CN110499378A - A method of detecting escherichia coli pathogen from clinical blood - Google Patents
A method of detecting escherichia coli pathogen from clinical blood Download PDFInfo
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Abstract
The method that the present invention provides a kind of to detect escherichia coli pathogen from clinical blood, comprising the following steps: the extraction of escherichia coli genomic DNA in S1, blood；Escherichia coli PCR is detected in S2, blood: S2.1, the PCR primer that specificity is designed using escherichia coli genomic dna sequence, forward primer is as shown in SEQ ID NO.1；Reverse primer is as shown in SEQ ID NO.2；The expanding fragment length of primer is 200bp；S2.2, using escherichia coli genomic DNA as template, using specificity PCR primer carry out PCR amplification；S2.3, agarose gel electrophoresis and detection are carried out to pcr amplification product, amplifies the segment that length is 200bp and is then judged to detecting escherichia coli.The present invention reaches 100CFU/mL to the detectable limit of escherichia coli in blood, by directly to the rapidly extracting of escherichia coli genomic DNA in clinical blood and carry out specific gene fast PCR detect, it can be achieved that timely and effectively being diagnosed to infection due to Escherichia coli in blood.
Description
Technical field
The present invention relates to the detection technique fields of blood infection pathogen, and in particular to one kind detects big from clinical blood
The method of the uncommon bacterium pathogen of intestines angstrom.
Background technique
Escherichia coli is commonly called as Escherichia coli, is bacterial species common in human body.General escherichia coli is not
With pathogenic, but some other type such as Diarrhoea-causing Escherichia E.coli class can cause human diseases, children's septicemia also with disease
Originality escherichia coli is closely related.Bloodstream infection caused by escherichia coli also has become the microbial blood flow of Gram-negative
The arch-criminal of infection.
Currently, the conventional inspection method of hospital is still needed to by culture etc. for the detection of pathogenic microorganism in clinical blood
The intermediate steps of complicated and time consumption, the strain for being easy to cultivate as escherichia coli is this also need coated plate scribing line to be incubated overnight, then after arriving
Continuous Physiology and biochemistry or drug resistance analysis, generally may require that 3-5 days or so.It will lead to the treatment of infected patient so not in time etc.
A series of problems, What is more can directly result in the situation of physician-patient relationship tense.
So the method for finding escherichia coli in a kind of quick and easy and efficient detection blood sample has great face
Bed meaning and wide application market.
Summary of the invention
The method that the purpose of the present invention is to provide a kind of to detect escherichia coli pathogen from clinical blood, by straight
The fast PCR detection for connecing to the rapidly extracting of escherichia coli genomic DNA in clinical blood and carrying out its specific gene, can be real
Now to the timely and effectively diagnosis of infection due to Escherichia coli in blood, clinical detection cycle time is 3-5h and behaviour by the present invention
Make simple and convenient, greatly improve the timeliness of clinical examination, there is good value for clinical application.
In order to achieve the above object, the present invention is achieved by the following technical programs:
A method of detecting escherichia coli pathogen from clinical blood, comprising the following steps:
The extraction of escherichia coli genomic DNA in S1, blood；
Escherichia coli PCR is detected in S2, blood:
S2.1, one section of specific PCR primer, the PCR primer packet are designed using escherichia coli genomic dna sequence
Include forward primer and reverse primer；
The forward primer is as shown in SEQ ID NO.1: 5 '-TAATGTTCTGCGACGCTCAC-3 '；
The reverse primer is as shown in SEQ ID NO.2: 5 '-CCCGGCTAACGTATCCAC-3 '；
The expanding fragment length of primer is 200bp；
S2.2, using the escherichia coli genomic DNA extracted from blood in step S1 as template, using in step S2.1
Specificity PCR primer carry out PCR amplification；
S2.3, agarose gel electrophoresis and detection are carried out to pcr amplification product, amplifies segment that length is 200bp then
It is judged to detecting escherichia coli.
Preferably, in step S1, escherichia coli extracting genome DNA in blood sample specifically includes the following steps:
S1.1, blood sample mix mixing with enzymatic lysis liquid in equal volume, and 37 DEG C are incubated for 15 minutes；
The blood sample that enzymatic lysis liquid is handled in S1.2, step S1.1 mixes in equal volume with alkaline lysis liquid, mixes gently, room
Temperature stands cracking 5 minutes；
In S1.3, step S1.2 cracking treated blood sample and impurity elimination liquid by product than being that 2:1 is mixed, addition impurity elimination
It is mixed immediately after liquid until precipitating, 12000 revs/min of room temperature centrifugation 10 minutes in placement centrifuge；
S1.4, gentle aspiration upper layer suspension adsorb column sleeve in 2mL centrifuge tube into DNA adsorption column, and by DNA；12000
Rev/min centrifugation 1 minute, remove centrifuge tube in waste liquid, 2mL centrifuge tube is inserted in DNA adsorption column again；
S1.5, the 0.4M KI and isopropanol mixed liquor that 500 μ L are added into DNA adsorption column；12000 revs/min of centrifugations 1
Minute, the waste liquid in centrifuge tube is removed, 2mL centrifuge tube is inserted in DNA adsorption column again；
S1.6, the 80%v/v dehydrated alcohol and 10mM Tris-HCl (pH8.0) that 700 μ L are added into DNA adsorption column again
Mixed liquor；12000 revs/min are centrifuged 1 minute, remove the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in DNA adsorption column again；
S1.7, it repeats step S1.6 1 time；
S1.8,12000 revs/min of adsorption column of sky DNA be centrifuged 5 minutes, and DNA adsorption column is transferred to 1.5mL centrifuge tube
It is interior；
S1.9,30 μ L 10mM Tris-HCl and 1mM EDTA mixed liquors are added to empty DNA adsorption column film center, pH is
8.0-8.5；12000 revs/min are centrifuged 2 minutes, collect the genomic DNA eluted.
Preferably, in step S1.1, contain in enzymatic lysis liquid: 0.1mg/mL ribonuclease A, 1mg/mL Proteinase K,
0.05mg/mL wide spectrum lyases ClyH, 10mg/mL lysozyme, 25mM Tris-HCl, 10mM EDTA, pH 8.0-8.5.
Preferably, in step S1.2, contain in alkaline lysis liquid: 0.2M NaOH, 1%SDS, pH 12-14.
Preferably, in step S1.3, contain in impurity elimination liquid: 4M NH4Ac-HAc, pH 4.5-4.8.
Preferably, in step S2.2, the reaction system of PCR when PCR amplification are as follows: 10 times of PCR buffer (plus Mg2+)1μ
The 1 μ L of reverse primer of forward primer 1 the μ L, 2 μm of ol/L of L, dNTPs substrate (2.5mM each) 0.8 μ L, 2 μm of ol/L,
EasyTaq DNA polymerase (Transgen Biology) 0.5 μ L, 1 μ L of genomic DNA template, distilled water complement to 10
μL。
The response procedures of PCR are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 50 DEG C of annealing 20s, 72 DEG C of extension 10s, 30
Secondary circulation；Last 72 DEG C of extensions 10min, 16 DEG C of preservations.
Preferably, in step S2.2, bacterial content >=100CFU/mL of escherichia coli extracting genome DNA is provided.
The beneficial effects of the present invention are:
The present invention is different from infection due to Escherichia coli detection technique in traditional clinical blood, have on detection efficiency compared with
Big promotion.The invention firstly uses specific genome DNA extraction reagent, it is uncommon quickly and easily to extract large intestine angstrom in blood sample
The genomic DNA of bacterium；The specific gene of escherichia coli is recycled, design optimization is used for the specific primer of PCR amplification, then
Infection due to Escherichia coli in clinical blood is detected by PCR quick and high efficient reaction.The present invention overcomes clinically traditional
Culture increases the time-consuming and laborious and complicated detection methods such as bacterium.Meanwhile the present invention reaches the detectable limit of escherichia coli in blood
To 100CFU/mL, the requirement for clinically detecting application is substantially conformed to.If PCR detection method established by the present invention is applied
In clinical practice, the efficiency and sensitivity clinically detected to escherichia coli in blood will be increased substantially.The method of the present invention
With simple to operate, detection speed is fast, high specificity, high sensitivity, stability equal significant advantages by force.
Detailed description of the invention
It in order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will be in embodiment
Required attached drawing is briefly described, it should be apparent that, drawings discussed below is only some embodiments of the present invention,
To those skilled in the art, without any creative labor, it can also obtain according to these attached drawings
Obtain other attached drawings.
Fig. 1 is the specificity experiments result of escherichia coli primer detection；Wherein, 1:Marker (100bp)；2: negative right
According to；3: escherichia coli；4: staphylococcus aureus；5: salmonella；6: Pseudomonas aeruginosa；7: single hyperplasia Listeria；8: lung
Scorching klebsiella.
Fig. 2 is the sensitivity experiment result of escherichia coli detection in blood；Wherein, 1:Marker (100bp)；2: negative
Control；3: negative blood sample；4: positive control；5-13: being respectively 108, 107, 106, 105, 104, 103, 102, 10,100CFU/mL is big
The uncommon bacterium of intestines angstrom.
Fig. 3 is the clinical application example of escherichia coli detection method in blood.Wherein, 1:Marker (100bp)；2: yin
Property control group；3: positive controls；4-9: being respectively clinical blood experimental group.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention,
Technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is the present invention one
Divide embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making
Every other embodiment obtained, shall fall within the protection scope of the present invention under the premise of creative work.
Embodiment one: specific PCR primer design
One section of specific PCR primer, specific PCR primer packet are designed using escherichia coli genomic dna sequence
Include forward primer and reverse primer.Forward primer is as shown in SEQ IDNO.1: 5 '-TAATGTTCTGCGACGCTCAC-3 '.Reversely
Primer is as shown in SEQ ID NO.2: 5 '-CCCGGCTAACGTATCCAC-3 '.The expanding fragment length of primer is 200bp.
Embodiment two:
The escherichia coli being added in blood, staphylococcus aureus, sramana are detected using escherichia coli special primer
The pathogenic bacteria such as Salmonella, Pseudomonas aeruginosa, Listeria monocytogenes and Klebsiella Pneumoniae.The present embodiment is to every 100 μ L blood
OD is added in liquid600For 0.1 above-mentioned all kinds of pathogens.This implementation column include Bacteria in Blood genomic DNA extraction and
PCR amplification detection.This method the following steps are included:
(1) OD of 100 μ L culture is collected600=0.1 escherichia coli, staphylococcus aureus, salmonella, green pus
Bacillus, Listeria monocytogenes and Klebsiella Pneumoniae.12000 revs/min are centrifuged 1 minute, collect the thallus of precipitating.
(2) blood for taking 6 group of 100 μ L fresh is separately added into 6 group of 100 μ L fresh blood through the big of step (1) precipitating
The uncommon bacterium thallus of intestines angstrom, staphylococcus aureus thallus, salmonella thallus, Pseudomonas aeruginosa thallus, monocyte hyperplasia Liszt
Bacterium thallus and Klebsiella Pneumoniae thallus, are resuspended the thallus of precipitating, and sufficiently piping and druming is uniformly mixed.
(3) 100 μ L enzymatic lysis liquid are added into every group of blood, 37 DEG C are incubated for 15 minutes.Wherein contain in enzymatic lysis liquid:
0.1mg/mL ribonuclease A, 1mg/mL Proteinase K, 0.05mg/mL wide spectrum lyases ClyH, 10mg/mL lysozyme, 25mM
Tris-HCl, 10mM EDTA, pH 8.0-8.5.
(4) 200 μ L alkaline lysis liquid are added, are mixed gently, cracking 5 minutes is stored at room temperature.Wherein contain in alkaline lysis liquid:
0.2M NaOH, 1%SDS, pH 12-14.
(5) 200 μ L impurity elimination liquid are added, are mixed immediately, places in centrifuge and is centrifuged 10 minutes for 12000 revs/min of room temperature.
Wherein contain in impurity elimination liquid: 4M NH4Ac-HAc, pH 4.5-4.8.
(6) gentle aspiration supernatant adsorbs column sleeve in 2mL centrifuge tube into DNA adsorption column, and by DNA.12000 turns/
Minute centrifugation 1 minute, removes the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in DNA adsorption column again.
(7) be added into DNA adsorption column 500 μ L volumes 0.4M KI and isopropanol mixed liquor (KI is dissolved in isopropanol,
The concentration of KI is 0.4M).12000 revs/min are centrifuged 1 minute, remove the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in again
DNA adsorption column.
(8) 80%v/v dehydrated alcohol and the 10mM Tris-HCl (pH8.0) that 700 μ L are added into DNA adsorption column again are mixed
Close liquid (concentration of Tris-HCl is 10mM in 80%v/v dehydrated alcohol).12000 revs/min are centrifuged 1 minute, remove centrifuge tube
2mL centrifuge tube is inserted in DNA adsorption column by interior waste liquid again.
(9) it is primary that step (8) are repeated.
(10) it is centrifuged 5 minutes for 12000 revs/min of adsorption column of sky DNA, and DNA adsorption column is transferred to 1.5mL centrifuge tube
It is interior.
(11) 30 μ L 10mM Tris-HCl and 1mM EDTA mixed liquor (mixed liquors are added to empty DNA adsorption column film center
The concentration of middle Tris-HCl is 10mM, and the concentration of EDTA is 1mM), pH8.0-8.5.12000 revs/min are centrifuged 2 minutes, collect
The genomic DNA eluted.
(12) using from the extracted escherichia coli genomic DNA of step (11) as template, using the PCR of above-mentioned specificity
Primer carries out PCR amplification, specifically:
PCR reaction system: 10 times of PCR buffer (plus Mg2+) 1 μ L, dNTPs substrate (2.5mM each) 0.8 μ L, 2 μ
Reverse primer (SEQ ID NO.2) 1 the μ L, EasyTaq of forward primer (SEQ ID NO.1) 1 the μ L, 2 μm of ol/L of mol/L
DNApolymerase (Transgen Biology) 0.5 μ L, 1 μ L of genomic DNA template, distilled water complement to 10 μ L.
PCR response procedures: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 50 DEG C of annealing 20s, 72 DEG C of extension 10s are followed for 30 times
Ring；Last 72 DEG C of extensions 10min；16 DEG C of pcr amplification product preservations.
(13) 1.5% agarose gel electrophoresis detect pcr amplification product (see Fig. 1), amplify the segment that length is 200bp
Then it is judged to detecting escherichia coli.As seen from Figure 1, escherichia coli specific primer only amplifies specific large intestine
Angstrom uncommon bacterium gene and do not amplify purpose band in other pathogenic bacteria.Illustrate the escherichia coli primer designed in the present invention
It is specific high.
Embodiment three:
Utilize the escherichia coli being added in escherichia coli special primer detection blood.The present embodiment is to every 100 μ L blood
10 are separately added into liquid8, 107, 106, 105, 104, 103,
102, 101, 100The escherichia coli of CFU.This implementation column include Bacteria in Blood genomic DNA extraction and
PCR amplification detection.This method the following steps are included:
(1) the 10 of culture are collected8, 107, 106, 105, 104, 103, 102, 101, 100The escherichia coli of CFU.12000 turns/
Minute centrifugation 1 minute, collects the thallus of precipitating.
(2) blood for taking 9 group of 100 μ L fresh is separately added into 9 group of 100 μ L fresh blood through step (1) precipitating not
With the escherichia coli thallus of colony count, the thallus of precipitating is resuspended, sufficiently piping and druming is uniformly mixed.
(3) 100 μ L enzymatic lysis liquid are added thereto, 37 DEG C are incubated for 15 minutes.Wherein contain in enzymatic lysis liquid: 0.1mg/mL
Ribonuclease A, 1mg/mL Proteinase K, 0.05mg/mL wide spectrum lyases ClyH, 10mg/mL lysozyme, 25mM Tris-
HCl, 10mM EDTA, pH 8.0-8.5.
(4) 200 μ L alkaline lysis liquid are added, are mixed gently, cracking 5 minutes is stored at room temperature.Contain in alkaline lysis liquid: 0.2M
NaOH, 1%SDS, pH 12-14.
(5) 200 μ L impurity elimination liquid are added, are mixed immediately, places in centrifuge and is centrifuged 10 minutes for 12000 revs/min of room temperature.
Contain in impurity elimination liquid: 4M NH4Ac-HAc, pH 4.5-4.8.
(6) gentle aspiration supernatant adsorbs column sleeve in 2mL centrifuge tube into DNA adsorption column, and by DNA.12000 turns/
Minute centrifugation 1 minute, removes the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in DNA adsorption column again.
(7) the 0.4M KI and isopropanol mixed liquor of 500 μ L are added into DNA adsorption column.12000 revs/min are centrifuged 1 point
Clock removes the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in DNA adsorption column again.
(8) 80%v/v dehydrated alcohol and the 10mM Tris-HCl (pH8.0) that 700 μ L are added into DNA adsorption column again are mixed
Close liquid.12000 revs/min are centrifuged 1 minute, remove the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in DNA adsorption column again.
(9) it is primary that step (8) are repeated.
(10) it is centrifuged 5 minutes for 12000 revs/min of adsorption column of sky DNA, and DNA adsorption column is transferred to 1.5mL centrifuge tube
It is interior.
(11) 30 μ L 10mM Tris-HCl and 1mM EDTA mixed liquors, pH8.0- is added to empty DNA adsorption column film center
8.5.12000 revs/min are centrifuged 2 minutes, collect the genomic DNA eluted.
(12) using from the extracted escherichia coli genomic DNA of step (11) as template, using the PCR of above-mentioned specificity
Primer carries out PCR amplification, specifically:
PCR reaction system: 10 times of PCR buffer (plus Mg2+) 1 μ L, dNTPs substrate (2.5mM each) 0.8 μ L, 2 μ
Reverse primer (SEQ ID NO.2) 1 the μ L, EasyTaq of forward primer (SEQ ID NO.1) 1 the μ L, 2 μm of ol/L of mol/L
DNApolymerase (Transgen Biology) 0.5 μ L, 1 μ L of genomic DNA template, distilled water complement to 10 μ L.
PCR response procedures: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 50 DEG C of annealing 20s, 72 DEG C of extension 10s are followed for 30 times
Ring；Last 72 DEG C of extensions 10min；16 DEG C of pcr amplification product preservations.
(14) 1.5% agarose gel electrophoresis detect pcr amplification product (see Fig. 2), amplify the segment that length is 200bp
Then it is judged to detecting escherichia coli.It is available by Fig. 2, escherichia coli Monitoring lower-cut value in clinical blood of the invention
It can achieve 100CFU/mL.
Example IV:
The quick detection example of infection due to Escherichia coli in 6 groups of different clinical blood samples,
Specifically includes the following steps:
(1) the clinical blood sample for taking 6 groups of 100 different μ L fresh.
(2) 100 μ L enzymatic lysis liquid are added into every group of fresh clinical blood sample, 37 DEG C are incubated for 15 minutes.Wherein enzyme is split
Contain in solution liquid: 0.1mg/mL ribonuclease A, 1mg/mL Proteinase K, 0.05mg/mL wide spectrum lyases ClyH, 10mg/mL
Lysozyme, 25mM Tris-HCl, 10mM EDTA, pH 8.0-8.5.
(3) 200 μ L alkaline lysis liquid are added, are mixed gently, cracking 5 minutes is stored at room temperature.Contain in alkaline lysis liquid: 0.2M
NaOH, 1%SDS, pH 12-14.
(4) 200 μ L impurity elimination liquid are added, are mixed immediately, places in centrifuge and is centrifuged 10 minutes for 12000 revs/min of room temperature.
Contain in impurity elimination liquid: 4M NH4Ac-HAc, pH 4.5-4.8.
(5) gentle aspiration supernatant adsorbs column sleeve in 2mL centrifuge tube into DNA adsorption column, and by DNA.12000 turns/
Minute centrifugation 1 minute, removes the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in DNA adsorption column again.
(6) the 0.4M KI and isopropanol mixed liquor of 500 μ L are added into DNA adsorption column.12000 revs/min are centrifuged 1 point
Clock removes the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in DNA adsorption column again.
(7) 80%v/v dehydrated alcohol and the 10mM Tris-HCl (pH8.0) that 700 μ L are added into DNA adsorption column again are mixed
Close liquid.12000 revs/min are centrifuged 1 minute, remove the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in DNA adsorption column again.
(8) it is primary that step (7) are repeated.
(9) it is centrifuged 5 minutes for 12000 revs/min of adsorption column of sky DNA, and DNA adsorption column is transferred in 1.5mL centrifuge tube.
(10) 30 μ L 10mM Tris-HCl and 1mM EDTA mixed liquors, pH8.0- is added to empty DNA adsorption column film center
8.5.12000 revs/min are centrifuged 2 minutes, collect the genomic DNA eluted.
(11) using from the extracted escherichia coli genomic DNA of step (10) as template, using the PCR of above-mentioned specificity
Primer carries out PCR amplification, specifically:
PCR reaction system: 10 times of PCR buffer (plus Mg2+) 1 μ L, dNTPs substrate (2.5mM each) 0.8 μ L, 2 μ
Reverse primer (SEQ ID NO.2) 1 the μ L, EasyTaq of forward primer (SEQ ID NO.1) 1 the μ L, 2 μm of ol/L of mol/L
DNApolymerase (Transgen Biology) 0.5 μ L, 1 μ L of genomic DNA template, distilled water complement to 10 μ L.
PCR response procedures: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 50 DEG C of annealing 20s, 72 DEG C of extension 10s are followed for 30 times
Ring；Last 72 DEG C of extensions 10min；16 DEG C of pcr amplification product preservations.
(12) 1.5% agarose gel electrophoresis detect pcr amplification product (see Fig. 3), amplify the segment that length is 200bp
Then it is judged to detecting escherichia coli.It can be found out with Fig. 3,4,5,7 and No. 8 hole clinical blood experimental groups detect that 200bp is big
The uncommon bacterium specific gene of intestines angstrom.6 and No. 9 hole clinical blood experimental groups do not detect escherichia coli.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these modification or
Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.
Sequence table
<110>Zhuo Yuan health Science and Technology Ltd.
<120>a kind of method that escherichia coli pathogen is detected from clinical blood
<160> 2
<170> PatentIn Version 3.3
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<213>artificial sequence
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<212> DNA
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Claims (8)
1. a kind of method for detecting escherichia coli pathogen from clinical blood, which comprises the following steps:
The extraction of escherichia coli genomic DNA in S1, blood；
Escherichia coli PCR is detected in S2, blood:
S2.1, one section of specific PCR primer is designed using escherichia coli genomic dna sequence, the PCR primer includes just
To primer and reverse primer；
The forward primer is as shown in SEQ ID NO.1: 5 '-TAATGTTCTGCGACGCTCAC-3 '；
The reverse primer is as shown in SEQ ID NO.2: 5 '-CCCGGCTAACGTATCCAC-3 '；
The expanding fragment length of primer is 200bp；
S2.2, using the escherichia coli genomic DNA extracted from blood in step S1 as template, using the spy in step S2.1
Anisotropic PCR primer carries out PCR amplification；
S2.3, agarose gel electrophoresis and detection are carried out to pcr amplification product, amplifies the segment that length is 200bp and then determines
To detect escherichia coli.
2. the method according to claim 1 for detecting escherichia coli pathogen from clinical blood, which is characterized in that step
In rapid S1, in blood sample escherichia coli genomic DNA extraction specifically includes the following steps:
S1.1, blood sample mix mixing with enzymatic lysis liquid in equal volume, and 37 DEG C are incubated for 15 minutes；
The blood sample that enzymatic lysis liquid is handled in S1.2, step S1.1 mixes in equal volume with alkaline lysis liquid, mixes gently, room temperature is quiet
Set cracking 5 minutes；
In S1.3, step S1.2 cracking treated blood sample and impurity elimination liquid by product than being that 2:1 is mixed, after addition impurity elimination liquid
It is mixed immediately until precipitating, 12000 revs/min of room temperature centrifugation 10 minutes in placement centrifuge；
S1.4, gentle aspiration upper layer suspension adsorb column sleeve in 2mL centrifuge tube into DNA adsorption column, and by DNA；12000 turns/
Minute centrifugation 1 minute, removes the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in DNA adsorption column again；
S1.5, the 0.4M KI and isopropanol mixed liquor that 500 μ L are added into DNA adsorption column；12000 revs/min are centrifuged 1 minute,
The waste liquid in centrifuge tube is removed, 2mL centrifuge tube is inserted in DNA adsorption column again；
The Tris-HCl of S1.6, the 80%v/v dehydrated alcohol that 700 μ L are added into DNA adsorption column again and 10mM pH8.0 are mixed
Liquid；12000 revs/min are centrifuged 1 minute, remove the waste liquid in centrifuge tube, 2mL centrifuge tube is inserted in DNA adsorption column again；
S1.7, it repeats step S1.6 1 time；
S1.8,12000 revs/min of adsorption column of sky DNA be centrifuged 5 minutes, and DNA adsorption column is transferred in 1.5mL centrifuge tube；
S1.9,30 μ L 10mM Tris-HCl and 1mM EDTA mixed liquors, pH 8.0- is added to empty DNA adsorption column film center
8.5；12000 revs/min are centrifuged 2 minutes, collect the genomic DNA eluted.
3. the method according to claim 2 for detecting escherichia coli pathogen from clinical blood, which is characterized in that step
In rapid S1.1, contain in enzymatic lysis liquid: 0.1mg/mL ribonuclease A, 1mg/mL Proteinase K, the cracking of 0.05mg/mL wide spectrum
Enzyme, 10mg/mL lysozyme, 25mM Tris-HCl, 10mM EDTA, pH 8.0-8.5.
4. the method according to claim 2 for detecting escherichia coli pathogen from clinical blood, which is characterized in that step
In rapid S1.2, contain in alkaline lysis liquid: 0.2M NaOH, 1%SDS, pH 12-14.
5. the method according to claim 2 for detecting escherichia coli pathogen from clinical blood, which is characterized in that step
In rapid S1.3, contain in impurity elimination liquid: 4M NH4Ac-HAc, pH 4.5-4.8.
6. the method according to claim 1 for detecting escherichia coli pathogen from clinical blood, which is characterized in that step
In rapid S2.2, the reaction system of PCR when PCR amplification are as follows: 10 times of PCR buffer (plus Mg2+) 1 μ L, dNTPs substrate (2.5mM
Each) 1 μ L, the EasyTaq DNA polymerase of reverse primer of forward primer 1 the μ L, 2 μm of ol/L of 0.8 μ L, 2 μm of ol/L
(Transgen Biology) 0.5 μ L, 1 μ L of genomic DNA template, distilled water complement to 10 μ L.
7. the method according to claim 1 for detecting escherichia coli pathogen from clinical blood, which is characterized in that step
In rapid S2.2, the response procedures of PCR are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 50 DEG C of annealing 20s, 72 DEG C of extension 10s,
30 circulations；Last 72 DEG C of extensions 10min, 16 DEG C of preservations.
8. the method according to claim 1 for detecting escherichia coli pathogen from clinical blood, which is characterized in that step
In rapid S2, bacterial content >=100CFU/mL of escherichia coli extracting genome DNA is provided.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0643140A1 (en) * | 1993-09-13 | 1995-03-15 | Canon Kabushiki Kaisha | Determination of nucleic acid by PCR, measurement of number of microbial cells, genes, or gene-copies by PCR, and measuring-kit employed for the same |
| CN101629207A (en) * | 2008-12-12 | 2010-01-20 | 武汉大学 | Fluorescent quantitative PCR kit for rapidly testing Escherichia coli in urine |
| CN101928773A (en) * | 2010-05-14 | 2010-12-29 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Oligonucleotide primer for detecting common pathogenic bacteria by adopting fluorescent quantitation PCR (Rich Client Platform) technology, method thereof for detecting common pathogenic bacteria and application thereof |
| CN102703588A (en) * | 2011-08-09 | 2012-10-03 | 中国人民解放军后勤工程学院 | Multiplex PCR-based synchronous and rapid method for detecting 13 pathogenic microorganisms in water |
| EP2749642A1 (en) * | 2011-11-30 | 2014-07-02 | National Cancer Center | Induced malignant stem cells |
-
2019
- 2019-09-12 CN CN201910861066.2A patent/CN110499378A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0643140A1 (en) * | 1993-09-13 | 1995-03-15 | Canon Kabushiki Kaisha | Determination of nucleic acid by PCR, measurement of number of microbial cells, genes, or gene-copies by PCR, and measuring-kit employed for the same |
| CN101629207A (en) * | 2008-12-12 | 2010-01-20 | 武汉大学 | Fluorescent quantitative PCR kit for rapidly testing Escherichia coli in urine |
| CN101928773A (en) * | 2010-05-14 | 2010-12-29 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Oligonucleotide primer for detecting common pathogenic bacteria by adopting fluorescent quantitation PCR (Rich Client Platform) technology, method thereof for detecting common pathogenic bacteria and application thereof |
| CN102703588A (en) * | 2011-08-09 | 2012-10-03 | 中国人民解放军后勤工程学院 | Multiplex PCR-based synchronous and rapid method for detecting 13 pathogenic microorganisms in water |
| EP2749642A1 (en) * | 2011-11-30 | 2014-07-02 | National Cancer Center | Induced malignant stem cells |
Non-Patent Citations (3)
| Title |
|---|
| CLAUDIA TOMA ET AL.: ""Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli"", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| OMEGE: ""E.Z.N.A.Bacterial DNA Kit"", 《E.Z.N.A.BACTERIAL DNA KIT》 * |
| 张冬会等: ""外周血DNA提取方法及其特点"", 《中国组织工程研究与临床康复》 * |
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