CN106434996A - Kit and method for detecting Acinetobacter baumannii DNA - Google Patents
Kit and method for detecting Acinetobacter baumannii DNA Download PDFInfo
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Abstract
The invention discloses a kit and method for detecting Acinetobacter baumannii DNA; the kit comprises: a nucleic acid release agent, a PCR (polymerase chain reaction) liquid, an enzyme mixed liquid, an Acinetobacter baumannii positive control, and an Acinetobacter baumannii negative control, wherein the PCR liquid is made from PCR buffer solution, deoxyribonucleoside, primers for amplifying targeted polynucleotide, a probe for amplifying targeted polynucleotide, primers for amplifying internally labeled fragments and a probe for detecting an internal label. By optimizing conditions in the embodiment of the invention, primers for amplifying targeted polynucleotide and probes for amplifying the targeted polynucleotide, both having optimal amplification, are screened out, the kit is capable of detecting not pathogens of Acinetobacter baumannii but various infections of Acinetobacter baumannii but, is highly specific, and accordingly has greatly improved detection sensitivity, accuracy and stability.
Description
Technical field
The present invention relates to technical field of biological, more particularly to a kind of test kit of detection Acinetobacter bauamnnii DNA and
Detection method.
Background technology
The laboratory diagnostic method of Acinetobacter bauamnnii mainly uses business-like Bacteria Identification system, such as API at present
20NE, VITED 2, Phoenix and MicroScan WalkAway, but its error rate is up to 25%.Based on biochemical and antibacterial
The authentication method of culture, not only sensitivity is low for the authentication method of antibacterial culturing, and its cultivation cycle is up to 24-72 hour, often makes
Patient misses optimal treatment period.
Clinically detection Acinetobacter bauamnnii-DNA mainly uses real-time fluorescence PCR technology.The improvement of this technology and should
It is used in recent years and develops rapidly.This technology hybridizes PCR, molecule and photochemistry organically combines together, makes PCR amplification and product
The overall process of analysis is all carried out under single tube sealing condition.A kind of with some specific band transmittings using quantitative real time PCR Instrument
Light source and the PCR amplification instrument of fluorescence detection device, collect detection fluorescence signal, by corresponding analysis software, glimmering by detected
Optical signal is corresponding with period, can automatically analyze acquisition amplification curve after off-test；According to amplification curve and fluorescence threshold line
Intersection point (i.e. Ct value) and amplification curve shape, it can be determined that yin and yang attribute result.Compare with traditional PCR, which is anti-
Answer the probe that a two ends difference mark fluorescent reporter group and quenching group is increased in system.When probe structure is complete, glimmering
The fluorescent energy that light reporter group sends is quenched group absorptions, assumes quenching effect；If there is target sequence in amplification procedure
Exist, with the extension of target fragment, probe molecule is gradually by Taq enzyme hydrolysis cut-out, fluorescent reporter group and quenching group phase
Mutually dissociate, fluorescence energy transfer effect between the two has been blocked, the fluorescence signal that fluorescent reporter group sends is by fluorescence detection device
Collect.With the carrying out of amplification, fluorescence signal assumes linear enhancing with the amplification of purpose fragment.After off-test, permissible
The software for being carried by fluorescent PCR instrument automatically analyzes data, it is possible to obtain the definite value result of yin and yang attribute result and concentration of specimens, because
This, the technology gradually replaces traditional PCR method, obtains very in the detection and quantitative analyses of target polynucleotide sample
It is widely applied.
Domestic existing several test kits based on real-time fluorescence PCR technology for detection Acinetobacter bauamnnii DNA are applied to clinic
In detection, Acinetobacter bauamnnii-DNA extraction method that these test kits are provided is mainly boiling method.In practical clinical
In, the method exposes many weak points, and nucleic acid extraction process is more complicated, and time-consuming for sample process, and when sample is processed,
Through multiple steps such as boiling lysis, high speed centrifugation enrichment DNAs, DNA loss is serious, and the sample of high concentration is present cracking and
Enrichment is insufficient, and the accuracy of testing result is low.
Content of the invention
The goal of the invention of the present invention is to provide a kind of test kit of detection Acinetobacter bauamnnii DNA and detection method, with
Solve the existing test kit based on real-time fluorescence PCR technology for detection Acinetobacter bauamnnii DNA, detection process, accuracy in detection
Low problem.
First aspect embodiments provides a kind of test kit of detection Acinetobacter bauamnnii DNA, the test kit
Including：Nucleic acid releasing agent, PCR reactant liquor, enzyme mixation, Acinetobacter bauamnnii positive control, Acinetobacter bauamnnii negative control；
Wherein, the PCR reactant liquor includes：PCR reaction buffer, dideoxyribonucleotide triphosphate, for many nucleoside of target
The primer of acid amplification, for the probe of target polynucleotide detection, primer for internal standard fragment amplification and for detecting interior target
Probe；
The primer for target polynucleotide amplification includes：Acinetobacter bauamnnii-F and Acinetobacter bauamnnii-R；
The base sequence of the Acinetobacter bauamnnii-F is：5’-GCTTCCGCTATTCCARTTTATCA-3’；
The base sequence of the Acinetobacter bauamnnii-R is；5’-AACCAACACGCTTCACTTCMTTA-3’；
The base sequence of the probe for target polynucleotide detection is：5’-TTAGCTCGTCGTATTGGACTTGARC
TCATGT-3’.
Further, the primer for internal standard fragment amplification includes：Forward primer IC-F and downstream primer IC-R；
The base sequence of the forward primer IC-F is：5’-CCGACGAGCCGAACCACA-3’
The base sequence of the downstream primer IC-R is：5’-CACACCACGGACACACAAAGGA-3’；
The base sequence for detecting interior target probe is：5'-CAAGACGGATTGCGAGCCTTACAGC-3'.
Further, described interior it is designated as inserting the DNA artificial sequence synthetic that a section long of pMD18T carrier is 177 base pairs
Recombinant；177 base-pair sequence of interior target is：
5-ATGCCATGAAAGCTTCCGCTATTCCATTTATCAAGATTTAGCTCGTCGTATTGGACTTGACTCATGT
CTAAGGAAGTGAAGCGTGTTGGTTATGGCAATGCAGATATCGGTACCCAAGTCGATAATTTTTGGCTGTGGGTCCTT
AAAAATTACTCCTCAGCAAGAGGCACATTTGCT-3’.
Further, the enzyme mixation is by 0.2U/ μ l～5U/ μ l hot resistant DNA polymerase and 0.02U/ μ l～0.2U/ μ l
Uracil dna glycosylase constitutes.
Further, the nucleic acid releasing agent includes：Concentration be 0.01～0.1mM/L Buddhist Sha is graceful, concentration be 200～
The potassium chloride of 500mM/L, concentration are 0.01%～2%g/mL dodecyl sodium sulfate and volume/volume is 0.02%～5%
Ethanol.
Further, the PCR buffer by concentration for 200mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride solution, dense
Spend for 20mmol/L magnesium chloride solution, concentration be 500mmol/L Klorvess Liquid, volume/volume be 0.2% TritonX solution and
Volume/volume constitutes for 10% formamide solution.
Further, the pH of the Tri(Hydroxymethyl) Amino Methane Hydrochloride solution is 7.5.
Further, the Acinetobacter bauamnnii strong positive sample that the Acinetobacter bauamnnii positive control is collected for clinical hospitals
This；
The Acinetobacter bauamnnii negative control is the inactivation feminine gender clinical sample for not containing Acinetobacter bauamnnii.
Embodiment of the present invention second aspect illustrates a kind of detection method of detection Acinetobacter bauamnnii DNA, methods described bag
Include：
S101：2～5 μ l of nucleic acid releasing agent is all added in each PCR reaction tube, and sequentially adds sample to be tested, negative
Control, positive control, obtain PCR reaction tube to be measured；
S102；PCR reactant liquor 38～43 μ l, 3000rpm centrifugation 30 is all added in PCR reaction tube to be measured each described
Second, it is placed on fluorescent quantitative PCR instrument and is tested.
Third aspect present invention illustrates a kind of detection method of detection Acinetobacter bauamnnii DNA, and methods described includes：
S201：100～500 μ l samples to be tested are taken, 13,000rpm centrifugations 5 minutes remove clear liquid, add 30～50 μ l release
Agent, standing obtained the sample to be tested of pretreatment after 10 minutes；
S202：Add the sample to be tested of pretreatment described in 5～10 μ l, negative control, the positive right in each PCR reaction tube
According to obtaining PCR reaction tube to be measured；
S203：PCR reactant liquor 38～43 μ l, 3000rpm is all added to be centrifuged in each described PCR reaction tube to be measured 30 seconds,
It is placed on fluorescent quantitative PCR instrument and is tested.
From above technical scheme, the embodiment of the present invention illustrate a kind of test kit of detection Acinetobacter bauamnnii DNA and
Detection method, the test kit includes：Nucleic acid releasing agent, PCR reactant liquor, enzyme mixation, Acinetobacter bauamnnii positive control, Bao
Graceful acinetobacter calcoaceticus negative control；Wherein, the PCR reactant liquor includes：PCR reaction buffer, dideoxyribonucleotide triphosphate, use
In the primer of target polynucleotide amplification, for the primer of target polynucleotide amplification, primer for internal standard fragment amplification and it is used for
Target probe in detection.SeqMan and MegAlign software in application DNAStar software kit of the present invention is to retrieving in Genbank
To Acinetobacter bauamnnii genome sequence carry out tetraploid rice, find out homologous conservative section, in Acinetobacter bauamnnii most
Conservative region devises 5 pairs of primers and probe, optimized, filtered out expanding effect best for target polynucleotide amplification
Primer and the probe for target polynucleotide amplification, the test kit can detect that the various infection of Acinetobacter bauamnnii, but can not
Detect non-Acinetobacter bauamnnii pathogen, with higher specificity, therefore substantially increase detection sensitivity, accuracy and
Stability；While a kind of detection method of the detection Acinetobacter bauamnnii DNA shown in the embodiment of the present invention adopts strong albumen
Denaturant, rapid damage pathogen adventitia, pathogen nucleic acid is discharged, without the need for heating, without the need for centrifugation, only a pipettor is needed,
Release and the extraction of DNA can be completed；Methods described specifically has accuracy in detection height, and operation is quick, the simple and linear model of method
The advantages of enclosing width.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to institute in embodiment
The accompanying drawing for using is needed to be briefly described, it should be apparent that, drawings in the following description are only some enforcements of the present invention
Example, for those of ordinary skill in the art, on the premise of not paying creative work, can also obtain according to these accompanying drawings
Obtain other accompanying drawings.
Fig. 1 is the flow chart of the detection method according to an a kind of detection Acinetobacter bauamnnii DNA for being preferable to carry out exemplifying；
Fig. 2 is a kind of flow process of the detection method of the detection Acinetobacter bauamnnii DNA according to another preferred embodiment
Figure；
Fig. 3 is the amplification curve for randomly selecting 10 positive reference product；
Fig. 4 is the amplification curve for randomly selecting 10 negative reference product；
Fig. 5 is 2015001 batches of reagent withinrun precision testing results；
Fig. 6 is 2015002 batches of reagent withinrun precision testing results；
Fig. 7 is 2015003 batches of reagent withinrun precision testing results；
Fig. 8 is that Candida albicans, staphylococcus aureuses, escherichia coli, chlamydia trachomatiss and urea urea substance are examined
The amplification curve that tests；
The detection method of detection Acinetobacter bauamnnii DNA of the Fig. 9 shown in the embodiment of the present invention is measured under 6 Concentraton gradient
Acinetobacter bauamnnii DNA detection amplification curve；
Figure 10 measures the amplification curve of the detection of the Acinetobacter bauamnnii DNA under 6 Concentraton gradient for conventional heating methods；
The amplification curve of Figure 11 test kit detection positive sample amplification for the first time；
The amplification curve of Figure 12 test kit detection second amplification of positive sample；
The amplification curve of Figure 13 test kit detection negative sample amplification for the first time；
The amplification curve of Figure 14 test kit detection second amplification of negative sample.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Whole description, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
Embodiment of the present invention first aspect illustrates a kind of test kit of detection Acinetobacter bauamnnii DNA, the test kit bag
Include：Nucleic acid releasing agent, PCR reactant liquor, enzyme mixation, Acinetobacter bauamnnii positive control, Acinetobacter bauamnnii negative control；
Wherein, the PCR reactant liquor includes：PCR reaction buffer, dideoxyribonucleotide triphosphate, for many nucleoside of target
The primer of acid amplification, for the probe of target polynucleotide detection, primer for internal standard fragment amplification and for detecting interior target
Probe；
The primer for target polynucleotide amplification includes：Acinetobacter bauamnnii-F and Acinetobacter bauamnnii-R；
The base sequence of the Acinetobacter bauamnnii-F is：5’-GCTTCCGCTATTCCARTTTATCA-3’；
The base sequence of the Acinetobacter bauamnnii-R is；5’-AACCAACACGCTTCACTTCMTTA-3’；
The base sequence of the probe for target polynucleotide detection is：5’-TTAGCTCGTCGTATTGGACTTGARC
TCATGT-3’.
Specifically, PCR reactant liquor includes 10 × PCR reaction buffer, 5 μ l, 0.1～0.5mmol/L dezyribonucleoside three
Phosphoric acid, the Acinetobacter bauamnnii-F of 0.1 μm of ol/L～0.5 μm ol/L and Acinetobacter bauamnnii-R, 0.1 μm of ol/L～0.5 μm ol/
The probe for target polynucleotide detection of L.10 × PCR reaction buffer includes the 200mmol/L trihydroxy methyl of pH7.5
Aminomethane hydrochloride solution, 20mmol/L magnesium chloride solution, 500mmol/L Klorvess Liquid, 0.2% (volume/volume) song
Draw and lead to solution and 10% (volume/volume) formamide solution；The dideoxyribonucleotide triphosphate be dAUP, dCUP, dUUP,
DGUP or dTUP；Acinetobacter bauamnnii-F, the Acinetobacter bauamnnii-R and the probe for target polynucleotide detection are derived from
The primer of the conservative region of Acinetobacter bauamnnii and probe.
SeqMan and MegAlign software in embodiment of the present invention application DNAStar software kit is to retrieving in Genbank
To Acinetobacter bauamnnii genome sequence carry out tetraploid rice, find out homologous conservative section, in Acinetobacter bauamnnii most
Conservative region devises 5 pairs of primers and probe, optimized, filtered out expanding effect best for target polynucleotide amplification
Primer (Acinetobacter bauamnnii-F and Acinetobacter bauamnnii-R) and the probe for target polynucleotide amplification, the wherein Bao Man is not
The base sequence of lever bacterium-F is：5’-GCTTCCGCTATTCCARTTTATCA-3’；
The base sequence of the Acinetobacter bauamnnii-R is；5’-AACCAACACGCTTCACTTCMTTA-3’；
The base sequence of the probe for target polynucleotide detection is：5’-TTAGCTCGTCGTATTGGACTTGARC
TCATGT-3’.The testing cassete can detect that the various infection of Acinetobacter bauamnnii, but can not detect non-Acinetobacter bauamnnii disease
Substance, with higher specificity, therefore substantially increases detection sensitivity, accuracy and stability.
Further, described interior it is designated as inserting the DNA artificial sequence synthetic that a section long of pMD18T carrier is 177 base pairs
Recombinant；177 base-pair sequence of interior target is：
5-ATGCCATGAAAGCTTCCGCTATTCCATTTATCAAGATTTAGCTCGTCGTATTGGACTTGACTCATGT
CTAAGGAAGTGAAGCGTGTTGGTTATGGCAATGCAGATATCGGTACCCAAGTCGATAATTTTTGGCTGTGGGTCCTT
AAAAATTACTCCTCAGCAAGAGGCACATTTGCT-3’.
Preferably, the primer for internal standard fragment amplification includes：Forward primer IC-F and downstream primer IC-R；
The base sequence of the forward primer IC-F is：5’-CCGACGAGCCGAACCACA-3’；
The base sequence of the downstream primer IC-R is：5’-CACACCACGGACACACAAAGGA-3’；
The base sequence for detecting interior target probe is：5'-CAAGACGGATTGCGAGCCTTACAGC-3'.
The testing cassete increased internal standard during sample extraction, the internal standard：One section for insertion pMD18T carrier
The recombinant of the DNA artificial sequence synthetic of a length of 177 base pair, concentration is 2.00E+05copies/ml～2.00E+
06copies/ml, as the positive internal reference in PCR amplification system using internal standard monitoring DNA extraction and PCR course of reaction, prison
Whether effectively control reaction system, prevents pattern detection false negative.After fluorescent quantitative PCR terminates, by curve shape and Ct
Value judges Acinetobacter bauamnnii-DNA yin and yang attribute, and testing result can be used for the auxiliary diagnosis of Acinetobacter bauamnnii infection and medicine
The observation of curative effect.
The embodiment of the present invention through substantial amounts of experiment, to each reagent in kind of the test kit of detection Acinetobacter bauamnnii DNA
Densitometer component is optimized.
Preferably, the enzyme mixation is by 0.2U/ μ l～5U/ μ l hot resistant DNA polymerase and 0.02U/ μ l～0.2U/ μ l
Uracil dna glycosylase (UNG enzyme) constitutes.
Can be degraded using UNG enzyme the feature of the DNA containing dU, UNG enzyme and dUTP is with the addition of in PCR system, permissible
The pollution of prevention previous PCR product, prevents pattern detection false positive；
Preferably, the Acinetobacter bauamnnii strong positive sample that the Acinetobacter bauamnnii positive control is collected for clinical hospitals
This；
The Acinetobacter bauamnnii negative control is the inactivation feminine gender clinical sample for not containing Acinetobacter bauamnnii.
Preferably, the nucleic acid releasing agent includes：Concentration be 0.01～0.1mM/L Buddhist Sha is graceful, concentration be 200～
The potassium chloride of 500mM/L, concentration are 0.01%～2%g/mL dodecyl sodium sulfate and volume/volume is 0.02%～5%
Ethanol.
Preferably, the PCR buffer by concentration for 200mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride solution, dense
Spend for 20mmol/L magnesium chloride solution, concentration be 500mmol/L Klorvess Liquid, volume/volume be 0.2% TritonX solution and
Volume/volume constitutes for 10% formamide solution.
Preferably, the pH of the Tri(Hydroxymethyl) Amino Methane Hydrochloride solution is 7.5.
Embodiment of the present invention second aspect illustrates a kind of detection method of detection Acinetobacter bauamnnii DNA, as shown in figure 1,
Methods described includes：
S101：2～5 μ l of nucleic acid releasing agent is all added in each PCR reaction tube, and sequentially adds sample to be tested, negative
Control, positive control, obtain PCR reaction tube to be measured；
Specifically, first, sample to be tested is numbered, then, peek PCR reaction tube, the PCR reaction tube includes
Sample to be tested PCR reaction tube, positive control PCR reaction tube and negative control PCR reaction tube；The number ratio of the PCR reaction tube
The number of the sample to be tested is many two, and the two PCR reaction tubes are respectively as positive control PCR reaction tube and negative control
PCR reaction tube；PCR reaction tube is numbered, and the nucleic acid releasing agent of 4～20 μ l will be all added in each PCR reaction tube, by institute
State the PCR reaction tube that sample to be tested sequentially adds reference numeral；Positive control is added in positive control PCR reaction tube, in feminine gender
Control PCR reaction tube adds negative control.
S102；PCR mixed liquor 38～43 μ l, 3000rpm centrifugation 30 is all added in PCR reaction tube to be measured each described
Second, supernatant is taken, is placed on fluorescent quantitative PCR instrument and is tested.
Add wherein in each PCR reaction tube PCR mixed liquor be 38～43 μ l of PCR reactant liquor, 1～2 μ l of enzyme mixation and
01 μ l of internal standard.
Third aspect present invention illustrates a kind of detection method of detection Acinetobacter bauamnnii DNA, as shown in Fig. 2 the side
Method includes：
S201：100～500 μ l samples to be tested are taken, 13,000rpm centrifugations 5 minutes remove clear liquid, add 30～50 μ l release
Agent, standing obtained the sample to be tested of pretreatment after 10 minutes；
S202：Add the sample to be tested of pretreatment described in 5～10 μ l, negative control, the positive right in each PCR reaction tube
According to obtaining PCR reaction tube to be measured；
S203：PCR mixed liquor 38～43 μ l, 3000rpm is all added to be centrifuged in each described PCR reaction tube to be measured 30 seconds,
Supernatant is taken, is placed on fluorescent quantitative PCR instrument and is tested.
Add wherein in each PCR reaction tube PCR mixed liquor be 38～43 μ l of PCR reactant liquor, 1～2 μ l of enzyme mixation and
01 μ l of internal standard.
Supernatant after centrifugation is transferred in the sample slot of fluorescent quantitative PCR instrument, by correspondence by the embodiment of the present invention
Order arranges sample to be tested title and qualitative reference product concentration.
Fluorescence detection channel is selected：Select FAM passage (Reportere:FAM,Quencher:None) detection Bao Man is motionless
Bacillus；Select HEX or VIC passage (Reporter:VIC,Quencher:None) internal standard is detected；Reference fluorescent (Passive
Reference) none is set to.
Quantitative fluorescent PCR reaction condition is：
Interpretation of result：
After reaction terminates, instrument automatically saves result, it is possible to use the software that instrument is carried is automatically analyzed (can also
Initial value, end value and the threshold line value for adjusting baseline manually is analyzed), then record sample Ct value result.Amplification is bent
The intersection point of line and threshold line, (i.e. cycle threshold refers to that the fluorescence signal in PCR reaction tube reaches the threshold of setting to referred to as Ct
The cycling numerical value for being experienced during value)；Instrument software is according to each sample Ct value size, it can be determined that testing result.For measure Ct
The sample of value≤38, is reported as the Acinetobacter bauamnnii-DNA positive；For the sample for determining no Ct value, meanwhile, internal standard is detected as
Positive (Ct value≤40), are reported as Acinetobacter bauamnnii-DNA feminine gender.If internal standard Ct value>40 or no show, then the inspection of the sample
Survey result invalid, should search and exclude reason, and this sample is carried out repeating test.
The detection method of the detection Acinetobacter bauamnnii DNA shown in the embodiment of the present invention, in application DNAStar software kit
SeqMan and MegAlign software carries out tetraploid rice to the Acinetobacter bauamnnii genome sequence for retrieving in Genbank,
Homologous conservative section is found out, and 5 pairs of primers and probe is devised in the most conservative region of Acinetobacter bauamnnii, optimized, filter out
The best pair of primers of expanding effect and probe, can detect that the various infection of Acinetobacter bauamnnii, but can not detect non-Bao
Graceful acinetobacter calcoaceticus pathogen, illustrates that test kit of the present invention has good specificity.Compared with the available reagent box of market, the present invention
Have the advantage that：1) sample process simplicity, extraction efficiency height, the extracting method of Acinetobacter bauamnnii-DNA is compared and
Optimize, the method that have selected nucleic acid release, using strong protein denaturant, rapid damage pathogen coat protein structure, release
Pathogen nucleic acid is released, and release and the extraction of DNA can be completed without the need for heating；2) need not heat, be difficult to cause aerosol dirt
Dye；3) detection sensitivity is up to 500copies/m；4) combination is optimized to PCR reaction system, can be dropped using UNG enzyme
The feature of DNA of the solution containing dU, with the addition of UNG enzyme and dUTP in PCR system, can prevent the pollution of previous PCR product, prevent
Only pattern detection false positive；5) internal standard being increased during sample extraction, was reacted using internal standard monitoring DNA extraction and PCR
Journey, whether effectively monitoring reaction system, prevents pattern detection false negative.After fluorescent quantitative PCR terminates, by curve shape
And Ct value judges Acinetobacter bauamnnii-DNA yin and yang attribute, testing result can be used for Acinetobacter bauamnnii infection auxiliary diagnosis and
The observation of curative effect of medication.
In order to verify that the present invention is to implement the detection test kit of Acinetobacter bauamnnii DNA for exemplifying and the inspection of detection method
The accuracy of survey, the embodiment of the present invention is carried out to 10 kinds of enterprise work positive reference product and 10 kinds of enterprise work negative reference product
Test.Testing result is as shown in Figure 3 and Figure 4.Wherein Fig. 3 is the amplification curve of positive reference product, and Fig. 4 is negative reference product.
Test result shows, all can detect that the various infection of Acinetobacter bauamnnii, in negative reference in positive reference product
Kind all not can detect that the various infection of Acinetobacter bauamnnii, the examination of the detection Acinetobacter bauamnnii DNA that the present invention is illustrated in real time
Agent box and detection method carry out detecting that yin and yang attribute reference material meets in rate to be 100% visible reality of the present invention to enterprise work reference material
The test kit and detection method for exemplifying detection Acinetobacter bauamnnii DNA is applied, test kit detection enterprise work reference material is carried out
It is 100%, to illustrate that test kit of the present invention has good specificity that detection yin and yang attribute reference material meets in rate.
The embodiment of the present invention is carried out to detecting the test kit of Acinetobacter bauamnnii DNA and the precision of detection method further
Checking, the present embodiment is respectively adopted three batches of test kits (2015001,2015002,2015003) and detects high, medium and low three concentration
Sample, respectively 8 repetitions of detection of each sample, the increasing curve of detection is as shown in Fig. 5～Fig. 7, and wherein Fig. 5 is 2015001 batches of reagents
Withinrun precision testing result, Fig. 6 is that 2015002 batches of reagent withinrun precision testing results, Fig. 7 is criticized for 2015003 batches of reagents
Interior precision testing result.
As a result show, the embodiment of the present invention was being tested to the test kit and detection method that detect Acinetobacter bauamnnii DNA
In in journey batch and batch between reproducible, the coefficient of variation of testing result Ct value<10%.
Further.The embodiment of the present invention is entered to detecting the test kit of Acinetobacter bauamnnii DNA and the specificity of detection method
Checking is gone.
The embodiment of the present invention is to common clinical substance, Candida albicans, staphylococcus aureuses, escherichia coli, trachoma
Chlamydia (CT) and urea urea substance (UU) are checked, and assay is as shown in Figure 8.
Visible white candidiasises, staphylococcus aureuses, escherichia coli, chlamydia trachomatiss (CT) and ureaplasma urealyticum (UU)
Deng common causative no cross reaction, testing result is feminine gender.It can be seen that the detection Acinetobacter bauamnnii shown in the embodiment of the present invention
The test kit of DNA and detection method, can detect that the various infection of Acinetobacter bauamnnii, but can not detect non-Acinetobacter bauamnnii
Pathogen, illustrates that test kit of the present invention has good specificity.
Further, impact test of the embodiment of the present invention to DNA Different Extraction Method to Acinetobacter bauamnnii-DNA detection
Verified.
The embodiment of the present invention selects 6 gradient samples, respectively with the nucleic acid quick release method shown in the embodiment of the present invention and
Boiling method extracts nucleic acid, detects, testing result is as shown in Figure 9 and Figure 10 with this detectable：
As a result show, test result indicate that, by while detecting that gradient dilution sample to be tested finds, the embodiment of the present invention is shown
Go out to detect the detection method of Acinetobacter bauamnnii DNA, the inspection of nucleic acid is extracted using nucleic acid quick release method and traditional boiling method
Surveying result does not have notable difference, and the inventive method operation is more easy to be quick, without the need for heating.
Further, the preventive effect that the embodiment of the present invention is polluted to PCR primer is tested.
Specifically, 2 positive sample are taken respectively and 2 negative sample is detected to its amplified production, and record respectively
The negative sample and the positive sample amplification curve and secondary amplification curve result are as shown in figures 11-14；
As a result show:Add appropriate UNG enzyme that PCR primer can be prevented to pollute in PCR reaction system；
From above technical scheme, the embodiment of the present invention illustrate a kind of test kit of detection Acinetobacter bauamnnii DNA and
Detection method, the test kit includes：Nucleic acid releasing agent, PCR reactant liquor, enzyme mixation, Acinetobacter bauamnnii positive control, Bao
Graceful acinetobacter calcoaceticus negative control；Wherein, the PCR reactant liquor includes：PCR reaction buffer, dideoxyribonucleotide triphosphate, use
In the primer of target polynucleotide amplification, for the primer of target polynucleotide amplification, primer for internal standard fragment amplification and it is used for
Target probe in detection.SeqMan and MegAlign software in application DNAStar software kit of the present invention is to retrieving in Genbank
To Acinetobacter bauamnnii genome sequence carry out tetraploid rice, find out homologous conservative section, in Acinetobacter bauamnnii most
Conservative region devises 5 pairs of primers and probe, optimized, filtered out expanding effect best for target polynucleotide amplification
Primer and the probe for target polynucleotide amplification, the test kit can detect that the various infection of Acinetobacter bauamnnii, but can not
Detect non-Acinetobacter bauamnnii pathogen, with higher specificity, therefore substantially increase detection sensitivity, accuracy and
Stability；While a kind of detection method of the detection Acinetobacter bauamnnii DNA shown in the embodiment of the present invention adopts strong albumen
Denaturant, rapid damage pathogen adventitia, pathogen nucleic acid is discharged, without the need for heating, without the need for centrifugation, only a pipettor is needed,
Release and the extraction of DNA can be completed；Methods described specifically has accuracy in detection height, and operation is quick, the simple and linear model of method
The advantages of enclosing width.
Those skilled in the art will readily occur to its of the present invention after considering description and putting into practice invention disclosed herein
Its embodiment.The application is intended to any modification, purposes or the adaptations of the present invention, these modifications, purposes or
Person's adaptations follow the general principle of the present invention and including the undocumented common knowledge in the art of the present invention
Or conventional techniques.Description and embodiments are considered only as exemplary, and true scope and spirit of the invention are by following
Claim is pointed out.
It should be appreciated that the precision architecture for being described above and being shown in the drawings is the invention is not limited in, and
And various modifications and changes can carried out without departing from the scope.The scope of the present invention is only limited by appended claim.
Sequence table sample
SEQUENCE LISTING
<110>Hunan Shengxiang Biological Technology Co., Ltd.
<120>A kind of test kit of detection Acinetobacter bauamnnii DNA and detection method
<130> 1
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213>Acinetobacter bauamnnii
<400> 1
gcttccgcta ttccarttta tca 23
<210> 2
<211> 23
<212> DNA
<213>Acinetobacter bauamnnii
<400> 2
aaccaacacg cttcacttcm tta 23
<210> 3
<211> 31
<212> DNA
<213>Acinetobacter bauamnnii
<400> 3
ttagctcgtc gtattggact tgarctcatg t 31
<210> 4
<211> 18
<212> DNA
<213>Internal standard
<400> 4
ccgacgagcc gaaccaca 18
<210> 5
<211> 22
<212> DNA
<213>Internal standard
<400> 5
cacaccacgg acacacaaag ga 22
<210> 6
<211> 25
<212> DNA
<213>Internal standard
<400> 6
caagacggat tgcgagcctt acagc 25
<210> 7
<211> 177
<212> DNA
<213>Internal standard
<400> 7
atgccatgaa agcttccgct attccattta tcaagattta gctcgtcgta ttggacttga 60
ctcatgtcta aggaagtgaa gcgtgttggt tatggcaatg cagatatcgg tacccaagtc 120
gataattttt ggctgtgggt ccttaaaaat tactcctcag caagaggcac atttgct 177
Claims (10)
1. a kind of test kit of detection Acinetobacter bauamnnii DNA, it is characterised in that the test kit includes：Nucleic acid releasing agent,
PCR reactant liquor, enzyme mixation, Acinetobacter bauamnnii positive control, Acinetobacter bauamnnii negative control；
Wherein, the PCR reactant liquor includes：PCR reaction buffer, dideoxyribonucleotide triphosphate, expands for target polynucleotide
The primer of increasing, for the probe of target polynucleotide detection, primer for internal standard fragment amplification and for detecting interior target probe；
The primer for target polynucleotide amplification includes：Acinetobacter bauamnnii-F and Acinetobacter bauamnnii-R；
The base sequence of the Acinetobacter bauamnnii forward primer Ab-F is：5’-GCTTCCGCTATTCCARTTTATCA-3’；
The base sequence of the Acinetobacter bauamnnii downstream primer Ab-R is；5’-AACCAACACGCTTCACTTCMTTA-3’；
The base sequence of the probe Ab-P for target polynucleotide detection is：5’-TTAGCTCGTCGTATTGGACTTGARC
TCATGT-3’.
2. a kind of test kit of detection Acinetobacter bauamnnii DNA according to claim 1, it is characterised in that the internal standard
For inserting the recombinant of the one section long DNA artificial sequence synthetic for being 177 base pairs of pMD18T carrier；177 alkali of interior target
Base to sequence is：
5-ATGCCATGAAAGCTTCCGCTATTCCATTTATCAAGATTTAGCTCGTCGTATTGGACTTGACTCATGTCTAA
GGAAGTGAAGCGTGTTGGTTATGGCAATGCAGATATCGGTACCCAAGTCGATAATTTTTGGCTGTGGGTCCTTAAAA
ATTACTCCTCAGCAAGAGGCACATTTGCT-3’.
3. the test kit of a kind of detection Acinetobacter bauamnnii DNA according to claim 2, it is characterised in that described be used for
The primer of internal standard fragment amplification includes：Forward primer IC-F and downstream primer IC-R；
The base sequence of the forward primer IC-F is：5’-CCGACGAGCCGAACCACA-3’
The base sequence of the downstream primer IC-R is：5’-CACACCACGGACACACAAAGGA-3’；
The base sequence for detecting interior target probe I C-P is：5'-CAAGACGGATTGCGAGCCTTACAGC-3'.
4. the test kit of a kind of detection Acinetobacter bauamnnii DNA according to any one of claim 1-3, it is characterised in that
The enzyme mixation is by 0.2U/ μ l～5U/ μ l hot resistant DNA polymerase and the Uracil DNA Glycosylase of 0.02U/ μ l～0.2U/ μ l
Enzyme constitutes.
5. the test kit of a kind of detection Acinetobacter bauamnnii DNA according to any one of claim 1-3, it is characterised in that
The nucleic acid releasing agent includes：Concentration be 0.01～0.1mM/L Buddhist Sha is graceful, potassium chloride that concentration is 200～500mM/L, dense
Spend for 0.01%～2%g/mL dodecyl sodium sulfate and ethanol that volume/volume is 0.02%～5%.
6. the test kit of a kind of detection Acinetobacter bauamnnii DNA according to any one of claim 1-3, it is characterised in that
The PCR buffer by concentration be the Tri(Hydroxymethyl) Amino Methane Hydrochloride solution of 200mmol/L, concentration be
Magnesium solution, concentration are 500mmol/L Klorvess Liquid, volume/volume be 0.2% TritonX solution and volume/volume is 10%
Formamide solution constitutes.
7. a kind of test kit of detection Acinetobacter bauamnnii DNA according to claim 6, it is characterised in that three hydroxyl
The pH of aminomethane HCI solution is 7.5.
8. the test kit of a kind of detection Acinetobacter bauamnnii DNA according to any one of claim 1-3, it is characterised in that
The Acinetobacter bauamnnii strong positive sample that the Acinetobacter bauamnnii positive control is collected for clinical hospitals；
The Acinetobacter bauamnnii negative control is the inactivation feminine gender clinical sample for not containing Acinetobacter bauamnnii.
9. a kind of detection method of detection Acinetobacter bauamnnii DNA, it is characterised in that methods described includes：
S101：2～5 μ l of nucleic acid releasing agent is all added in each PCR reaction tube, and sequentially adds sample to be tested, negative control,
Positive control, obtains PCR reaction tube to be measured；
S102；To in PCR reaction tube to be measured each described, all add PCR reactant liquor 38～43 μ l, 3000rpm to be centrifuged 30 seconds, take
Supernatant, is placed on fluorescent quantitative PCR instrument and is tested.
10. a kind of detection method of detection Acinetobacter bauamnnii DNA, it is characterised in that methods described includes：
S201：100～500 μ l samples to be tested are taken, 13,000rpm centrifugations 5 minutes remove clear liquid, 30～50 μ l releasing agents are added,
Standing obtained the sample to be tested of pretreatment after 10 minutes；
S202：The sample to be tested of pretreatment, negative control, positive control described in 5～10 μ l is added in each PCR reaction tube, is obtained
To PCR reaction tube to be measured；
S203：All add PCR reactant liquor 38～43 μ l, 3000rpm to be centrifuged in each described PCR reaction tube to be measured 30 seconds, take
Clear liquid, is placed on fluorescent quantitative PCR instrument and is tested.
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| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
| CN110079623A (en) * | 2019-05-27 | 2019-08-02 | 深圳市人民医院 | A kind of detection method suitable for Acinetobacter bauamnnii virulence |
| CN111187828A (en) * | 2020-02-11 | 2020-05-22 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of human folate metabolism gene |
| CN111254188A (en) * | 2020-02-06 | 2020-06-09 | 成都导胜生物技术有限公司 | Tissue fluid for direct PCR amplification, amplification system and kit |
| CN111996239A (en) * | 2019-05-27 | 2020-11-27 | 复旦大学附属中山医院 | Primers and probes for detecting acinetobacter baumannii and detection method thereof |
| WO2021103641A1 (en) * | 2019-11-25 | 2021-06-03 | 南开大学 | Detection method for molecular typing acinetobacter baumannii sv4 serotype o antigen |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
| CN110079623A (en) * | 2019-05-27 | 2019-08-02 | 深圳市人民医院 | A kind of detection method suitable for Acinetobacter bauamnnii virulence |
| CN111996239A (en) * | 2019-05-27 | 2020-11-27 | 复旦大学附属中山医院 | Primers and probes for detecting acinetobacter baumannii and detection method thereof |
| CN110079623B (en) * | 2019-05-27 | 2022-11-11 | 深圳市人民医院 | Method for detecting toxicity of acinetobacter baumannii |
| WO2021103641A1 (en) * | 2019-11-25 | 2021-06-03 | 南开大学 | Detection method for molecular typing acinetobacter baumannii sv4 serotype o antigen |
| CN111254188A (en) * | 2020-02-06 | 2020-06-09 | 成都导胜生物技术有限公司 | Tissue fluid for direct PCR amplification, amplification system and kit |
| CN111187828A (en) * | 2020-02-11 | 2020-05-22 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of human folate metabolism gene |
| CN111187828B (en) * | 2020-02-11 | 2023-09-15 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of human folic acid metabolism gene |
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