CN107513582A - Authentication chip, kit and the authentication method of positive blood culture - Google Patents
Authentication chip, kit and the authentication method of positive blood culture Download PDFInfo
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Abstract
The invention discloses a kind of authentication chip of positive blood culture, kit and authentication method.The authentication chip has multiple reative cells, including positive quality control room, negative Quality Control room and at least one Bacteria Identification room；Isothermal duplication primer is respectively and fixedly provided with the Bacteria Identification room and the positive quality control room.The authentication chip, kit and authentication method can be used at least one of 8 kinds of common bacteriums, specially staphylococcus aureus, EHEC, pseudomonas aeruginosa, Klebsiella Pneumoniae, enterococcus faecalis, Acinetobacter bauamnnii, streptococcus pneumonia and haemophilus influenzae in identification positive blood culture.With reference to LAMP reaction reagents etc., whole qualification process only needs about 1.5h, can also be applied without PCR instrument, therefore in the poor laboratories of appointed condition；Compared with existing various methods, authentication chip of the invention has the advantages such as easy quick, sensitivity is high, specificity good, cheap and strong adaptability when identifying positive blood culture.
Description
Technical field
The present invention relates to biochemistry detection field, more particularly, to a kind of authentication chip of positive blood culture, kit and
Authentication method.
Background technology
The incidence of Present Global health field bloodstream infection is still very high, and it is to cause patient dead that bloodstream infection, which remains on,
One of the main reason for dying.In view of infection progress is serious with caused consequence rapidly, research of the monitoring for bloodstream infection in real time is outstanding
To be important.Up to the present, blood culture, which remains, judges the most basic and most important experimental method of bloodstream infection.Blood culture with positive bacteria
Afterwards it is generally necessary to spend time of 2 days to identify bloodstream infection pathogen, wherein the transferred species culture for being used for pathogen in 1 day, in addition 1
Its then identification for pathogen and susceptibility detection.Because larger difference be present to the sensitiveness of medicine in different pathogens, thus it is sick
Research of the identification of substance for bloodstream infection is very crucial.If pathogen can be identified within the shorter time, then
Can be just that clinical treatment strives for more intervention times and chance of success.
At present have certain methods can be used for positive blood culture Rapid identification, such as probe hybridization, real-time PCR,
MALDI-TOF methods etc., these methods all possess each different advantages, but the part that also all comes with some shortcomings.Such as probe
Hybridization technique operation is relatively complicated, and sensitiveness is relatively low；Real-time PCR needs PCR instrument, and cost is higher；MALDI-TOF methods
Special installation is needed, and sensitiveness and accuracy be not high.Therefore, a kind of quick, easy, accurate and lower-cost positive of research and development
Blood culture thing authentication method and kit have turned into the common aspiration of numerous scientific research personnel.
The content of the invention
Based on this, for above-mentioned technical problem, it is necessary to provide a kind of quick, easy, accurate and lower-cost positive
Authentication chip, kit and the authentication method of blood culture thing.
A kind of authentication chip of positive blood culture, the authentication chip have multiple reative cells, including positive quality control room,
Negative Quality Control room and Bacteria Identification room；The Bacteria Identification room includes streptococcus pneumonia authentication room, enterococcus faecalis authentication room, Bao Man
Acinetobacter calcoaceticus authentication room, pseudomonas aeruginosa authentication room, Klebsiella Pneumoniae authentication room, EHEC authentication room, golden yellow
At least one of staphylococcus authentication room and haemophilus influenzae authentication room, the Bacteria Identification room and the positive quality control
Interior is respectively and fixedly provided with isothermal duplication primer, wherein,
Sequence is fixed with the enterococcus faecalis authentication room respectively such as SEQ ID No:1~SEQ ID No:It is more shown in 6
The combination of bar primer；
Sequence is fixed with the staphylococcus aureus authentication room respectively such as SEQ ID No:7~SEQ ID No:12 institutes
The combination of a plurality of primer shown；
Sequence is fixed with the EHEC authentication room respectively such as SEQ ID No:13~SEQ ID No:Shown in 18
A plurality of primer combination；
Sequence is fixed with the pseudomonas aeruginosa authentication room respectively such as SEQ ID No:19~SEQ ID No:24 institutes
The combination of a plurality of primer shown；
Sequence is fixed with the streptococcus pneumonia authentication room respectively such as SEQ ID No:25~SEQ ID No:Shown in 29
A plurality of primer combination；
Sequence is fixed with the Klebsiella Pneumoniae authentication room respectively such as SEQ ID No:30~SEQ ID No:34 institutes
The combination of a plurality of primer shown；
Sequence is fixed with the Acinetobacter bauamnnii authentication room respectively such as SEQ ID No:35~SEQ ID No:40 institutes
The combination of a plurality of primer shown；
Sequence is fixed with the haemophilus influenzae authentication room respectively such as SEQ ID No:41~SEQ ID No:45 institutes
The combination of a plurality of primer shown；
Sequence is fixed with the positive quality control room respectively such as SEQ ID No:46~SEQ ID No:It is a plurality of shown in 49
The combination of primer.
In one of the embodiments, the Bacteria Identification room include streptococcus pneumonia authentication room, enterococcus faecalis authentication room,
Acinetobacter bauamnnii authentication room, pseudomonas aeruginosa authentication room, Klebsiella Pneumoniae authentication room, EHEC authentication room, gold
Totally eight kinds of staphylococcus aureus authentication room and haemophilus influenzae authentication room.A kind of identification kit of positive blood culture,
Include the authentication chip of the positive blood culture described in any of the above-described embodiment.
In one of the embodiments, the identification kit of the positive blood culture also includes DNA extracts reagents, LAMP
At least one of reaction reagent, Bst archaeal dna polymerases, nitrite ion, fluorescent dye and stoning sour water reagent.
The LAMP reaction reagents include reaction buffer, dNTP, MgSO4And glycine betaine.
Containing being added with positive quality control DNA fragmentation in the LAMP reaction reagents, the positive quality control DNA fragmentation be sequence such as
SEQ ID No:The partial gene fragments of zebra fish ccna1 genes shown in 50.
The nitrite ion is hydroxynaphthol blue (HNB) solution；The fluorescent dye is SYBR green.
A kind of authentication method of positive blood culture, comprises the following steps：
Extract the DNA of positive blood culture；
Using the DNA of extraction as template, added after it is mixed with LAMP reaction reagents described in any of the above-described embodiment
In the Bacteria Identification room of the authentication chip of positive blood culture, and in the positive quality control room of the authentication chip and negative Quality Control room
It is interior to add corresponding reagent；
Nucleic acid amplification reaction is carried out under isothermal conditions, and the color or fluorescence for observing each reative cell of the authentication chip become
Change.
In one of the embodiments, the DNA of the extraction positive blood culture is added into the positive blood culture
The DNA is extracted by the way of boiling after entering DNA extracts reagents.
In one of the embodiments, the nucleic acid amplification reaction that carries out under isothermal conditions is prior to 65 by reaction system
DEG C reaction 50min, then 80 DEG C reaction 10min, finally 25 DEG C cool down 1min.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) is being carried out
Without transformation temperature during nucleic acid amplification, whole amplified reaction can be completed under isothermal conditions, and the judgement of final result can
Observe by the naked eye color change to can be achieved, therefore LAMP does not need PCR instrument etc. being capable of accurate temperature controlling and progress fluorescence monitoring
Special installation.Authentication chip, kit and the authentication method of above-mentioned positive blood culture are reflected using LAMP technology principle
Regular inspection is surveyed, and is had the advantage such as quick, easy, accurate and sensitive, on this basis with reference to chip technology, can further be highlighted
High flux and it is inexpensive the advantages that.
Brief description of the drawings
Fig. 1 is the structural representation of the authentication chip of the positive blood culture of an embodiment；
Fig. 2 is that common bacteria (Acinetobacter bauamnnii, Klebsiella Pneumoniae and staphylococcus aureus) mixes in embodiment
The qualification result of thing, chip reative cell is observed from left to right, bacterial reaction room 134,136,138 is the positive, and other reative cells are
Feminine gender, yin and yang attribute Quality Control result is normal, thus can interpretation be Acinetobacter bauamnnii, Klebsiella Pneumoniae and staphylococcus aureus mix
Symphysis is grown.
Fig. 3 is that common bacteria (streptococcus pneumonia bacterium, Klebsiella Pneumoniae and staphylococcus aureus) mixes in embodiment
The qualification result of thing, chip reative cell is observed from left to right, bacterial reaction room 131,136,138 is the positive, and other reative cells are
Feminine gender, yin and yang attribute Quality Control result is normal, thus can interpretation be streptococcus pneumonia bacterium, Klebsiella Pneumoniae and staphylococcus aureus mix
Symphysis is grown.
Embodiment
For the ease of understanding the present invention, the present invention is described more fully below with reference to relevant drawings.In accompanying drawing
Give presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this paper institutes
The embodiment of description.On the contrary, the purpose for providing these embodiments is to make the understanding to the disclosure more thorough
Comprehensively.
Unless otherwise defined, all of technologies and scientific terms used here by the article is with belonging to technical field of the invention
The implication that technical staff is generally understood that is identical.Term used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
The arbitrary and all combination of the Listed Items of pass.
As shown in figure 1, the authentication chip 100 of the positive blood culture of an embodiment has multiple reative cells, including sun
Property control room 110, negative Quality Control room 120 and at least one Bacteria Identification room 130.In the present embodiment, Bacteria Identification room 110
And isothermal duplication primer is respectively and fixedly provided with positive quality control room 130.Fixed isothermal duplication draws in Bacteria Identification room 110 not of the same race
Thing is different, the one kind of fixed isothermal duplication primer in the combination of following eight kinds of primers in each Bacteria Identification room 110：
Sequence is respectively such as SEQ ID No:1~SEQ ID No:The combination of a plurality of primer shown in 6,
Sequence is respectively such as SEQ ID No:7~SEQ ID No:The combination of a plurality of primer shown in 12,
Sequence is respectively such as SEQ ID No:13~SEQ ID No:The combination of a plurality of primer shown in 18,
Sequence is respectively such as SEQ ID No:19~SEQ ID No:The combination of a plurality of primer shown in 24,
Sequence is respectively such as SEQ ID No:25~SEQ ID No:The combination of a plurality of primer shown in 29,
Sequence is respectively such as SEQ ID No:30~SEQ ID No:The combination of a plurality of primer shown in 34,
Sequence is respectively such as SEQ ID No:35~SEQ ID No:The combination of a plurality of primer shown in 40 and
Sequence is respectively such as SEQ ID No:41~SEQ ID No:The combination of a plurality of primer shown in 45；
Sequence is fixed with positive quality control room respectively such as SEQ ID No:46~SEQ ID No:A plurality of primer shown in 49
Combination.
In a specific embodiment, Bacteria Identification room 130 has eight kinds, in Fig. 1, respectively pneumonia chain from left to right
Coccus authentication room 131, enterococcus faecalis authentication room 132, Acinetobacter bauamnnii authentication room 133, pseudomonas aeruginosa authentication room 134,
Klebsiella Pneumoniae authentication room 135, EHEC authentication room 136, staphylococcus aureus authentication room 137 and influenza are bloodthirsty
Bacillus authentication room 138.Streptococcus pneumonia authentication room 131, enterococcus faecalis authentication room 132, Acinetobacter bauamnnii authentication room 133, copper
Green pseudomonad authentication room 134, Klebsiella Pneumoniae authentication room 135, EHEC authentication room 136, staphylococcus aureus
Authentication room 137 and haemophilus influenzae authentication room 138 are arranged in order setting, and various Bacteria Identification rooms 130 only have one respectively.
It is understood that in other embodiments, various Bacteria Identification rooms 130 can also have it is multiple, to carry out parallel laboratory test detection, to enter one
Step improves the accuracy of experiment detection.
More specifically, in the embodiment shown in fig. 1, the sequence of fixed primer combination in streptococcus pneumonia authentication room 131
Row are respectively such as SEQ ID No:25~SEQ ID No:Shown in 29；The sequence of fixed primer combination in enterococcus faecalis authentication room 132
Row are respectively such as SEQ ID No:1~SEQ ID No:Shown in 6；Fixed primer combination in Acinetobacter bauamnnii authentication room 133
Sequence is respectively such as SEQ ID No:35~SEQ ID No:Shown in 40；Fixed primer sets in pseudomonas aeruginosa authentication room 134
The sequence of conjunction is respectively such as SEQ ID No:19~SEQ ID No:Shown in 24；It is fixed in Klebsiella Pneumoniae authentication room 135 to draw
The sequence of thing combination is respectively such as SEQ ID No:30~SEQ ID No:Shown in 34；Fixed in EHEC authentication room 136
The sequence of primer combination is respectively such as SEQ ID No:13~SEQ ID No:Shown in 18；In staphylococcus aureus authentication room 137
The sequence of fixed primer combination is respectively such as SEQ ID No:7~SEQ ID No:Shown in 12；Haemophilus influenzae authentication room 138
The sequence of the primer combination of interior fixation is respectively such as SEQ ID No:41~SEQ ID No:Shown in 45.
Present embodiment additionally provides a kind of identification kit of positive blood culture, and it includes above-mentioned positive blood culture
Authentication chip 100.
In one embodiment, changing the identification kit of positive blood culture also includes DNA extracts reagents, LAMP reaction examinations
At least one of agent, Bst archaeal dna polymerases, nitrite ion, fluorescent dye and stoning sour water reagent.
Specifically, the composition of DNA extracts reagents is：8wt% sucrose, 50mM EDTA, 50mM TrisHCl (pH8.0).
LAMP reaction reagents include reaction buffer, dNTP, MgSO4And glycine betaine.Wherein, reaction buffer is 2 times dense
Degree, dNTP, MgSO4Distinguish 1.6mM, 8mM and 1mM with the concentration of glycine betaine.LAMP reaction reagents equimultiple can be diluted during use.
Further, in one embodiment, contain in the LAMP reaction reagents and be added with positive quality control DNA fragmentation, positive matter
It is sequence such as SEQ ID No to control DNA fragmentation:The partial gene fragments of zebra fish ccna1 genes shown in 50.
The concentration of Bst archaeal dna polymerases is 0.16U/ μ L.
Nitrite ion is hydroxynaphthol blue (HNB) solution；Fluorescent dye is SYBR green.
Further, present embodiment additionally provides a kind of authentication method of positive blood culture, and it comprises the following steps：
Extract the DNA of positive blood culture；
Using the DNA of extraction as template, the mirror of above-mentioned positive blood culture is added after it is mixed with LAMP reaction reagents
Determine in the Bacteria Identification room 130 of chip 100, and add in the positive quality control room 110 of authentication chip 100 and negative Quality Control room 120
Enter corresponding reagent；
Nucleic acid amplification reaction is carried out under isothermal conditions, observes the color or change in fluorescence of each reative cell of authentication chip.
In one embodiment, the DNA for extracting positive blood culture is addition DNA extracts reagents in heliotropism blood culture thing
DNA is extracted by the way of boiling afterwards.The temperature and time of heating is preferably the 15min at 100 DEG C.More specifically, it can use
But following steps are not limited to extract：
(1) 1mL positive blood cultures are taken into 1.5mL microcentrifugal tubes, are centrifuged in the case where centrifugal force is 10000g
5min；
(2) supernatant is removed after centrifuging, adds 40 μ L DNA extracts reagents, 100 DEG C are heated 15min after vibration mixes, cold
But 13000g centrifugations 5min after；
(3) supernatant comprising DNA profiling is transferred into another after centrifuging to go in the 1.5mL microcentrifugal tubes of nucleic acid,
- 20 DEG C are put to save backup.
Before carrying out nucleic acid amplification reaction, in addition to the step of preparation reaction system, specifically, 80 μ L reaction systems can wrap
Include 10 μ L template DNAs, 40 μ L LAMP reaction reagents, 3.2 μ L Bst archaeal dna polymerases, 2.8 μ L nitrite ions (or fluorescent dye) and
24 μ L are enucleated sour water.
In one of the embodiments, it is that reaction system is anti-prior to 65 DEG C to carry out nucleic acid amplification reaction under isothermal conditions
50min, then 80 DEG C of reaction 10min are answered, finally cool down 1min at 25 DEG C.
When carrying out fluoroscopic examination, fluorescence detection device can be used, FAM passages may be selected in passage.
The result interpretation mode of above-mentioned authentication method is：Visual results, navy blue is negative for amplification, light blue for expansion
Increase positive.Using the monitoring of real-time fluorescence detection device and analysis of fluorescence signal, sample amplification curve is S-type, and signal value is higher than
Threshold value is just determined as the positive, and no amplification curve or amplification curve are straight to be determined as feminine gender.If bacterium corresponding to certain bacterium
Authentication room 130 is the positive, and the result of positive quality control room 110 and negative Quality Control room 120 is normal, can report positive blood culture
Turn out certain bacterium.When two or more Bacteria Identification rooms 130 is positive, show there are two kinds or more bacteriums mixing lifes
It is long.
Above-mentioned authentication chip 100, kit and authentication method can be used for 8 kinds of common bacteriums in identification positive blood culture
At least one of, specially staphylococcus aureus, EHEC, pseudomonas aeruginosa, Klebsiella Pneumoniae, excrement intestines ball
Bacterium, Acinetobacter bauamnnii, streptococcus pneumonia and haemophilus influenzae, preferably eight kinds can identify.With reference to LAMP reaction reagents
Deng whole qualification process only needs about 1.5h, can also be applied without PCR instrument, therefore in the poor laboratories of appointed condition；With
Existing various methods are compared, and authentication chip 100 of the invention has easy quick, sensitivity when identifying positive blood culture
The advantages such as high, specific good, cheap and strong adaptability.
It is specific embodiment part below.
1st, design of primers
According to staphylococcus aureus, EHEC, pseudomonas aeruginosa, Klebsiella Pneumoniae, enterococcus faecalis, Bao Man
Acinetobacter calcoaceticus, streptococcus pneumonia and the genomic DNA of haemophilus influenzae and positive quality control genetic fragment, 49 are designed altogether specifically
Property isothermal duplication primer, it is specific such as SEQ ID No in sequence table:1~SEQ ID No:Shown in 49.Wherein, sequence is respectively such as
SEQ ID No:25~SEQ ID No:Primer sets shown in 29 are shared in amplification streptococcus pneumonia；Sequence is respectively such as SEQ ID
No:1~SEQ ID No:Primer sets shown in 6 are shared in amplification enterococcus faecalis；Sequence is respectively such as SEQ ID No:35~SEQ
ID No:Primer sets shown in 40 are shared in amplification Acinetobacter bauamnnii；Sequence is respectively such as SEQ ID No:19~SEQ ID No:
Primer sets shown in 24 are shared in amplification pseudomonas aeruginosa；Sequence is respectively such as SEQ ID No:30~SEQ ID No:Shown in 34
Primer sets share in amplification Klebsiella Pneumoniae；Sequence is respectively such as SEQ ID No:13~SEQ ID No:Primer shown in 18
Combine for expanding EHEC；Sequence is respectively such as SEQ ID No:7~SEQ ID No:Primer sets shown in 12 share in
Expand staphylococcus aureus；Sequence is respectively such as SEQ ID No:41~SEQ ID No:Primer sets shown in 45 are shared in amplification
Haemophilus influenzae.Sequence is respectively such as SEQ ID No:46~SEQ ID No:Primer sets shown in 49 are shared in the positive matter of amplification
Control DNA fragmentation.
2nd, chip designs
It is cloudy successively from left to right in Fig. 1 specifically as shown in figure 1, the authentication chip 100 has 10 reative cells altogether
Property control room 120, streptococcus pneumonia authentication room 131, enterococcus faecalis authentication room 132, Acinetobacter bauamnnii authentication room 133, verdigris
Pseudomonad authentication room 134, Klebsiella Pneumoniae authentication room 135, EHEC authentication room 136, staphylococcus aureus mirror
Determine room 137, haemophilus influenzae authentication room 138, positive quality control room 110.Each Bacteria Identification room 130 and positive quality control room 110
The primer combination that designs is fixed in above-mentioned steps 1 respectively.
3rd, Evaluation on specificity is analyzed
Choose common 20 kinds of common bacterias and carry out evaluation analysis specificity, specifically include staphylococcus aureus, large intestine angstrom
Uncommon bacterium, pseudomonas aeruginosa, Klebsiella Pneumoniae, enterococcus faecalis, Acinetobacter bauamnnii, streptococcus pneumonia, haemophilus influenzae,
MRSE, Streptococcusagalactiae, Salmonella typhi, micrococcus scarlatinae, VREF, stenotrophomonas maltophilia,
Klebsiella oxytoca, proteus mirabilis, serratia marcesens, bacillus alcalophilus, mycobacterium tuberculosis and Candida albicans.Remove
Outside 8 kinds of purpose bacteriums, the DNA of remaining bacterium can not produce positive findings (partial results are as shown in Figures 2 and 3).
4th, the identification of positive blood culture
4.1st, DNA is extracted
(1) 1mL positive blood cultures are taken into 1.5mL microcentrifugal tubes, are centrifuged in the case where centrifugal force is 10000g
5min。
(2) supernatant is removed after centrifuging, adds 40 μ L DNA extract solutions, 100 DEG C of heating 15min after vibration mixes, cooling
13000g centrifuges 5min afterwards.
(3) supernatant comprising DNA profiling is transferred into another after centrifuging to go in the 1.5mL microcentrifugal tubes of nucleic acid,
- 20 DEG C are put to save backup.
4.2nd, chip loading
(1) sample is added in reaction system, specific reaction composition and addition see the table below：
| Ingredient names | Addition (μ L) |
| LAMP reaction reagents | 40 |
| Developer/fluorescent dye | 2.8 |
| It is enucleated sour water | 24 |
| Bst archaeal dna polymerases | 3.2 |
| Template DNA | 10 |
(2) tear the film on the reative cell of authentication chip off, 80 μ L mixed liquors are drawn with micro sample-adding rifle, then by pipette tips
In intercalation reaction room and mixed liquor is added, is careful not to produce bubble.
4.3rd, amplification identification
(1) reative cell and steam vent of authentication chip are closed, authentication chip is placed in simple heater (such as dry bath pot)
Or expanded in real-time fluorescence monitoring device, reaction condition is 65 DEG C and reacts 50min, 80 DEG C of reaction 10min, 25 DEG C of coolings
1min, carry out channel selecting FAM passages during fluoroscopic examination.
(2) visual results, navy blue is negative for amplification, light blue positive to expand.Set using real-time fluorescence detection
Standby monitoring and analysis of fluorescence signal, sample amplification curve is S-type, and signal value is just determined as the positive, no amplification song higher than threshold value
Line or amplification curve is straight is determined as feminine gender.If authentication room corresponding to certain bacterium is the positive, and yin and yang attribute Quality Control room
Result it is normal, can report that positive blood culture turns out certain bacterium.When two or more authentication rooms is positive, show
There are two kinds or more bacterium mixed growths.
5th, qualification result
As shown in Fig. 2 Acinetobacter bauamnnii, Klebsiella Pneumoniae and the mixture of staphylococcus aureus are reflected
Determine, observe chip reative cell from left to right, bacterial reaction room 134,136,138 is the positive, and other reative cells is negative, yin and yang attribute
Quality Control result is normal, thus can interpretation be Acinetobacter bauamnnii, Klebsiella Pneumoniae and staphylococcus aureus mixed growth.
As shown in figure 3, the mixture of streptococcus pneumonia, Klebsiella Pneumoniae and staphylococcus aureus is identified,
Chip reative cell is observed from left to right, and bacterial reaction room 131,136,138 is the positive, and other reative cells is negative, negative and positive property
Control result it is normal, therefore can interpretation be streptococcus pneumonia, Klebsiella Pneumoniae and staphylococcus aureus mixed growth.
Each technical characteristic of above example can be combined arbitrarily, to make description succinct, not to above-described embodiment
In each technical characteristic it is all possible combination be all described, as long as however, lance is not present in the combination of these technical characteristics
Shield, all it is considered to be the scope of this specification record.
Above example only expresses the several embodiments of the present invention, and its description is more specific and detailed, but can not
Therefore it is construed as limiting the scope of the patent.It should be pointed out that for the person of ordinary skill of the art,
On the premise of not departing from present inventive concept, various modifications and improvements can be made, these belong to protection scope of the present invention.
Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
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<213>Artificial sequence (Artificial Sequence)
<400> 12
gaagtcgagt ttgacaaagg tc 22
<210> 13
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
gccatctcct gatgacgc 18
<210> 14
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
atttaccgca gccagacg 18
<210> 15
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
cattttgcag ctgtacgctc gcagcccatc atgaatgttg ct 42
<210> 16
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
ctggggcgag gtcgtggtat tccgacaaac accacgaatt 40
<210> 17
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
ctttgtaaca acctgcatcg aca 23
<210> 18
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
atcaatctcg atatccatga aggtg 25
<210> 19
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
gcgttgccgc caacaatg 18
<210> 20
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
catgcgggca acctctc 17
<210> 21
<211> 35
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
gttgtcaccc cacctccggg cggcaacgtt cctcc 35
<210> 22
<211> 34
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
ctccgtgcag ggcgaactgc aggcgagcca actc 34
<210> 23
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
acctgccgtg ccatacc 17
<210> 24
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
gttcatgcag ctccagcag 19
<210> 25
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
gcgtgcaacc atataggcaa 20
<210> 26
<211> 15
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
agcattccaa ccgcc 15
<210> 27
<211> 41
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 27
ccgccagtga taatccgctt cacactcaac tgggaatccg c 41
<210> 28
<211> 43
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 28
tctcgcacat tgttgggaac ggccaggcac cattatcaac agg 43
<210> 29
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 29
tgcatcatgc aggtagga 18
<210> 30
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 30
gcctcgctgt atctgccaa 19
<210> 31
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 31
cgatgtggtt ttcccaggt 19
<210> 32
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 32
cggtatgctc tgcgccaaac aagggttgag cggctacc 38
<210> 33
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 33
ctgaaggtcg gggccgactt gatcgctgac ccagacatga 40
<210> 34
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 34
cctcaaacgt tactttcctg agtc 24
<210> 35
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 35
cggtaattag tgtgatctga c 21
<210> 36
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 36
catttcagtt tagagcactg t 21
<210> 37
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 37
ttgcttaacc taaactcttg agtgagaaga cacattaact cattaacaga 50
<210> 38
<211> 43
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 38
agcaaattaa ctgaatcaag cgtttactta agcaccgtac agc 43
<210> 39
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 39
aatttatttc agactcaatt ttgccaa 27
<210> 40
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 40
tggtatgtga atttagattg aa 22
<210> 41
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 41
tctggtattc cggagggc 18
<210> 42
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 42
agtcctacct gatttgaggt 20
<210> 43
<211> 41
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 43
ctaccgtctt tcaagcaaac ccatgagcgt cgtttctccc t 41
<210> 44
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 44
tacgctaaca ctgcacga 18
<210> 45
<211> 47
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 45
acacctttac cagctaaata acctttggta caccagaata caacatc 47
<210> 46
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 46
aggcacagta tcttacggtg aatatgcagc ttcatcatga cc 42
<210> 47
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 47
tgcacgacgt tggcctaa 18
<210> 48
<211> 48
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 48
ttgacaatgg cttaggtcta accaaaagat atacgtggtg gacgttac 48
<210> 49
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 49
ctcaacacca aacccagcgg 20
<210> 50
<211> 1176
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 50
atgtcttcgc gcagttttgc tcctctttcc agtcgtcagg agaacatttt aggaagagcg 60
gaaggcctgc gccagctcaa gcccggccaa agaattgtgc tcggtgtttt gaccgagaat 120
gagcagcata atcgaatatt tggacaggtt gcgtccaaat atgagacaac gctccaggag 180
gtgtctaagc gtgacgtcag tacgttcaga gcgtcaattg gtgatagtgt gattgagaca 240
gccactgtac agtcagtcaa gtcaacatca ttcctgctgc cttcagaact gctagtactc 300
gacgatgctg ttcaagatat tggctcaggg tcatttatgg attcatccat gcattcattg 360
gctgatgaag aagctgcttc ctctgaggac gtcttgtgtg tttctgaata tgctgaggac 420
atccacagat acctgcgtga atgcgaagtt aaatacagac caaaacccgg ctatatgaga 480
aagcagcctg acataaccaa ctgcatgagg gtcattcttg tggactggct ggtggaagtt 540
ggcgaagaat acaagctgtg ctctgagacc ctgtacctgg ctgtcaatta cctggaccgc 600
tttctttcgt gcatgtctgt cctgagagga aaactgcagc ttgtggggac ggctgcaata 660
ctcctggctg ccaaatacga ggaagtgtat cctccagagg tggatgagtt tgtctacatc 720
acggatgaca cctatacaaa gaaacagctg cttcggatgg agcagcacct gctccgagtg 780
cttgcgtttg acatgaccgc tcccacaatc caccagtttc tgatgcagta cagtttggag 840
gagcatgtct gcgccagaac tctaaacctc gctctgtatc tctcagaact gagcttgctt 900
gaggtggatc cttttgtgca atatctgcca tccaagaccg ctgctgcagc atattgtctg 960
gccaactaca ctctaaatgg ggctttgtgg cctgagaatc tgtatgcctt tactggttac 1020
tctctggctg tgattggccc gtgtttgaaa gagcttcata agctccatct cggggctgga 1080
agtcgccctc aacaggccat ccaggagaag tacaagagct cgaaatatca tggagtgtcc 1140
cagttggaac ctgtagagac tctgccgctg ccctga 1176
Claims (10)
1. a kind of authentication chip of positive blood culture, it is characterised in that the authentication chip has multiple reative cells, including sun
Property control room, negative Quality Control room and Bacteria Identification room；The Bacteria Identification room includes streptococcus pneumonia authentication room, enterococcus faecalis reflects
Determine room, Acinetobacter bauamnnii authentication room, pseudomonas aeruginosa authentication room, Klebsiella Pneumoniae authentication room, EHEC identification
At least one of room, staphylococcus aureus authentication room and haemophilus influenzae authentication room, the Bacteria Identification room and institute
State and isothermal duplication primer is respectively and fixedly provided with positive quality control room, wherein,
Sequence is fixed with the enterococcus faecalis authentication room respectively such as SEQ ID No:1~SEQ ID No:A plurality of shown in 6 is drawn
The combination of thing；
Sequence is fixed with the staphylococcus aureus authentication room respectively such as SEQ ID No:7~SEQ ID No:Shown in 12
The combination of a plurality of primer；
Sequence is fixed with the EHEC authentication room respectively such as SEQ ID No:13~SEQ ID No:It is more shown in 18
The combination of bar primer；
Sequence is fixed with the pseudomonas aeruginosa authentication room respectively such as SEQ ID No:19~SEQ ID No:Shown in 24
The combination of a plurality of primer；
Sequence is fixed with the streptococcus pneumonia authentication room respectively such as SEQ ID No:25~SEQ ID No:It is more shown in 29
The combination of bar primer；
Sequence is fixed with the Klebsiella Pneumoniae authentication room respectively such as SEQ ID No:30~SEQ ID No:Shown in 34
The combination of a plurality of primer；
Sequence is fixed with the Acinetobacter bauamnnii authentication room respectively such as SEQ ID No:35~SEQ ID No:Shown in 40
The combination of a plurality of primer；
Sequence is fixed with the haemophilus influenzae authentication room respectively such as SEQ ID No:41~SEQ ID No:Shown in 45
The combination of a plurality of primer；
Sequence is fixed with the positive quality control room respectively such as SEQ ID No:46~SEQ ID No:A plurality of primer shown in 49
Combination.
2. the authentication chip of positive blood culture as claimed in claim 1, it is characterised in that the Bacteria Identification room includes lung
Scorching streptococcus authentication room, enterococcus faecalis authentication room, Acinetobacter bauamnnii authentication room, pseudomonas aeruginosa authentication room, kerekou pneumonia
Totally eight kinds of primary dientification of bacteria room, EHEC authentication room, staphylococcus aureus authentication room and haemophilus influenzae authentication room.
3. a kind of identification kit of positive blood culture, it is characterised in that including positive blood as claimed in claim 1 or 2
The authentication chip of culture.
4. the identification kit of positive blood culture as claimed in claim 3, it is characterised in that also including DNA extracts reagents,
At least one of LAMP reaction reagents, Bst archaeal dna polymerases, nitrite ion, fluorescent dye and stoning sour water reagent.
5. the identification kit of positive blood culture as claimed in claim 4, it is characterised in that the LAMP reaction reagents bag
Include reaction buffer, dNTP, MgSO4And glycine betaine.
6. the identification kit of positive blood culture as claimed in claim 4, it is characterised in that in the LAMP reaction reagents
Containing added with positive quality control DNA fragmentation, the positive quality control DNA fragmentation is sequence such as SEQ ID No:Zebra fish shown in 50
The partial gene fragments of ccna1 genes.
7. the identification kit of positive blood culture as claimed in claim 4, it is characterised in that the nitrite ion is hydroxyl naphthalene
Phenol indigo plant solution；The fluorescent dye is SYBR green.
8. a kind of authentication method of positive blood culture, it is characterised in that comprise the following steps：
Extract the DNA of positive blood culture；
Added using the DNA of extraction as template, after it is mixed with LAMP reaction reagents as claimed in claim 1 or 2 positive
In the Bacteria Identification room of the authentication chip of blood culture thing, and add in the positive quality control room of the authentication chip and negative Quality Control room
Enter corresponding reagent；
Nucleic acid amplification reaction is carried out under isothermal conditions, observes the color or change in fluorescence of each reative cell of the authentication chip.
9. the authentication method of positive blood culture as claimed in claim 8, it is characterised in that the extraction positive blood culture
DNA be into the positive blood culture add DNA extracts reagents after the DNA is extracted by the way of boiling.
10. the authentication method of positive blood culture as claimed in claim 8, it is characterised in that described to enter under isothermal conditions
Row nucleic acid amplification reaction is by prior to 65 DEG C reaction 50min of reaction system, then 80 DEG C of reaction 10min, is finally cooled down at 25 DEG C
1min。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710994468.0A CN107513582A (en) | 2017-10-23 | 2017-10-23 | Authentication chip, kit and the authentication method of positive blood culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710994468.0A CN107513582A (en) | 2017-10-23 | 2017-10-23 | Authentication chip, kit and the authentication method of positive blood culture |
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| Publication Number | Publication Date |
|---|---|
| CN107513582A true CN107513582A (en) | 2017-12-26 |
Family
ID=60727523
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710994468.0A Pending CN107513582A (en) | 2017-10-23 | 2017-10-23 | Authentication chip, kit and the authentication method of positive blood culture |
Country Status (1)
| Country | Link |
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| CN (1) | CN107513582A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235288A (en) * | 2020-03-12 | 2020-06-05 | 上海蕴箬生物科技有限公司 | Micro-fluidic chip kit for rapidly detecting pathogenic bacteria on wound surface |
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