mixed liquor Mix.
3. other operating procedures are identical as 3 in embodiment 7 as reaction system as condition.
As a result such as Figure 10.Blue absorption peak represents multiple MLPA reaction results.Judge Prb1-17 from the peak absorbance result The absorption peak for generating and meeting design length can be reacted, illustrates that this 17 pairs of probe designs are successful.
9 MLPA probe sensitivity of embodiment is verified
By taking the sensitivity technique of probe Prb17 on the right side of No. 17 microorganism haemophilus influenzaes of detection as an example, steps are as follows：
1. the plasmid containing positive target spot DNA is done following gradient dilution with TE buffer solutions：101, 102, 103, 104, 105, 106, 107, 108, 109, 1010(Figure 11, swimming lane 1-10).Probe does following dilution：101, 102, 103, 104, 105, 106, 107, 108。
2. the experiment to be run of design template detection sensitivity：To dilute the template of various concentration as reaction target site, It is reacted using the probe of 1 μm of ol as reactant MLPA, passes through 1% gel electrophoresis analysis.
3. designing probe sensitivity operation test：By taking longest Prb17 probes as an example, does and detected without the dilution of concentration Standard target sequence runs MLPA reactions, 1% detected through gel electrophoresis of reaction product.
4. doing the repeated reactivity worth of repeated experiment verification minimum sensitivity after detected through gel electrophoresis result.MLPA is produced Object uses 1% gel detection.
1) examination criteria product DNA concentration is analyzed
Figure 11 Gel electrophoresis results show Prb17 probe standard target spots dilution 107(swimming lane 7) can clearly see reaction item again Band, 108Band is already close to fuzzy when times (swimming lane 8) concentration dilution.Then the repeated experiment of two dilutions has been done respectively, As (A1-20 swimming lanes are dilution 10 to Figure 128Times, no product detection arrives), standard form does 107(B1-20 swimming lanes are dilution 10 again7 Times, product is steadily detected) dilution after reproducibility still reach 100%, illustrate the detection sensitivity of MLPA probes very Height, can reach the other detection limit of pieck stage and PCR detection sensitivities belong to same rank.
2) MLPA concentration and probe concentrations detection sensitivity is analyzed
Concentration and probe concentration is an important factor for influencing MLPA reaction detection limits, and as shown in figure 13, the Prb17 standards of preparation are done MLPA reaction gel electrophoretograms are carried out after the dilution of various concentration, are had obviously with the concentration gradient dilution testing result of probe Variation, when diluted concentration gradient reaches 104(swimming lane 5) dilution rear electrophoresis band completely disappears.Do 10 simultaneously4With 103Times concentration ladder It spends diluted 20 parallel repetitions to test, dilution 104Product (see Figure 14, A 1-20 swimming lanes) cannot be detected again, and dilutes 103Times It can steadily detect product (see Figure 14, B 1-20 swimming lanes).Illustrate the detectable limit of concentration and probe concentration in nanogram rank, therefore The concentration of probe affects MLPA reaction detections, while can also control different concentration and probe concentrations to control in multiple MLPA reactions Probe peak absorbance obtain relatively good detection result.
10 MLPA probe repeatability of embodiment is verified
Take standard items as reactant, with probe Prb1 (No. 1 microorganism Neisseria meningitidis, the length detected point Wei 144bp), 271) and Prb17 (No. 17 micro- lifes (No. 8 microorganism serratia marcescens, the length detected are respectively to Prb8 Probe Prb17 on the right side of logistics haemophilus influenza, the length detected is respectively 512bp) probes of short, medium and long three length is Example, each probe carries out 20 MLPA and repeats to test, then carries out 1% detected through gel electrophoresis.As shown in figure 15, short, medium and long three The probe MLPA reactions of length have bright single band, tentatively illustrate that probe Repeatability is very good.
This MLPA diagnostic kit demonstrated above has higher probe specificity, sensibility, repeatability.In addition, through meter It calculates, long probe prepared by 1mL PrbX-M13 bacteriophage liquid mediums can theoretically carry out 160,000 MLPA reactions enough, So that the manufacturing cost of probe substantially reduces.It can be seen that MLPA technologies are applied to neurosurgery encephalic bacterial infection Disposable more pathogenic bacteria diagnosis have good market development potential.
Embodiment 11
Following sequences are that company is sent to carry out chemical synthesis and obtain
Encephalic bacterial infection common causative bacterium MLPA detection kits after a kind of neurosurgery, including first group Probe is sequentially detection Neisseria meningitidis, Pseudomonas aeruginosa, Salmonella, thermophilic malt widow to the 17th group of probe Support monad, staphylococcus aureus, staphylococcus epidermis, Klebsiella pneumoniae, serratia marcescens, Lu Shi not lever Bacterium, corynebacterium diphtheriae, streptococcus mitis, Acinetobacter baumannii, streptococcus pneumonia, propionibacterium acnes, listeria spp, cloaca intestines The probe of bacillus and haemophilus influenzae contains left side probe LPO and right side probe RPO in every group of probe respectively：
The left side probe LPO of first group of probe contains sequence shown in SEQ ID NO 1, and right side probe RPO contains SEQ ID Sequence shown in NO 2；
The left side probe LPO of second group of probe contains sequence shown in SEQ ID NO 3, and right side probe RPO contains SEQ ID Sequence shown in NO 4；
The left side probe LPO of third group probe contains sequence shown in SEQ ID NO 5, and right side probe RPO contains SEQ ID Sequence shown in NO 6；
The left side probe LPO of 4th group of probe contains sequence shown in SEQ ID NO 7, and right side probe RPO contains SEQ ID Sequence shown in NO 8；
The left side probe LPO of 5th group of probe contains sequence shown in SEQ ID NO 9, and right side probe RPO contains SEQ ID Sequence shown in NO 10；
The left side probe LPO of 6th group of probe contains sequence shown in SEQ ID NO 11, and right side probe RPO contains SEQ ID Sequence shown in NO 12；
The left side probe LPO of 7th group of probe contains sequence shown in SEQ ID NO 13, and right side probe RPO contains SEQ ID Sequence shown in NO 14；
The left side probe LPO of 8th group of probe contains sequence shown in SEQ ID NO 15, and right side probe RPO contains SEQ ID Sequence shown in NO 16；
The left side probe LPO of 9th group of probe contains sequence shown in SEQ ID NO 17, and right side probe RPO contains SEQ ID Sequence shown in NO 18；
The left side probe LPO of tenth group of probe contains sequence shown in SEQ ID NO 19, and right side probe RPO contains SEQ ID Sequence shown in NO 20；
The left side probe LPO of 11st group of probe contains sequence shown in SEQ ID NO 21, and right side probe RPO contains SEQ Sequence shown in ID NO 22；
The left side probe LPO of 12nd group of probe contains sequence shown in SEQ ID NO 23, and right side probe RPO contains SEQ Sequence shown in ID NO 24；
The left side probe LPO of 13rd group of probe contains sequence shown in SEQ ID NO 25, and right side probe RPO contains SEQ Sequence shown in ID NO 26；
The left side probe LPO of 14th group of probe contains sequence shown in SEQ ID NO 27, and right side probe RPO contains SEQ Sequence shown in ID NO 28；
The left side probe LPO of 15th group of probe contains sequence shown in SEQ ID NO 29, and right side probe RPO contains SEQ Sequence shown in ID NO 30；
The left side probe LPO of 16th group of probe contains sequence shown in SEQ ID NO 31, and right side probe RPO contains SEQ Sequence shown in ID NO 32；
The left side probe LPO of 17th group of probe contains sequence shown in SEQ ID NO 33, and right side probe RPO contains SEQ Sequence shown in ID NO 34.
Above-mentioned probe is used to be hybridized as template using the DNA of measuring samples.
5 ' ends of first group of probe to each right side probe of the 17th group of probe are connected just like SEQ ID NO 35 Shown in BsmI restriction enzyme site sequences, 3 ' end connection just like EcoR V restriction enzyme site sequences shown in SEQ ID NO 36.
The kit further includes the left side probe LPO and right side probe to being connected after hybridization using a pair of of universal primer RPO carries out PCR amplification, and the sense primer and downstream primer of the universal primer contain SEQ ID NO 37 and SEQ ID respectively Sequence shown in NO 38.
Further, which further includes Ligase-65, Ligase buffer, Probe Mix and PCR Primer Mix, wherein
The formula of Ligase-65 is：1U/ μ L, storing liquid be 20mM Tris, pH 7.5,50mM KCl, 1mM DTT, 0.1mM EDTA and 50% (v/v) glycerine；
The formula of Ligase buffer is：10×Ligation Buffer：400mM Tris, pH 7.8,100mM MgCl2, 100mM DTT, 5mM ATP；
The formula of Probe Mix is：A concentration of 4fM of each probe, working solution are 10mM Tris-Cl, pH 8.0,1mM EDTA；
The formula of PCR Primer Mix is：Sense primer containing the universal primer and downstream primer, each primer A concentration of 10pM, sense primer be Cy 5.0 mark, dNTPs 2.5mM, working solution be 10mM Tris-Cl, pH 8.0,1mM EDTA。
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The foregoing is only a preferred embodiment of the present invention, therefore cannot limit the scope of implementation of the present invention according to this, i.e., According to equivalent changes and modifications made by the scope of the claims of the present invention and description, all should still belong in the range of the present invention covers.

Claims (4)

1. encephalic bacterial infection common causative bacterium MLPA detection probes after a kind of neurosurgery, it is characterised in that：Packet First group of probe is included to the 17th group of probe, be sequentially detection Neisseria meningitidis, Pseudomonas aeruginosa, Salmonella, Germ oligotrophy unit cell, staphylococcus aureus, staphylococcus epidermis, Klebsiella pneumoniae, serratia marcescens, Lu Shi Acinetobacter calcoaceticus, corynebacterium diphtheriae, streptococcus mitis, Acinetobacter baumannii, streptococcus pneumonia, propionibacterium acnes, listeria spp, The probe of enterobacter cloacae and haemophilus influenzae, every group of probe are made of left side probe LPO and right side probe RPO respectively, institute Left side probe is stated by hybridization sequences and the upstream universal primer sequence as shown in SEQ ID NO 37 for being connected to the end of hybridization sequences 5 ' Row composition, the right side probe by hybridization sequences, be connected to hybridization sequences 5 ' end the BsmI enzymes as shown in SEQ ID NO 35 Enzyme site sequence is connected to EcoR V restriction enzyme site sequence GATATC and be connected to EcoR V digestions position that hybridization sequences 3 ' are held The reverse complementary sequence composition for the SEQ ID NO 38 that point sequence 3 ' is held, wherein
The hybridization sequences of the left side probe LPO of first group of probe are as shown in SEQ ID NO 1, the hybridization sequences of right side probe RPO As shown in SEQ ID NO 2；
The hybridization sequences of the left side probe LPO of second group of probe are as shown in SEQ ID NO 3, the hybridization sequences of right side probe RPO As shown in SEQ ID NO 4；
The hybridization sequences of the left side probe LPO of third group probe are as shown in SEQ ID NO 5, the hybridization sequences of right side probe RPO As shown in SEQ ID NO 6；
The hybridization sequences of the left side probe LPO of 4th group of probe are as shown in SEQ ID NO 7, the hybridization sequences of right side probe RPO As shown in SEQ ID NO 8；
The hybridization sequences of the left side probe LPO of 5th group of probe are as shown in SEQ ID NO 9, the hybridization sequences of right side probe RPO As shown in SEQ ID NO 10；
The hybridization sequences of the left side probe LPO of 6th group of probe are as shown in SEQ ID NO 11, the hybridization sequences of right side probe RPO As shown in SEQ ID NO 12；
The hybridization sequences of the left side probe LPO of 7th group of probe are as shown in SEQ ID NO 13, the hybridization sequences of right side probe RPO As shown in SEQ ID NO 14；
The hybridization sequences of the left side probe LPO of 8th group of probe are as shown in SEQ ID NO 15, the hybridization sequences of right side probe RPO As shown in SEQ ID NO 16；
The hybridization sequences of the left side probe LPO of 9th group of probe are as shown in SEQ ID NO 17, the hybridization sequences of right side probe RPO As shown in SEQ ID NO 18；
The hybridization sequences of the left side probe LPO of tenth group of probe are as shown in SEQ ID NO 19, the hybridization sequences of right side probe RPO As shown in SEQ ID NO 20；
The hybridization sequences of the left side probe LPO of 11st group of probe are as shown in SEQ ID NO 21, the hybridization sequence of right side probe RPO Row are as shown in SEQ ID NO 22；
The hybridization sequences of the left side probe LPO of 12nd group of probe are as shown in SEQ ID NO 23, the hybridization sequence of right side probe RPO Row are as shown in SEQ ID NO 24；
The hybridization sequences of the left side probe LPO of 13rd group of probe are as shown in SEQ ID NO 25, the hybridization sequence of right side probe RPO Row are as shown in SEQ ID NO 26；
The hybridization sequences of the left side probe LPO of 14th group of probe are as shown in SEQ ID NO 27, the hybridization sequence of right side probe RPO Row are as shown in SEQ ID NO 28；
The hybridization sequences of the left side probe LPO of 15th group of probe are as shown in SEQ ID NO 29, the hybridization sequence of right side probe RPO Row are as shown in SEQ ID NO 30；
The hybridization sequences of the left side probe LPO of 16th group of probe are as shown in SEQ ID NO 31, the hybridization sequence of right side probe RPO Row are as shown in SEQ ID NO 32；
The hybridization sequences of the left side probe LPO of 17th group of probe are as shown in SEQ ID NO 33, the hybridization sequence of right side probe RPO Row are as shown in SEQ ID NO 34.
2. encephalic bacterial infection common causative bacterium MLPA detection kits after a kind of neurosurgery, it is characterised in that： Including probe described in claim 1.
3. detection kit as claimed in claim 2, it is characterised in that：Further include a pair of of universal primer, the universal primer Sense primer and downstream primer respectively as shown in SEQ ID NO 37 and SEQ ID NO 38.
4. detection kit as claimed in claim 3, it is characterised in that：Further include Ligase-65, Ligase buffer, Probe Mix and PCR Primer Mix, wherein
The formula of Ligase-65 is：1U/ μ L, storing liquid are 20mM Tris, 50mM KCl, the 1mM DTT of pH 7.5, 0.1mM EDTA and 50% (v/v) glycerine；
The formula of Ligase buffer is：10×Ligation Buffer：400mM Tris, the 100mM MgCl of pH 7.82, 100mM DTT, 5mM ATP；
The formula of Probe Mix is：A concentration of 4fM of each probe, working solution are the 10mM Tris-Cl, 1mM of pH 8.0 EDTA；
The formula of PCR Primer Mix is：Sense primer containing the universal primer and downstream primer, dNTPs 2.5mM； Working solution is 10mM Tris-Cl, the 1mM EDTA of pH 8.0；A concentration of 10pM of each primer, sense primer are marked for Cy 5.0 Note.
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