CN108753930A - The method of encephalic bacterium infection after the quick detection techniques of multiple real time fluorescence PCR - Google Patents
The method of encephalic bacterium infection after the quick detection techniques of multiple real time fluorescence PCR Download PDFInfo
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Abstract
The present invention provides a kind of methods of encephalic bacterium infection after quick detection techniques of multiple real time fluorescence PCR, belong to technical field of molecular biology.This method is detected encephalic surgical operation fever patient cerebrospinal fluid using multiple fluorescence PCR technology, realizes and carries out parting detection to a variety of common intracranial infection bacteriums.Whether encephalic has bacterium infection after the method for the present invention can realize quick detection technique, it being capable of the common bacteria types of Rapid identification surgical site infections patients with intracranial infection, and solve the problems, such as that cultivation cannot identify novel bacterial and can not cultivate bacterium, its detection method specificity and high sensitivity, reduce the false positive of PCR detections, shorten detection cycle, can quick diagnosis encephalic bacterium infection early, for blocking clinical infection and direction of medication usage to have significance.
Description
Technical field
The invention belongs to molecular biology and medicine technology field, are related to a kind of fluorescence detection method, and in particular to a kind of
The method of encephalic bacterium infection after the quick detection techniques of multiple real time fluorescence PCR.
Background technology
Intracranial infection is common one of the severe complication of neurosurgery Craniotomy, can cause nervous function
Damage, incidence is about 2.6%~30%.Once generation not only seriously affects the prognosis of patient, but also symptom is not easy to control,
Usually threat to life.Since encephalic bacterium infection usually involves brain, spinal cord and neighbouring anatomical tissue, patient is often accompanied by high cranium
The symptoms such as pressure, the disturbance of consciousness, brain edema, epilepsy, patient's prognosis is totally poor, and case fatality rate is up to 27.4%~39.2%, to it
Family and society cause serious burden.And these patients as can in time, correctly diagnose its pathogen, majority has well
Prognosis.Therefore, the pathogen of bacterium infection carries out quick and accurate detection and identification then in split cranium postoperative brain spinal fluid
Seem particularly significant.
Central nervous system is not susceptible to infect due to organization protections such as blood-brain barrier, meninx, skull and scalps.Especially
It is the special protection function of blood-brain barrier, the incidence of intracranial infection is less than body other systems, but operation of opening cranium can make these
Barrier is destroyed, and brain tissue is made to be communicated with the external world, therefore increases the occurrence probability of intracranial infection.Jiang Fei etc. opens 579
Cranium postoperative patient cerebrospinal fluid bacterial growth distribution experiment and drug sensitivity test result carry out analysis and find, cerebrospinal fluid Bacteria Culture
Positive bacterium is numerous, and first 15 are followed successively by：Coagulase-negative staphylococci, staphylococcus aureus, streptococcus pneumonia, B
Group streptococcus, corynebacterium diphtheroides, streptococcus fecalis, bargen's streptococcus, aerococcus viridans, Acinetobacter lwoffii, Bao Man not lever
Bacterium, Pseudomonas aeruginosa, escherichia coli, enterobacter cloacae, clostridium perfringen and Klebsiella Pneumoniae.
In neurosurgery Craniotomy, patient's high fever is most common symptom, but whether there is intracranial infection, which kind of is
Bacterium infection is that neurosurgeon is concerned about and urgent problem the most.Intracranial infection common gram positive pathogenic bacterium has
Staphylococcus aureus, enterococcus faecalis, streptococcus pneumonia, gram negative pathogenic bacteria have escherichia coli, Pseudomonas aeruginosa,
Klebsiella Pneumoniae and Acinetobacter bauamnnii.It there is no the Rapid identification means for the above-mentioned source of infection in clinical at present, and cultivate
The identification method period is longer, and the source of infection has the characteristic of fast breeding, can not be easy to delay treatment while accurate medication
Best opportunity causes the heavy losses of sufferer.If can start it is a kind of can quickly, Precise Diagnosis Craniotomy fever patient
With the presence or absence of bacterium infection, the clinical testing procedure of further qualitative Species of Pathogens will provide for clinician's rational use of medicines
Strong foundation.
At present at home and abroad in larger medical mechanism, simultaneously to the pathogeny detection method of Craniotomy encephalic bacterium infection
There is no important breakthrough.It is still for the detection most popular method of encephalic bacterium infection after operations on cranium and brain both at home and abroad：Cerebrospinal fluid disease
Opportunistic pathogen dyeing microscopic examination and culture, wherein bacterial cultivation are using its high specific by as " goldstandard ", but bacterial cultivation exists
The time-consuming, disadvantages such as susceptibility is low.Then it is biochemical to also need to certain time progress firstly the need of at least incubation of 8h for bacterial cultivation
Or immunological testing identifies bacterium, influences its early diagnosis and timely, the effective use of antibiotic.In addition, latex agglutination, exempting from
Though the experimental techniques such as epidemic disease electrophoresis, ELISA and cerebrospinal fluid cell count and biochemical analysis respectively have feature, general lack of
Sensibility and specificity, it is difficult to meet clinical requirement, seriously lag behind clinical treatment, the death rate of such disease is caused to occupy height not
Under.
Since the 1990s, as molecular biology develops rapidly, condition is provided to solve this problem, especially
The biochip technology occurred in recent years, be highly miniaturized with it, intensive and volume is small, it is easy to operate, can be simultaneously
The advantages that specific detection various biomolecules, has broad application prospects in the detection of pathogenic microorganism.But it is previous all kinds of
Biochip is mainly limited to basic research and the drug of the life science in laboratory because of characteristics applications such as its high throughput, quotient's density
Development field.In recent years, it using round pcr as the diagnostic technique in molecular biology of representative, is increasingly becoming in biomedical sector most
Valuable research means.Various round pcrs have attracted tremendous attention in diagnosis field immediately since foundation, more next
More amplifications and detection for being applied to pathogen DNA in central nervous system (CNS) infection, morning is provided for bacterium infection
Phase, detection means quickly, sensitive.But general regular-PCR operation is cumbersome and is easy pollution.
Real-time fluorescent PCR technology is expected to solve problem above, and the researchs such as Henry M Wu find fluorescent quantitative PCR technique inspection
Pathogens In Cerebrospinal Fluid is surveyed than cultivation high sensitivity, in 451 parts of CSF samples, cultivation finds 80 bacterium infection positives
(17.7%), as a result the positive germs (25.1%) of real-time fluorescent PCR technology discovery 113 are confirmed equal by Gram's staining
It is correct.The Real-Time Fluorescent Quantitative PCR Technique probe of K.GREISEN and Henry M Wu designs is primarily directed in bacterium ribose
Body 16S rRNA genes are realized, qualitative analysis can go out whether have bacterium infection very well by three probes, tentatively telling is
Gram-positive bacteria and Gram-negative bacteria, but which kind of common bacteria cannot specifically identify is, is not easy to further instruct clinical
Doctor is administered, therefore the technology has certain limitation.
Multiple real time fluorescence round pcr qualitative detection encephalic surgical site infections bacterium infection can be utilized, and is realized special
Property a variety of common pathogens of identification, can quickly, Precise Diagnosis Craniotomy fever patient whether there is bacterium infection, and it is accurate
Certainty Species of Pathogens will provide stronger foundation for clinician's rational use of medicines.Current such method is at home and abroad still
Without relevant report.
Invention content
The purpose of the present invention is exactly in order to solve the above-mentioned technical problem, and to provide a kind of multiple real time fluorescence PCR and quickly examine
The method for surveying postoperative encephalic bacterium infection, whether encephalic has bacterium infection, energy after method of the invention can realize quick detection technique
It is enough rapidly and efficiently to detect that corresponding bacterial species, the specificity and high sensitivity of detection method reduce the vacation sun of PCR detections
Property, shorten detection cycle, can quick diagnosis encephalic bacterium infection early, and solve cultivation cannot identify novel bacterial and
The problem of bacterium can not be cultivated, for blocking clinical infection and direction of medication usage to have significance.
To achieve the goals above, the technical solution adopted by the present invention is：
The method of encephalic bacterium infection, includes the following steps after a kind of quick detection techniques of multiple real time fluorescence PCR：
(1) the bacterial strain hybrid dna of pathogen sample is extracted；
(2) fluorescence quantitative PCR reaction solution is prepared：Mg2+4mM、dNTP 0.3mM、10×Hot Start PCR buffer、
Each 200nM of upstream and downstream primer, specific probe 100nM, Hot Start Taq DNA Ploymerase 3.5U；Then it uses
EcoR I and DNase I digestions handle reaction solution；
(3) following three groups of reaction solutions are chosen, quantitative fluorescent PCR inspection is carried out to the bacterial strain hybrid dna template of pathogen sample
It surveys；
In first group of reaction solution, primer and probe sequence is：
Sense primer：5 '-AYAAACYGGAGGAAGGTG-3 ', downstream primer：5'-GATCCAARCGCARRTTCCG-3'；
Probe：G+ probes：5 '-FAM-TCAAATCATCATGCCCC-MGB, G- probe：5'-VIC-ATCATGGCCCTTACGACC-
MGB；
In second group of reaction solution, including following three pairs of primer and probes：
I) sense primer：5 '-ATTTATTACAATTAACGAAATGGGCA-3 ', downstream primer：5'-
CAACACCCTGAACTTCACC-3 ', probe：5'-FAM-ATTAACTGGATGGTACGCGCGAAGAATCGC-BHQ1-3'；
II) sense primer：5 '-TGGGTAGCGGAGAAATTCCAA-3 ', downstream primer：5'-
AGTCCAAACAGTGCTCTACCTCCA-3 ', probe：5'-JOE-CGCAGCGAAAGCGAGTCTGAATAGGG-BHQ1-3'；
III) sense primer：5 '-ACTCTTACGCAATCTAGCAGATG-3 ', downstream primer：5'-
TGCAATACTCGTGCGTTTTAA-3 ', probe：5'-TexasRed-CGAAAACGCTTGATACAGGGAGTTT-BHQ2-3'；
In third group reaction solution, including following four pairs of primer and probes：
IV) sense primer：5 '-TCCTGCTGATTGACGATCACCC-3 ', downstream primer：5'-
CCAGCGTTTCCAGACCGTTCAT-3 ', probe：5'-JOE-CCAGATATCACCGTGGTTGGCGAA-BHQ1-3'；
V) sense primer：5 '-CGACGCCTGAAGCGTGACGAAC-3 ', downstream primer：5'-
GGCTTGGTGATGTAGTCG-3 ', probe：5'-FAM-CATGCTCACCGCCAAGG-BHQ1-3'；
VI) sense primer：5 '-CTGATAGTTGAAGACGAACCCGT-3 ', downstream primer：5'-
AAATGTCGCAAATCATCAGGTCC-3 ', probe：5'-TexasRed-TGGCTGACCTCGCTGGGAGCAA-BHQ2-3'；
VII) sense primer：5 '-GGGCGAAGAAGCAATTGCT-3 ', downstream primer：5'-
GCGAGGCCGCTTACAGCAATG-3 ', probe：5'-CY5-CATCGGTGGCGTAGAAACAAC–BHQ3-3'；
The step of fluorescence quantitative PCR detection is：1) PCR inspections first are carried out to hybrid dna template with first group of reaction solution
It surveys；2) second group of reaction solution and third group reaction solution are used again, are carried out by arbitrary precedence or simultaneously to hybrid dna template
PCR is detected.
Primer probe sequence using the present invention is detected respectively as described above by three groups of reaction solutions, can be fast
Speed detects whether postoperative encephalic has a bacterium infection, and can in Rapid identification surgical site infections patients with intracranial infection 7 kinds it is common
Bacteria types, detection method specificity and high sensitivity, can quick diagnosis encephalic bacterium infection early, blocking is faced
Bed infection and direction of medication usage have significance.
Further, in step (3), using the bacterial strain hybrid dna of pathogen sample and sterile water as template, it is fixed to carry out fluorescence
PCR amplification is measured, amplification program is：Carry out 45 cycles after 95 DEG C of pre-degeneration 5min, each cycle includes 95 DEG C and is denaturalized 5s, 60 DEG C
Annealing extends 90s.
Further, in step (1), the DNA of cause of disease bacteria strain, nucleic acid extraction liquid used are extracted using pyrolysis method
For：5%Chelex-100,0.5%NP-40,0.5%Triton X-100,1mM EDTA.
Further, in step (3), when detecting each time, the volumetric usage ratio of the reaction solution and template is 9:1.
Further, second group of reaction solution can detect that staphylococcus aureus, enterococcus faecalis and streptococcus pneumonia.
Further, third group reaction solution can detect that escherichia coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae and Bao
Graceful acinetobacter calcoaceticus.
Further, further include the primer and probe of reference gene GAPDH in described second group and third group reaction solution,
Upstream primer sequence is：5 '-CGCTCCCTCTTTCTTTGCAG-3 ', downstream primer sequence are：5'-
TACCAAAGTTGTCATGGATGACC-3 ', probe sequence are：5'-TAMARA-TGCACCACCAACTGCTTAGCACCCC-
BHQ2-3’。
Further, in step (2), the dosage of the EcoR I enzymes is 2U, and the dosage of I enzymes of the DNase is 1.5U.
Further, by digestion treated reaction solution in 37 DEG C of water-bath 60min, then at 75~80 DEG C of water-baths 5~
Then 15min carries out fluorescence quantitative PCR detection.
Beneficial effects of the present invention are as follows：
(1) present invention establishes a kind of multiplex PCR fast diagnosis method can be used for the postoperative easy infection bacterium of encephalic, special
It is anisotropic good, to postoperative encephalic staphylococcus aureus, enterococcus faecalis, streptococcus pneumonia, escherichia coli, Pseudomonas aeruginosa,
The infection of this 7 kinds of bacterial strains of Klebsiella Pneumoniae and Acinetobacter bauamnnii can realize that Rapid identification detects；
(2) the parting detection to the source of infection can be rapidly completed in detection method fast and easy of the invention in 4 hours,
And lowest detection is limited to 102CFU/mL is identical as cultivation；
(3) multiple real time fluorescence PCR detection method specificity and high sensitivity of the invention, reduce the vacation of PCR detections
The positive shortens detection cycle, can quick diagnosis encephalic bacterium infection early, for blocking clinical infection and direction of medication usage to have
Significance.
Description of the drawings
Fig. 1 is the bacterium hybrid dna agarose gel electrophoresis figure extracted using nucleic acid extraction liquid；
Fig. 2 is the amplification figure for not removing endogenous nucleic acid pollution group amplification escherichia coli dna and sterile water；
Fig. 3 is the amplification figure for removing endogenous nucleic acid pollution group amplification escherichia coli dna and sterile water；
Fig. 4 is gram-positive bacteria primed probe specificity verification result figure；
Fig. 5 is Gram-negative bacteria primed probe specificity verification result figure；
Fig. 6 is amplification figure of the 1st group of reaction solution to staphylococcus aureus and escherichia coli hybrid dna；
Fig. 7 is amplification figure of the 2nd group of reaction solution to bacterium hybrid dna；
Fig. 8 is amplification figure of the 3rd group of reaction solution to bacterium hybrid dna.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments to the present invention
It is specifically described, it is necessary to, it is noted that following embodiment is used only for that the present invention is explained and illustrated, be not used to
Limit the present invention.Those skilled in the art still belong to according to some nonessential modifications and adaptations that foregoing invention content is made
In protection scope of the present invention.
Embodiment
1 material and instrument
1.1 material
Strains tested：Staphylococcus aureus, enterococcus faecalis, streptococcus pneumonia, escherichia coli, Pseudomonas aeruginosa,
Klebsiella Pneumoniae and Acinetobacter bauamnnii are given for 7 plants totally by Shanghai Disease Prevention and Control Centre, clinical separation strain tuberculosis
Mycobacteria, enterobacter cloacae, clostridium perfringen, herpes simplex virus and cytomegalovirus totally 5 plants by Guangzhou medicine third
Affiliated hospital provides.
All bacterial strains are inoculated in beef-protein medium, and 37 DEG C are cultivated 18h, spare after transferring 3 times.
1.2 reagents and instrument
Reagent：Hot Start Taq DNA Ploymerase,10×Hot Start PCR buffer,dNTP,
MgCl2, Hongene Biotechnology Co., Ltd；EcoR I, DNase I, precious bioengineering (Dalian) Co., Ltd；PCR draws
Object is synthesized by Invitrogen (Shanghai) Trading Co., Ltd.；293 human gene group DNAs, Nanjing Genscript Biotechnology Co., Ltd.；
Ⅰpattern herpesvirus DNA, is given by Shanghai Disease Prevention and Control Centre；Polystyrene divinyl chelating resin (Chelex-
100), Bole's life medical product (Shanghai) Co., Ltd.；NP-40, Triton X-100, lysozyme, life work bioengineering (on
Sea) limited liability company；Ethylenediamine tetra-acetic acid (EDTA), Sinopharm Chemical Reagent Co., Ltd. are pure to analyze.
Instrument：ABI 7500 Real-Time PCR System, ThermoFisher.
2 experimental methods
The extraction of 2.1 bacterial strain DNA
1. taking 1ml bacterium solutions in 1.5ml centrifuge tubes, room temperature 12000rpm is centrifuged 2 minutes, abandons supernatant.Add into centrifuge tube
Enter 100ul nucleic acid extractions liquid (5%Chelex-100,0.5%NP-40,0.5%Triton X-100,1mM EDTA), bacterium is resuspended
Body；2. 6ul 50mg/ml lysozymes are added, after 37 DEG C of water-bath 30min, 85 DEG C of water-bath 10min；3. 12000rpm is centrifuged 2 minutes,
Supernatant is detected for PCR.
All bacterial strain DNA are extracted according to above-mentioned steps, and the bacterial strain hybrid dna extracted is subjected to 1% Ago-Gel
Electrophoresis.
The agarose gel electrophoresis figure of the bacterial strain hybrid dna extracted is shown in Fig. 1.The corresponding bacteriums of 1-7 in figure are respectively
Staphylococcus aureus, enterococcus faecalis, streptococcus pneumonia, escherichia coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae and Bao Man
Acinetobacter calcoaceticus.As shown in Figure 1, all swimming lane bands are brighter and clear, illustrate the DNA of bacteria purity of said extracted method extraction
Height meets the requirement of quantitative fluorescent PCR.
2.2 prepare fluorescence quantitative PCR reaction solution
Quantitative fluorescent PCR reaction system is：Mg2+4mM, dNTP 0.3mM, 10 × Hot Start PCR buffer, up and down
Swim each 200nM of primer, specific probe 100nM, Hot Start Taq DNA Ploymerase 3.5U.
Take following three groups of reaction solutions：
In (one) the 1st group of reaction solution：
Upstream primer sequence is：5 '-AYAAACYGGAGGAAGGTG-3 ',
Downstream primer sequence is：5'-GATCCAARCGCARRTTCCG-3'；
Probe sequence is：
G+ probes：5'-FAM-TCAAATCATCATGCCCC-MGB；
G- probes：5'-VIC-ATCATGGCCCTTACGACC-MGB；
Contain following three pairs of primer and probe sequences in (two) the 2nd groups of reaction solutions：
I) sense primer：5 '-ATTTATTACAATTAACGAAATGGGCA-3 ', downstream primer：5'-
CAACACCCTGAACTTCACC-3 ', probe：5'-FAM-ATTAACTGGATGGTACGCGCGAAGAATCGC-BHQ1-3'；
II) sense primer：5 '-TGGGTAGCGGAGAAATTCCAA-3 ', downstream primer：5'-
AGTCCAAACAGTGCTCTACCTCCA-3 ', probe：5'-JOE-CGCAGCGAAAGCGAGTCTGAATAGGG-BHQ1-3'；
III) sense primer：5 '-ACTCTTACGCAATCTAGCAGATG-3 ', downstream primer：5'-
TGCAATACTCGTGCGTTTTAA-3 ', probe：5'-TexasRed-CGAAAACGCTTGATACAGGGAGTTT-BHQ2-3'；
Contain following four pairs of primer and probe sequences in (three) the 3rd groups of reaction solutions：
IV) sense primer：5 '-TCCTGCTGATTGACGATCACCC-3 ', downstream primer：5'-
CCAGCGTTTCCAGACCGTTCAT-3 ', probe：5'-JOE-CCAGATATCACCGTGGTTGGCGAA-BHQ1-3'；
V) sense primer：5 '-CGACGCCTGAAGCGTGACGAAC-3 ', downstream primer：5'-
GGCTTGGTGATGTAGTCG-3 ', probe：5'-FAM-CATGCTCACCGCCAAGG-BHQ1-3'；
VI) sense primer：5 '-CTGATAGTTGAAGACGAACCCGT-3 ', downstream primer：5'-
AAATGTCGCAAATCATCAGGTCC-3 ', probe：5'-TexasRed-TGGCTGACCTCGCTGGGAGCAA-BHQ2-3'；
VII) sense primer：5 '-GGGCGAAGAAGCAATTGCT-3 ', downstream primer：5'-
GCGAGGCCGCTTACAGCAATG-3 ', probe：5'-CY5-CATCGGTGGCGTAGAAACAAC–BHQ3-3'；
Wherein, in above-mentioned 2nd group and the 3rd group of reaction solution the also GAPDH containing reference gene primer and probe sequence, upstream
Primer sequence is：5 '-CGCTCCCTCTTTCTTTGCAG-3 ', downstream primer sequence are：5'-
TACCAAAGTTGTCATGGATGACC-3 ', probe sequence are：5'-TAMARA-TGCACCACCAACTGCTTAGCACCCC-
BHQ2-3’。
Endogenous nucleic acid pollutes in 2.3 removal reaction solutions
Above-mentioned three groups of reaction solutions are taken, handle reaction solution with EcoR I and DNase I digestions, wherein the dosage of I enzymes of DNase
Dosage for 1.5U, EcoR I enzymes is 2U, obtains endonuclease reaction liquid A and endonuclease reaction liquid B, endonuclease reaction liquid A is：Mg2+4mM、
dNTP 0.3mM,10×Hot Start PCR buffer,DNaseⅠ 1.5U；Endonuclease reaction liquid B is：Upstream and downstream primer is each
200nM, specific probe 100nM, Hot Start Taq DNA Ploymerase 3.5U, EcoR I 2U.
By endonuclease reaction liquid A in 37 DEG C of water-bath 60min, then at 75 DEG C of water-bath 15min；Simultaneously by endonuclease reaction liquid B in 37
DEG C water-bath 60min, then at 80 DEG C of water-bath 5min.The endogenous nucleic acid pollution in reaction solution is eliminated at this time.
30 μ L endonuclease reaction liquid A and 15 μ L endonuclease reaction liquid B mixings are taken, 5 μ L templates are added, carry out quantitative fluorescent PCR expansion
Increase, amplification program is：45 cycles are carried out after 95 DEG C of pre-degeneration 5min, each cycle includes 95 DEG C of denaturation 5s, and 60 DEG C of annealing are prolonged
Stretch 90s.
2.4 method validation
(1) removal that endogenous nucleic acid pollutes in reaction system
The 1st group of reaction solution is taken, respectively using escherichia coli dna and sterile water as template, carries out fluorescent quantitative PCR,
Comparison does not remove fluorescent quantitative PCR result between contaminating endogenous group and removal contaminating endogenous group.
Do not remove endogenous nucleic acid pollution group and remove endogenous nucleic acid pollution group fluorescent quantitative PCR result see Fig. 2 and
Fig. 3, wherein Fig. 2 are the amplification for not removing endogenous nucleic acid pollution group amplification escherichia coli dna and sterile water, and Fig. 3 is
Remove the amplification of endogenous nucleic acid pollution group amplification escherichia coli dna and sterile water.By Fig. 2 and Fig. 3 it is found that not removing
The result that endogenous nucleic acid pollution group expands sterile water is the positive, and removes the result of endogenous nucleic acid pollution group amplification sterile water
For feminine gender.Therefore, method using the present invention eliminates the pollution of the endogenous nucleic acid in reaction system, avoids false positive very well
As a result generation.
(2) specificity verification：
The 2nd group of reaction solution and the 3rd group of reaction solution are taken, fluorescence quantitative PCR detection gram-positive bacteria hybrid dna (gold is carried out
Staphylococcus aureus, enterococcus faecalis, streptococcus pneumonia), Gram-negative bacteria hybrid dna (escherichia coli, P. aeruginosa
Bacterium, Klebsiella Pneumoniae and Acinetobacter bauamnnii) and clinical separation strain mycobacterium tuberculosis, enterobacter cloacae, clostridium perfringen,
Herpes simplex virus and cytomegalovirus dna.
2nd group of reaction solution detection gram-positive bacteria hybrid dna has amplification curve, and detects Gram-negative bacteria mixing
DNA and clinical separation strain mycobacterium tuberculosis, enterobacter cloacae, clostridium perfringen, herpes simplex virus and cytomegalovirus dna
Without amplification curve, result is feminine gender, as a result sees Fig. 4.3rd group of reaction solution detection Gram-negative bacteria hybrid dna has amplification curve,
And other DNA are detected without amplification curve, result is feminine gender, as a result sees Fig. 5.
The above results show that the 2nd group of reaction solution of the invention and the 3rd group of reaction solution have most of intracranial infection bacteriums
The high feature of specificity.
2.5 fluorescence quantitative PCR detection
The 1st group of reaction solution is taken, using staphylococcus aureus, escherichia coli and 293 human genome hybrid dnas as template,
Fluorescence quantitative PCR detection result such as Fig. 6.It will be appreciated from fig. 6 that the 1st group of reaction solution can detect that gram-positive bacteria, Gram-negative
Bacterium and human genome.Wherein：The channels FAM result is shown as the gram-positive bacterium positive；The channels VIC result is shown as gram
Negative bacteria is positive；The channels TAMARA result is shown as in template that there are human genomes.
The 2nd group of reaction solution is taken, is with staphylococcus aureus, enterococcus faecalis, streptococcus pneumonia and human genome hybrid dna
Template, fluorescence quantitative PCR detection result such as Fig. 7.As shown in Figure 7, the 2nd group of reaction solution can accurately detect Staphylococcus aureus
Bacterium, enterococcus faecalis, streptococcus pneumonia and human genome.Wherein：The channels FAM result is shown as S. aureus-positive；
The channels TexasRed result is shown as the streptococcus pneumonia positive；The channels JOE result is shown as the enterococcus faecalis positive；The channels TAMARA
As a result it is shown as that there are human genomes in template.
The 3rd group of reaction solution is taken, with escherichia coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae, Acinetobacter bauamnnii and people
Genome hybrid dna is template, fluorescence quantitative PCR detection result such as Fig. 8.As shown in Figure 8, the 3rd group of reaction solution can be detected accurately
Go out escherichia coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae, Acinetobacter bauamnnii and human genome.Wherein：It ties in the channels FAM
Fruit is shown as the pseudomonas aeruginosa positive；The channels TexasRed result is shown as the Klebsiella Pneumoniae positive；The channels JOE result is aobvious
It is shown as the escherichia coli positive；The channels CY5 result is shown as the Acinetobacter bauamnnii positive；The channels TAMARA result is shown as template
In there are human genomes.
When carrying out parting detection to pathogen hybrid dna, following method may be used and be detected：1) first with the 1st group
Reaction solution detects pathogen sample DNA.If testing result is feminine gender, i.e., pattern detection result is feminine gender；If 2) 1. testing result
It is positive and VIC fluorescence channels are negative for FAM fluorescence channels, then be detected with the 2nd group of reaction solution；2. if testing result is FAM
Fluorescence channel is negative and VIC fluorescence channels are positive, then is detected with the 3rd group of reaction solution；3. if testing result is logical for FAM fluorescence
Road is positive and VIC fluorescence channels are positive, is detected respectively with the 2nd group and the 3rd group of reaction solution.
Wherein the 2) in step, and 1. 2. 3. step has no sequencing, only need to determine which is used according to corresponding testing result
Kind method is detected.
3 detection limit tests
3.1 test method
With sterile saline adjustment staphylococcus aureus, enterococcus faecalis, streptococcus pneumonia, escherichia coli, green pus
Pseudomonad, Klebsiella Pneumoniae and Acinetobacter bauamnnii liquid a concentration of 105、104、103、102、101CFU/ml respectively takes 200 μ
L bacterium solutions are separately added into isometric verified sterile CSF sample, as simulate CSF sample.According to National Standard Method to institute
There is the cerebrospinal fluid analog sample of concentration to carry out culture detection.The detection method of staphylococcus aureus is with reference to GB4789.10-
2010；The detection method of enterococcus faecalis is with reference to GB4789.35-2010；The detection method of streptococcus pneumonia is with reference to GB4789.11-
2014；The detection method of escherichia coli is with reference to GB4789.38-2012；The detection method reference of Pseudomonas aeruginosa
GB4789.29-2003；The detection method of Klebsiella Pneumoniae is with reference to GB4789.41-2016；The detection side of Acinetobacter bauamnnii
Method is with reference to GB4789.28-2013.Meanwhile nucleic acid is carried out to the simulation CSF sample of all concentration according to 2.1 method and is carried
It takes, the detection of fluorescent quantitation multiplex PCR.Fluorescence quantitative PCR detection result and cultivation testing result are compared, multiplex PCR skill is verified
Art detects the sensitivity and specificity of the postoperative easy infection bacterium of encephalic.
3.2 test result
Comparison cultivation and fluorescent quantitation multiplex PCR testing result, concrete outcome are shown in Table 1.In addition to streptococcus pneumonia, remaining
Two methods of the detection limit test result of bacterium is consistent, shows that the detection sensitivity of the present invention and specificity are good.Due to pneumonia chain
The condition of culture of coccus is more special, causes the detection of its cultivation to limit and is less than quantitative fluorescent PCR.
1 cultivation of table and fluorescent quantitation multiplex PCR detection limit test result
4 clinical performances are evaluated
4.1 material
Shenzhen Children's Hospital's in March, 2015 is collected to the cerebrospinal fluid mark of Craniotomy fever patient between in March, 2017
This, wherein man 64, female 41；1~14 years old age, average (3 ± 0.6) year；9~45kg of weight, average (15.6 ±
4.25)kg。
4.2 method
Using the above-mentioned fluorescent quantitation multi-PCR detection method of the present invention.
4.3 result
Detect samples of CSF 105 altogether, wherein G+ bacterium 65 (61.90%), G- bacterium 40 (38.10%), 2 kinds with
Upper bacteria mixed infection 5；As a result report consistency up to 100 with clinical laboratory cultivation.2 are the results are shown in Table after carrying out DNA sequencing：
DNA recall rates are up to 80%.
2 fluorescent quantitation multiplex PCR testing result of table
| Pathogen | Number of cases | Composition ratio % |
| G+ bacterium | 65 | 61.90 |
| Staphylococcus aureus | 24 | 22.85 |
| Enterococcus | 3 | 2.86 |
| Surface staphylococcus | 19 | 18.10 |
| Coagulase-negative staphylococci | 7 | 6.67 |
| G- bacterium | 40 | 38.10 |
| Escherichia coli | 9 | 8.57 |
| Klebsiella Pneumoniae | 3 | 2.86 |
| Enterobacter cloacae | 3 | 2.86 |
| Pseudomonas aeruginosa | 10 | 9.52 |
| Acinetobacter bauamnnii | 4 | 3.81 |
| Acinetobacter lwoffii | 2 | 1.90 |
Sequence table
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Claims (9)
1. a kind of method of encephalic bacterium infection after quick detection techniques of multiple real time fluorescence PCR, which is characterized in that the method packet
Include following steps：
(1) the bacterial strain hybrid dna of pathogen sample is extracted；
(2) fluorescence quantitative PCR reaction solution is prepared：Mg2+4mM, dNTP 0.3mM, 10 × Hot Start PCRbuffer, upstream and downstream
Each 200nM of primer, probe 100nM, Hot Start Taq DNAPloymerase 3.5U；Then EcoR I and DNase I are used
Digestion handles reaction solution；
(3) following three groups of reaction solutions are chosen, fluorescence quantitative PCR detection is carried out to the bacterial strain hybrid dna template of pathogen sample；
In first group of reaction solution, primer and probe sequence is：
Sense primer：5 '-AYAAACYGGAGGAAGGTG-3 ', downstream primer：5'-GATCCAARCGCARRTTCCG-3'；It visits
Needle：G+ probes：5 '-FAM-TCAAATCATCATGCCCC-MGB, G- probe：5'-VIC-ATCATGGCCCTTACGACC-MGB；
In second group of reaction solution, including following three pairs of primer and probes：
I) sense primer：5 '-ATTTATTACAATTAACGAAATGGGCA-3 ', downstream primer：5'-
CAACACCCTGAACTTCACC-3 ', probe：5'-FAM-ATTAACTGGATGGTACGCGCGAAGAATCGC-BHQ1-3'；
II) sense primer：5 '-TGGGTAGCGGAGAAATTCCAA-3 ', downstream primer：5'-
AGTCCAAACAGTGCTCTACCTCCA-3 ', probe：5'-JOE-CGCAGCGAAAGCGAGTCTGAATAGGG-BHQ1-3'；
III) sense primer：5 '-ACTCTTACGCAATCTAGCAGATG-3 ', downstream primer：5'-
TGCAATACTCGTGCGTTTTAA-3 ', probe：5'-TexasRed-CGAAAACGCTTGATACAGGGAGTTT-BHQ2-3'；
In third group reaction solution, including following four pairs of primer and probes：
IV) sense primer：5 '-TCCTGCTGATTGACGATCACCC-3 ', downstream primer：5'-
CCAGCGTTTCCAGACCGTTCAT-3 ', probe：5'-JOE-CCAGATATCACCGTGGTTGGCGAA-BHQ1-3'；
V) sense primer：5 '-CGACGCCTGAAGCGTGACGAAC-3 ', downstream primer：5'-GGCTTGGTGATGTAGTCG-
3 ', probe：5'-FAM-CATGCTCACCGCCAAGG-BHQ1-3'；
VI) sense primer：5 '-CTGATAGTTGAAGACGAACCCGT-3 ', downstream primer：5'-
AAATGTCGCAAATCATCAGGTCC-3 ', probe：5'-TexasRed-TGGCTGACCTCGCTGGGAGCAA-BHQ2-3'；
VII) sense primer：5 '-GGGCGAAGAAGCAATTGCT-3 ', downstream primer：5'-GCGAGGCCGCTTACAGCAATG-
3 ', probe：5'-CY5-CATCGGTGGCGTAGAAACAAC–BHQ3-3'；
The step of fluorescence quantitative PCR detection is：1) PCR detections first are carried out to hybrid dna template with first group of reaction solution；2)
Second group of reaction solution and third group reaction solution are used again, and PCR inspections are carried out by arbitrary precedence or simultaneously to hybrid dna template
It surveys.
2. according to claim 1 after the quick detection techniques of multiple real time fluorescence PCR encephalic bacterium infection method, feature exists
In, in step (3), using the bacterial strain hybrid dna of pathogen sample and sterile water as template, progress fluorescent quantitative PCR, amplification
Program is：45 cycles are carried out after 95 DEG C of pre-degeneration 5min, each cycle includes 95 DEG C of denaturation 5s, and 60 DEG C of annealing extend 90s.
3. according to claim 1 after the quick detection techniques of multiple real time fluorescence PCR encephalic bacterium infection method, feature exists
In in step (1), using the DNA of pyrolysis method extraction cause of disease bacteria strain, nucleic acid extraction liquid used is：5%Chelex-
100,0.5%NP-40,0.5%Triton X-100,1mM EDTA.
4. according to claim 3 after the quick detection techniques of multiple real time fluorescence PCR encephalic bacterium infection method, feature exists
In in step (3), the volumetric usage ratio of the reaction solution and template is 9:1.
5. according to claim 1 after the quick detection techniques of multiple real time fluorescence PCR encephalic bacterium infection method, feature exists
In second group of reaction solution can detect that staphylococcus aureus, enterococcus faecalis and streptococcus pneumonia.
6. according to claim 1 after the quick detection techniques of multiple real time fluorescence PCR encephalic bacterium infection method, feature exists
In third group reaction solution can detect that escherichia coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae and Acinetobacter bauamnnii.
7. according to claim 1 after the quick detection techniques of multiple real time fluorescence PCR encephalic bacterium infection method, feature exists
In further including the primer and probe of reference gene GAPDH, upstream primer sequence in described second group and third group reaction solution
For：5 '-CGCTCCCTCTTTCTTTGCAG-3 ', downstream primer sequence are：5 '-TACCAAAGTTGTCATGGATGACC-3 ' are visited
Needle sequence is：5'-TAMARA-TGCACCACCAACTGCTTAGCACCCC-BHQ2-3'.
8. according to claim 1 after the quick detection techniques of multiple real time fluorescence PCR encephalic bacterium infection method, feature exists
In in step (2), the dosage of the EcoR I enzymes is 2U, and the dosage of I enzymes of the DNase is 1.5U.
9. according to claim 8 after the quick detection techniques of multiple real time fluorescence PCR encephalic bacterium infection method, feature exists
In by digestion, treated then reaction solution carries out fluorescence in 37 DEG C of water-bath 60min then at 75~80 DEG C of 5~15min of water-bath
Quantitative PCR detection.
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Application publication date: 20181106 |