CN109536626A - For detecting the nucleic acid reagent, kit, system and method for Gram-negative bacteria and/or Gram-negative bacteria drug resistance - Google Patents
For detecting the nucleic acid reagent, kit, system and method for Gram-negative bacteria and/or Gram-negative bacteria drug resistance Download PDFInfo
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Abstract
This disclosure relates to a kind of for detecting the nucleic acid reagent, kit, system and method for Gram-negative bacteria and/or Gram-negative bacteria drug resistance, the nucleic acid reagent includes storage or mutually probe shown in primer and SEQ ID NO.63-92 shown in the SEQ ID NO.1-60 of any mixed storage independently of one another respectively.The disclosure establishes nucleic acid reagent, kit, the system and method for common 24 kinds of drug resistant genes of 6 kinds of pathogen such as detection kerekou pneumonia primary, escherichia coli, pseudomonas aeruginosa, bacillus cloacae, serratia marcesens, Acinetobacter bauamnnii and its carrying by above-described primer and probe, it can be realized quick, comprehensive, sensitive, special, automatic testing result to determine, significantly improve to a variety of Gram-negative bacterias and its drug resistance while the sensibility detected, specificity and simplicity.
Description
Technical field
This disclosure relates to field of biotechnology, and in particular, to one kind is for detecting Gram-negative bacteria and/or gram
Nucleic acid reagent, kit, the system and method for negative bacterium drug resistance.
Background technique
Gram-negative bacteria cause infection bacterial spectrum in account for 60% ratio, wherein predominantly kerekou pneumonia primary,
Escherichia coli, pseudomonas aeruginosa, bacillus cloacae, serratia marcesens, Acinetobacter bauamnnii.With largely making for antibiotic
With Gram-negative bacteria gradually produces drug resistance.Drug resistance is also known as drug resistance, generally refers to bacterium and drug multiple-contact
Afterwards, it even disappears to the decline of the sensibility of drug, drug is caused to reduce to the curative effect of drug-fast bacteria or invalid.After drug-fast bacteria occurs,
Quickly different geographical, it is of the same race transmitted between heterologous bactericidal, make that the individual of this kind of drug or area was not used to go out
The trend that existing antibody-resistant bacterium and resistant rate presentation rise year by year.And with the increase of medicament categories, the Antibiotic Resistance of drug-fast bacteria
Increasingly wider, this has become the very big puzzlement in clinical diagnosing and treating.
Currently, Gram-negative bacteria identification method be mainly bacterium be separately cultured, biochemical reaction and serological method.Entirely
Detection obtains final result and needs 2-5 days or so, cumbersome and time consuming.Clinically common bacterial drug resistance detection method has carefully
The detection of bacterium drug-resistant phenotype, drug sensitivity test, plasmid eliminate test, plasmid-fingerprint profile technology.Although these detection method energy
It is enough that system is carried out to drug resistant gene, is comprehensively identified, but all there is the drawbacks of respective technology.
Summary of the invention
Purpose of this disclosure is to provide a kind of fast and accurately detection Gram-negative bacteria and/or Gram-negative bacteria are resistance to
Nucleic acid reagent, kit, the system and method for pharmacological property.
To achieve the goals above, it disclosure first aspect: provides a kind of blue for detecting Gram-negative bacteria and/or leather
The nucleic acid reagent of family name's negative bacterium drug resistance, wherein the nucleic acid reagent includes storage or mutually any mixing independently of one another respectively
Probe shown in primer shown in the SEQ ID NO.1-60 of storage and SEQ ID NO.63-92.
Optionally, primer shown in the SEQ ID NO.1 relative to 0.5 μM, respectively as shown in SEQ ID NO.2-60
The content of primer is respectively 0.9~1.1M, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μ
M, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μ
M, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM,
0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM,
0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM,
0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM,
0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM,
0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM,
0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM and 0.9~1.1 μM, as shown in SEQ ID NO.63-92 probe respectively
Content is each independently 0.1~0.3 μM.
Optionally, the nucleic acid reagent further includes Quality Control in the positive；
Quality Control contains probe shown in primer shown in SEQ ID NO.61-62 and SEQ ID NO.93 in the positive.
Optionally, the nucleic acid reagent includes A pipe and B pipe；A pipe contains primer shown in SEQ ID NO.1-32,61-62
With probe shown in SEQ ID NO.63-78,93；B pipe contains primer and SEQ ID shown in SEQ ID NO.33-62
Probe shown in NO.79-93.
Optionally, probe shown in SEQ ID NO.63-66,79-82 has the first fluorescent marker；SEQ ID NO.67-
Probe shown in 70,83-86 has the second fluorescent marker；Probe shown in SEQ ID NO.71-74,87-89 has third glimmering
Signal；Probe shown in SEQ ID NO.75-78,90-93 has the 4th fluorescent marker；It is first fluorescent marker, described
Second fluorescent marker, the third fluorescent marker and the 4th fluorescent marker are different, and it is glimmering to be each independently selected from FAM
One of signal, VIC fluorescent marker, CY5 fluorescent marker and ROX fluorescent marker.
Optionally, the Gram-negative bacteria includes kerekou pneumonia primary, escherichia coli, pseudomonas aeruginosa, cloaca
At least one of enterobacteria, serratia marcescens, Acinetobacter bauamnnii, the Gram-negative bacteria drug resistance includes gram
Negative bacterium aminoglycoside resistant, Gram-negative bacteria penicillin cephalosporins drug resistance, Gram-negative bacteria sulfamido
At least one of drug resistance and Gram-negative bacteria trimethoprim class drug resistance.
Disclosure second aspect: it provides a kind of for detecting Gram-negative bacteria and/or Gram-negative bacteria drug resistance
Kit, the kit contain nucleic acid reagent described in disclosure first aspect, and optionally, and the kit also contains instead
Answer at least one of system buffer, archaeal dna polymerase, DMSO, magnesium ion, BSA, dNTP and water this
The open third aspect: nucleic acid reagent described in disclosure first aspect is provided in preparation for detecting Gram-negative
Purposes in the kit of bacterium and/or Gram-negative bacteria drug resistance.
Disclosure fourth aspect: it provides a kind of for detecting Gram-negative bacteria and/or Gram-negative bacteria drug resistance
System, the system include the PCR instrument with A pipe detector and B pipe detector, computing device and output device, the A pipe detection
Device and B pipe detector are respectively the nucleic acid reagent tank for being mounted with above-mentioned nucleic acid reagent, and the PCR instrument includes the first fluorescence
Channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel, first fluorescence channel, second fluorescence are logical
Road, the third fluorescence channel and the 4th fluorescence channel are different, and it is glimmering to be each independently FAM fluorescence channel, VIC
Optical channel, CY5 fluorescence channel or ROX fluorescence channel；The computing device includes memory and processor, is deposited in the memory
Computer program is contained, the processor is configured to the computer program stored in the memory is executed, it is as follows to realize
Differentiation:
If blank control, positive control and positive internal reference are set up, testing result is effective；
If the first fluorescence channel of A pipe has Tm value be 58 DEG C of corresponding dissolution peak curves if determine to wish in sample containing large intestine angstrom
Salmonella；If the first fluorescence channel of A pipe has Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain kerekou pneumonia primary in sample
Bacterium；If the first fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain Acinetobacter bauamnnii in sample；
If the first fluorescence channel of A pipe have Tm value be 69 DEG C of corresponding dissolution peak curves if determine to contain pseudomonas aeruginosa in sample；If A
It is that 58 DEG C of corresponding dissolution peak curves then determine to contain serratia marcescens in sample that the second fluorescence channel of pipe, which has Tm value,；If A is managed
Second fluorescence channel has 62 DEG C of Tm value corresponding dissolution peak curves then to determine to contain enterobacter cloacae in sample；If A pipe second is glimmering
It is that 66 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-1 in sample that optical channel, which has Tm value,；If the second fluorescence channel of A pipe
Having Tm value is that 70 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-10 in sample；If A pipe third fluorescence channel has Tm value
Then determine to contain blaIMP-4 in sample for 58 DEG C of corresponding dissolution peak curves；If A pipe third fluorescence channel have 61 DEG C of Tm value it is right
The dissolution peak curve answered then determines to contain blaIMP-6 in sample；If it is 65 DEG C corresponding molten that A pipe third fluorescence channel, which has Tm value,
Solution peak curve then determines to contain blaIMP-8 in sample；If A pipe third fluorescence channel has 69 DEG C of Tm value corresponding dissolution peak curves
Then determine to contain blaTEM in sample；If the 4th fluorescence channel of A pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine sample
Contain blaVEB in product；If the 4th fluorescence channel of A pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain in sample
blaSHV；If the 4th fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain blaVIM in sample；If
It is that 69 DEG C of corresponding dissolution peak curves then determine to contain blaCTX in sample that the 4th fluorescence channel of A pipe, which has Tm value,；If A pipe the 4th is glimmering
It is that 69 DEG C of corresponding dissolution peak curves are then determined as in the positive that Quality Control is qualified that optical channel, which has Tm value,；
If the first fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain aadA1 in sample；If
The first fluorescence channel of B pipe has 61 DEG C of Tm value corresponding dissolution peak curves then to determine to contain aadA2 in sample；If the first fluorescence of B pipe
It is that 65 DEG C of corresponding dissolution peak curves then determine to contain aadA3 in sample that, which there is Tm value in channel,；If the first fluorescence channel of B pipe has Tm value
Then determine to contain aadA4 in sample for 69 DEG C of corresponding dissolution peak curves；If it is 65 corresponding that the second fluorescence channel of B pipe, which has Tm value,
Dissolution peak curve then determines to contain sull in sample；If the second fluorescence channel of B pipe has 69 DEG C of Tm value corresponding dissolution peak curves
Determine to contain sul3 in sample；If the second fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine in sample
Contain aadA5；If the second fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain aadA6 in sample；
If B pipe third fluorescence channel have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain sut1 in sample；If B pipe third is glimmering
It is that 61 DEG C of corresponding dissolution peak curves then determine to contain sat1 in sample that optical channel, which has Tm value,；If B pipe third fluorescence channel has Tm
Value is that 65 DEG C of corresponding dissolution peak curves then determine to contain dfrA1 in sample；If the 4th fluorescence channel of B pipe has 58 DEG C of correspondences of Tm value
Dissolution peak curve then determine to contain dfrA14 in sample；If it is 65 DEG C of corresponding dissolution peaks that the 4th fluorescence channel of B pipe, which has Tm value,
Curve then determines to contain dfrA17 in sample；If the 4th fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if sentence
Contain dfrA15 in random sample product；If the 4th fluorescence channel of B pipe have Tm value be 69 DEG C of corresponding dissolution peak curves if be determined as the positive
Interior Quality Control is qualified；
If determining sample containing at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA6 in sample
Product have Gram-negative bacteria aminoglycoside resistant；If in sample containing blaOXA-1, blaOXA-10, blaIMP-4,
At least one of blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSHV, blaVIM and blaCTX then determine sample
With Gram-negative bacteria penicillin cephalosporins drug resistance；If in sample containing in sull, sul3, sut1 and sat1 extremely
Few one kind then determines that sample has Gram-negative bacteria sulfamido drug resistance；If containing dfrA1, dfrA14, dfrA17 in sample
At least one of with dfrA15, then determine that sample has Gram-negative bacteria trimethoprim class drug resistance.
The aspect of the disclosure the 5th: it provides a kind of for detecting Gram-negative bacteria and/or Gram-negative bacteria drug resistance
Method, wherein this method comprises: carrying out PCR amplification to the DNA of sample to be tested using above-mentioned nucleic acid reagent；Carry out the PCR
The PCR instrument of amplification includes the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel；Described first
Fluorescence channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are different, and respectively solely
It is on the spot FAM fluorescence channel, VIC fluorescence channel, CY5 fluorescence channel or ROX fluorescence channel；And carry out following differentiation:
If blank control, positive control and positive internal reference are set up, testing result is effective；
If the first fluorescence channel of A pipe has Tm value be 58 DEG C of corresponding dissolution peak curves if determine to wish in sample containing large intestine angstrom
Salmonella；If the first fluorescence channel of A pipe has Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain kerekou pneumonia primary in sample
Bacterium；If the first fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain Acinetobacter bauamnnii in sample；
If the first fluorescence channel of A pipe have Tm value be 69 DEG C of corresponding dissolution peak curves if determine to contain pseudomonas aeruginosa in sample；If A
It is that 58 DEG C of corresponding dissolution peak curves then determine to contain serratia marcescens in sample that the second fluorescence channel of pipe, which has Tm value,；If A is managed
Second fluorescence channel has 62 DEG C of Tm value corresponding dissolution peak curves then to determine to contain enterobacter cloacae in sample；If A pipe second is glimmering
It is that 66 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-1 in sample that optical channel, which has Tm value,；If the second fluorescence channel of A pipe
Having Tm value is that 70 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-10 in sample；If A pipe third fluorescence channel has Tm value
Then determine to contain blaIMP-4 in sample for 58 DEG C of corresponding dissolution peak curves；If A pipe third fluorescence channel have 61 DEG C of Tm value it is right
The dissolution peak curve answered then determines to contain blaIMP-6 in sample；If it is 65 DEG C corresponding molten that A pipe third fluorescence channel, which has Tm value,
Solution peak curve then determines to contain blaIMP-8 in sample；If A pipe third fluorescence channel has 69 DEG C of Tm value corresponding dissolution peak curves
Then determine to contain blaTEM in sample；If the 4th fluorescence channel of A pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine sample
Contain blaVEB in product；If the 4th fluorescence channel of A pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain in sample
blaSHV；If the 4th fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain blaVIM in sample；If
It is that 69 DEG C of corresponding dissolution peak curves then determine to contain blaCTX in sample that the 4th fluorescence channel of A pipe, which has Tm value,；If A pipe the 4th is glimmering
It is that 69 DEG C of corresponding dissolution peak curves are then determined as in the positive that Quality Control is qualified that optical channel, which has Tm value,；
If the first fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain aadA1 in sample；If
The first fluorescence channel of B pipe has 61 DEG C of Tm value corresponding dissolution peak curves then to determine to contain aadA2 in sample；If the first fluorescence of B pipe
It is that 65 DEG C of corresponding dissolution peak curves then determine to contain aadA3 in sample that, which there is Tm value in channel,；If the first fluorescence channel of B pipe has Tm value
Then determine to contain aadA4 in sample for 69 DEG C of corresponding dissolution peak curves；If it is 65 corresponding that the second fluorescence channel of B pipe, which has Tm value,
Dissolution peak curve then determines to contain sull in sample；If the second fluorescence channel of B pipe has 69 DEG C of Tm value corresponding dissolution peak curves
Determine to contain sul3 in sample；If the second fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine in sample
Contain aadA5；If the second fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain aadA6 in sample；
If B pipe third fluorescence channel have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain sut1 in sample；If B pipe third is glimmering
It is that 61 DEG C of corresponding dissolution peak curves then determine to contain sat1 in sample that optical channel, which has Tm value,；If B pipe third fluorescence channel has Tm
Value is that 65 DEG C of corresponding dissolution peak curves then determine to contain dfrA1 in sample；If the 4th fluorescence channel of B pipe has 58 DEG C of correspondences of Tm value
Dissolution peak curve then determine to contain dfrA14 in sample；If it is 65 DEG C of corresponding dissolution peaks that the 4th fluorescence channel of B pipe, which has Tm value,
Curve then determines to contain dfrA17 in sample；If the 4th fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if sentence
Contain dfrA15 in random sample product；If the 4th fluorescence channel of B pipe have Tm value be 69 DEG C of corresponding dissolution peak curves if be determined as the positive
Interior Quality Control is qualified；
If determining sample containing at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA6 in sample
Product have Gram-negative bacteria aminoglycoside resistant；If in sample containing blaOXA-1, blaOXA-10, blaIMP-4,
At least one of blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSHV, blaVIM and blaCTX then determine sample
With Gram-negative bacteria penicillin cephalosporins drug resistance；If in sample containing in sull, sul3, sut1 and sat1 extremely
Few one kind then determines that sample has Gram-negative bacteria sulfamido drug resistance；If containing dfrA1, dfrA14, dfrA17 in sample
At least one of with dfrA15, then determine that sample has Gram-negative bacteria trimethoprim class drug resistance.
The beneficial effect of the disclosure is:
The disclosure can fast implement 6 kinds of Gram-negative bacterias and 24 kinds of Gram-negative bacteria drug resistant genes in sample to be tested
Screening and identification, avoid the troublesome operation of the methods of serology, cause of disease culture, reach following detection effect:
(1) 2 pipe target pathogen and drug resistance detect simultaneously
The detection method that the disclosure is established can pass through 2 pipe screening, 6 kinds of Gram-negative bacterias and 24 kinds of gram-negatives
30 kinds of genes of the types such as property bacterium drug resistant gene.Fast and convenient acquisition related diseases original seed class and drug resistance information is obtained, when saving
Between, manpower and reagent cost.
(2) high sensitivity
The detection while detection method that the disclosure is established can be realized 30 kinds of genes, each target base in reaction system
The detection sensitivity of cause can reach 102CFU/ml is suitable with substance real-time fluorescence PCR detection susceptibility.
(3) specificity is high
In the detection method that the disclosure is established, all primers all pass through BLAST and compare analysis, the conservative with height
And specificity, target can not only be will test and mutually distinguished, and can also, living environment identical bacterium close with other kinds
It differentiates, this includes that Shigella, sramana, proteus, pertussis, citric acid bacillus, small intestine Yersinia ruckeri, thermophilic malt are narrow
Eat monad, citric acid bacillus etc..
(4) accuracy of sample results
Because of the special designing of sampler, may be implemented to be uniform into the sample of each reactor, while every pipe
Reaction has 2 repetitions, improves the accuracy of pattern detection result.
(5) it operates quick and convenient
Clinical sample can be placed directly in detection in the reactor of ParaDNA by sampler can be obtained reliable knot
Fruit avoids costly and time-consuming sample extraction step, realizes the Emergent detection in addition to Specialty Experiment room.
(6) emergency in-situ processing
ParaDNA system of the nucleic acid reagent of the disclosure based on Hybeacon technology, it is small in size, it is convenient for carrying, can be used for
Clinic emergency scene.
Other feature and advantage of the disclosure will the following detailed description will be given in the detailed implementation section.
Specific embodiment
The specific embodiment of the disclosure is described in detail below.It should be understood that described herein specific
Embodiment is only used for describing and explaining the disclosure, is not limited to the disclosure.
Disclosure first aspect: it provides a kind of for detecting Gram-negative bacteria and/or Gram-negative bacteria drug resistance
Nucleic acid reagent, wherein the nucleic acid reagent includes storage or mutually the SEQ ID of any mixed storage independently of one another respectively
Probe shown in primer shown in NO.1-60 and SEQ ID NO.63-92.
The disclosure detects Gram-negative bacteria and/or Gram-negative bacteria by ParaDNA and Hybeacon probe technique
Drug resistance, can simultaneously precise Identification kerekou pneumonia primary, escherichia coli, pseudomonas aeruginosa, bacillus cloacae, serratia marcesens,
6 kinds of pathogen such as Acinetobacter bauamnnii can also identify common 24 kinds of drug resistant genes that 6 kinds of pathogen carry simultaneously, mainly include
6 kinds of aminoglycoside resistant genes such as identification aadA1, aadA2, aadA3, aadA4, aadA5, aadA6, blaOXA-1,
10 kinds of blaOXA-10, blaIMP-4, blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSHV, blaVIM, blaCTX etc.
The resistance to drug target gene of penicillin cephalosporins, 4 kinds of Sulfonamides-resistant genes such as sull, sul3, sut1, Sat1, dfrA1,
4 kinds of trimethoprim class drug resistant genes such as dfrA14, dfrA17, dfrA15.For monitoring bacterial resistance dynamic, clinical rational is instructed
It is of great significance using antibacterials and effectively control infection.
For Hybeacon probe technique to the more demanding of probe, the Tm value of probe is particularly important；In addition, probe and primer
Combined effect also has important influence to expanding effect.Above-mentioned primer and probe in the design process, has considered not only difference
The primer and probe of target gene is in a reaction system the problem of coamplification, i.e. assessment Tm value, the target Tm that corresponds to probe
The difference of value, avoids the occurrence of situations such as hairpin structure and dimer at G/C content, and to guarantee alternative primer and probe section point
Above-mentioned a variety of enteroviruses can not be covered comprehensively, and specificity is good and coverage is high.
Further, primer shown in the SEQ ID NO.1 relative to 0.5 μM, respectively as shown in SEQ ID NO.2-60
Primer content respectively can for 0.9~1.1M, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM,
0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM,
0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM,
0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM,
0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM,
0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM,
0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM,
0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM,
0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM and 0.9~1.1 μM, respectively by SEQ ID NO.63-92 institute
The content of the probe shown can be each independently 0.1~0.3 μM.
According to the disclosure, to do good quality control, the nucleic acid reagent can also include Quality Control in the positive.Further,
Quality Control can contain probe shown in primer shown in SEQ ID NO.61-62 and SEQ ID NO.93 in the positive.At this moment,
Primer shown in SEQ ID NO.1 relative to 0.5 μM, the content of the primer as shown in SEQ ID NO.61-62 is respectively respectively
It can be 0.9~1.1 μM and 0.4~0.6 μM, the content of the probe as shown in SEQ ID NO.93 can be 0.1~0.3 μM.
By the way that Quality Control in the positive is added, can effectively prompt because of false negative caused by the reasons such as operation error, PCR mortifier
Testing result.
According to the disclosure, in order to enhance the accuracy of testing result, the nucleic acid reagent can be divided into two pipes, i.e., the described core
Acid reagent may include A pipe and B pipe；A pipe can contain primer and SEQ ID shown in SEQ ID NO.1-32,61-62
Probe shown in NO.63-78,93；B pipe can contain primer and SEQ ID NO.79-93 institute shown in SEQ ID NO.33-62
The probe shown.
It is possible to further carry out the permutation and combination of fluorescent marker according to the respective Tm value of probe, so that in same system
The amplification of different probe identified respectively.For example, as an implementation, shown in SEQ ID NO.63-66,79-82
Probe has the first fluorescent marker；Probe shown in SEQ ID NO.67-70,83-86 has the second fluorescent marker；SEQ ID
Probe shown in NO.71-74,87-89 has third fluorescent marker；Probe shown in SEQ ID NO.75-78,90-93 has
4th fluorescent marker；First fluorescent marker, second fluorescent marker, the third fluorescent marker and the 4th fluorescence
It marks different, and is each independently selected from FAM fluorescent marker, VIC fluorescent marker, CY5 fluorescent marker and ROX fluorescent marker
One of.As a kind of particularly preferred embodiment, probe shown in SEQ ID NO.63-66,79-82 has FAM glimmering
Signal；Probe shown in SEQ ID NO.67-70,83-86 has VIC fluorescent marker；SEQ ID NO.71-74,87-89 institute
The probe shown has CY5 fluorescent marker；Probe shown in SEQ ID NO.75-78,90-93 has ROX fluorescent marker.In order to increase
Strong dissolution peak effect, above-mentioned target-probe can be dual labelled probe.FAM is 6- Fluoresceincarboxylic acid, JOE 2,7- bis- in probe
The chloro- 6- Fluoresceincarboxylic acid of methyl -4,5 two, CY5 are 5H- indoles cyanines, and ROX is 6- carboxy-X-rhodamine, and VIC is purchased from ABI public affairs
The dyestuff of department.
According to the disclosure, the Gram-negative bacteria may include kerekou pneumonia primary, escherichia coli, P. aeruginosa
At least one of bacterium, enterobacter cloacae, serratia marcescens, Acinetobacter bauamnnii.The Gram-negative bacteria drug resistance can
To include Gram-negative bacteria aminoglycoside resistant, Gram-negative bacteria penicillin cephalosporins drug resistance, gram
At least one of negative bacterium sulfamido drug resistance and Gram-negative bacteria trimethoprim class drug resistance.
Disclosure second aspect: it provides a kind of for detecting Gram-negative bacteria and/or Gram-negative bacteria drug resistance
Kit, the kit contain nucleic acid reagent described in disclosure first aspect, and optionally, and the kit also contains instead
Answer at least one of system buffer, archaeal dna polymerase, DMSO, magnesium ion, BSA, dNTP and water.
The kit of the disclosure can be realized quick, accurate, sensitive, special, automatic testing result and determine, significantly improve
Sensibility, specificity and the simplicity that Gram-negative bacteria and/or Gram-negative bacteria drug resistance are detected simultaneously.
The disclosure third aspect: nucleic acid reagent described in disclosure first aspect is provided in preparation for detecting gram-negative
Purposes in the kit of property bacterium and/or Gram-negative bacteria drug resistance.
Disclosure fourth aspect: it provides a kind of for detecting Gram-negative bacteria and/or Gram-negative bacteria drug resistance
System, the system include the PCR instrument with A pipe detector and B pipe detector, computing device and output device, the A pipe detection
Device and B pipe detector are respectively the nucleic acid reagent tank for being mounted with the above-mentioned nucleic acid reagent including A pipe and B pipe, the PCR
Instrument include the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel, first fluorescence channel,
Second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are different, and are each independently FAM
Fluorescence channel, VIC fluorescence channel, CY5 fluorescence channel or ROX fluorescence channel；The computing device includes memory and processor,
It is stored with computer program in the memory, the processor is configured to executing the computer journey stored in the memory
Sequence, to realize following differentiation:
If blank control, positive control and positive internal reference are set up, testing result is effective；
If the first fluorescence channel of A pipe has Tm value be 58 DEG C of corresponding dissolution peak curves if determine to wish in sample containing large intestine angstrom
Salmonella；If the first fluorescence channel of A pipe has Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain kerekou pneumonia primary in sample
Bacterium；If the first fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain Acinetobacter bauamnnii in sample；
If the first fluorescence channel of A pipe have Tm value be 69 DEG C of corresponding dissolution peak curves if determine to contain pseudomonas aeruginosa in sample；If A
It is that 58 DEG C of corresponding dissolution peak curves then determine to contain serratia marcescens in sample that the second fluorescence channel of pipe, which has Tm value,；If A is managed
Second fluorescence channel has 62 DEG C of Tm value corresponding dissolution peak curves then to determine to contain enterobacter cloacae in sample；If A pipe second is glimmering
It is that 66 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-1 in sample that optical channel, which has Tm value,；If the second fluorescence channel of A pipe
Having Tm value is that 70 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-10 in sample；If A pipe third fluorescence channel has Tm value
Then determine to contain blaIMP-4 in sample for 58 DEG C of corresponding dissolution peak curves；If A pipe third fluorescence channel have 61 DEG C of Tm value it is right
The dissolution peak curve answered then determines to contain blaIMP-6 in sample；If it is 65 DEG C corresponding molten that A pipe third fluorescence channel, which has Tm value,
Solution peak curve then determines to contain blaIMP-8 in sample；If A pipe third fluorescence channel has 69 DEG C of Tm value corresponding dissolution peak curves
Then determine to contain blaTEM in sample；If the 4th fluorescence channel of A pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine sample
Contain blaVEB in product；If the 4th fluorescence channel of A pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain in sample
blaSHV；If the 4th fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain blaVIM in sample；If
It is that 69 DEG C of corresponding dissolution peak curves then determine to contain blaCTX in sample that the 4th fluorescence channel of A pipe, which has Tm value,；If A pipe the 4th is glimmering
It is that 69 DEG C of corresponding dissolution peak curves are then determined as in the positive that Quality Control is qualified that optical channel, which has Tm value,；
If the first fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain aadA1 in sample；If
The first fluorescence channel of B pipe has 61 DEG C of Tm value corresponding dissolution peak curves then to determine to contain aadA2 in sample；If the first fluorescence of B pipe
It is that 65 DEG C of corresponding dissolution peak curves then determine to contain aadA3 in sample that, which there is Tm value in channel,；If the first fluorescence channel of B pipe has Tm value
Then determine to contain aadA4 in sample for 69 DEG C of corresponding dissolution peak curves；If it is 65 corresponding that the second fluorescence channel of B pipe, which has Tm value,
Dissolution peak curve then determines to contain sull in sample；If the second fluorescence channel of B pipe has 69 DEG C of Tm value corresponding dissolution peak curves
Determine to contain sul3 in sample；If the second fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine in sample
Contain aadA5；If the second fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain aadA6 in sample；
If B pipe third fluorescence channel have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain sut1 in sample；If B pipe third is glimmering
It is that 61 DEG C of corresponding dissolution peak curves then determine to contain sat1 in sample that optical channel, which has Tm value,；If B pipe third fluorescence channel has Tm
Value is that 65 DEG C of corresponding dissolution peak curves then determine to contain dfrA1 in sample；If the 4th fluorescence channel of B pipe has 58 DEG C of correspondences of Tm value
Dissolution peak curve then determine to contain dfrA14 in sample；If it is 65 DEG C of corresponding dissolution peaks that the 4th fluorescence channel of B pipe, which has Tm value,
Curve then determines to contain dfrA17 in sample；If the 4th fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if sentence
Contain dfrA15 in random sample product；If the 4th fluorescence channel of B pipe have Tm value be 69 DEG C of corresponding dissolution peak curves if be determined as the positive
Interior Quality Control is qualified；
If determining sample containing at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA6 in sample
Product have Gram-negative bacteria aminoglycoside resistant；If in sample containing blaOXA-1, blaOXA-10, blaIMP-4,
At least one of blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSHV, blaVIM and blaCTX then determine sample
With Gram-negative bacteria penicillin cephalosporins drug resistance；If in sample containing in sull, sul3, sut1 and sat1 extremely
Few one kind then determines that sample has Gram-negative bacteria sulfamido drug resistance；If containing dfrA1, dfrA14, dfrA17 in sample
At least one of with dfrA15, then determine that sample has Gram-negative bacteria trimethoprim class drug resistance.
The aspect of the disclosure the 5th: it provides a kind of for detecting Gram-negative bacteria and/or Gram-negative bacteria drug resistance
Method, wherein this method comprises: carrying out PCR expansion to the DNA of sample to be tested using the above-mentioned nucleic acid reagent including A pipe and B pipe
Increase；The PCR instrument for carrying out the PCR amplification includes the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence
Channel；First fluorescence channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are respectively not
It is identical, and it is each independently selected from FAM fluorescence channel, VIC fluorescence channel, CY5 fluorescence channel or ROX fluorescence channel；And into
The following differentiation of row:
If blank control, positive control and positive internal reference are set up, testing result is effective；
If the first fluorescence channel of A pipe has Tm value be 58 DEG C of corresponding dissolution peak curves if determine to wish in sample containing large intestine angstrom
Salmonella；If the first fluorescence channel of A pipe has Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain kerekou pneumonia primary in sample
Bacterium；If the first fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain Acinetobacter bauamnnii in sample；
If the first fluorescence channel of A pipe have Tm value be 69 DEG C of corresponding dissolution peak curves if determine to contain pseudomonas aeruginosa in sample；If A
It is that 58 DEG C of corresponding dissolution peak curves then determine to contain serratia marcescens in sample that the second fluorescence channel of pipe, which has Tm value,；If A is managed
Second fluorescence channel has 62 DEG C of Tm value corresponding dissolution peak curves then to determine to contain enterobacter cloacae in sample；If A pipe second is glimmering
It is that 66 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-1 in sample that optical channel, which has Tm value,；If the second fluorescence channel of A pipe
Having Tm value is that 70 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-10 in sample；If A pipe third fluorescence channel has Tm value
Then determine to contain blaIMP-4 in sample for 58 DEG C of corresponding dissolution peak curves；If A pipe third fluorescence channel have 61 DEG C of Tm value it is right
The dissolution peak curve answered then determines to contain blaIMP-6 in sample；If it is 65 DEG C corresponding molten that A pipe third fluorescence channel, which has Tm value,
Solution peak curve then determines to contain blaIMP-8 in sample；If A pipe third fluorescence channel has 69 DEG C of Tm value corresponding dissolution peak curves
Then determine to contain blaTEM in sample；If the 4th fluorescence channel of A pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine sample
Contain blaVEB in product；If the 4th fluorescence channel of A pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain in sample
blaSHV；If the 4th fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain blaVIM in sample；If
It is that 69 DEG C of corresponding dissolution peak curves then determine to contain blaCTX in sample that the 4th fluorescence channel of A pipe, which has Tm value,；If A pipe the 4th is glimmering
It is that 69 DEG C of corresponding dissolution peak curves are then determined as in the positive that Quality Control is qualified that optical channel, which has Tm value,；
If the first fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain aadA1 in sample；If
The first fluorescence channel of B pipe has 61 DEG C of Tm value corresponding dissolution peak curves then to determine to contain aadA2 in sample；If the first fluorescence of B pipe
It is that 65 DEG C of corresponding dissolution peak curves then determine to contain aadA3 in sample that, which there is Tm value in channel,；If the first fluorescence channel of B pipe has Tm value
Then determine to contain aadA4 in sample for 69 DEG C of corresponding dissolution peak curves；If it is 65 corresponding that the second fluorescence channel of B pipe, which has Tm value,
Dissolution peak curve then determines to contain sull in sample；If the second fluorescence channel of B pipe has 69 DEG C of Tm value corresponding dissolution peak curves
Determine to contain sul3 in sample；If the second fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine in sample
Contain aadA5；If the second fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain aadA6 in sample；
If B pipe third fluorescence channel have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain sut1 in sample；If B pipe third is glimmering
It is that 61 DEG C of corresponding dissolution peak curves then determine to contain sat1 in sample that optical channel, which has Tm value,；If B pipe third fluorescence channel has Tm
Value is that 65 DEG C of corresponding dissolution peak curves then determine to contain dfrA1 in sample；If the 4th fluorescence channel of B pipe has 58 DEG C of correspondences of Tm value
Dissolution peak curve then determine to contain dfrA14 in sample；If it is 65 DEG C of corresponding dissolution peaks that the 4th fluorescence channel of B pipe, which has Tm value,
Curve then determines to contain dfrA17 in sample；If the 4th fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if sentence
Contain dfrA15 in random sample product；If the 4th fluorescence channel of B pipe have Tm value be 69 DEG C of corresponding dissolution peak curves if be determined as the positive
Interior Quality Control is qualified；
If determining sample containing at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA6 in sample
Product have Gram-negative bacteria aminoglycoside resistant；If in sample containing blaOXA-1, blaOXA-10, blaIMP-4,
At least one of blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSHV, blaVIM and blaCTX then determine sample
With Gram-negative bacteria penicillin cephalosporins drug resistance；If in sample containing in sull, sul3, sut1 and sat1 extremely
Few one kind then determines that sample has Gram-negative bacteria sulfamido drug resistance；If containing dfrA1, dfrA14, dfrA17 in sample
At least one of with dfrA15, then determine that sample has Gram-negative bacteria trimethoprim class drug resistance.
Wherein, the sample to be tested can be patient's anus swab sample, and the condition of the PCR amplification can be with are as follows: 98.0 DEG C,
1min (98.0 DEG C, 5s；58.0 DEG C, 5s；72.0 DEG C, 5s；49 circulations) 98.0 DEG C, 1min；35.0 DEG C, 1min；Melting curve
35.0 to 80.0 DEG C, liter rate is 0.5 DEG C/s.
Disclosed method quickly sensitive can specifically realize kerekou pneumonia primary, escherichia coli, pseudomonas aeruginosa, yin
The common drug resistant gene that 6 kinds of pathogen such as ditch bacillus, serratia marcesens, Acinetobacter bauamnnii and 6 kinds of pathogen carry is
It unites screening, testing process is simple, as a result automatic interpretation and reliable, saves time, manpower and reagent cost.
The disclosure is further elaborated by the following examples, but the disclosure is not therefore by any limit
System.
Reagent is commercial products in following embodiment, and primer, probe are synthesized in Biosearch (USA) company.
Embodiment
1, primer, probe synthesis
According to probe sequence shown in primer sequence shown in table 1 and table 2, sequent synthesis is carried out.FAM is 6- carboxylic in probe
Base fluorescein, JOE 2, the chloro- 6- Fluoresceincarboxylic acid of 7- dimethyl -4,5 two, CY5 are 5H- indoles cyanines, and ROX is 6- carboxyl-X- sieve
Red bright, VIC is the dyestuff purchased from ABI company.Bracket in the probe sequence of table 2 indicates that the base on the left of bracket has fluorescence mark
Remember, the selection of the content representation fluorescent marker in bracket.
Table 1
Table 2
2, sample process
After conventional method acquires patient's anus swab sample, handled with the extracts kit of commercialization.
Using samplers sample matched with ParaDNA treated anus swab sample, it is placed directly within the reaction of ParaDNA
It is i.e. amplifiable in device.
3, Hybeacon probe technique detection architecture is constructed
Polymerase Phire Hot Start II DNA Polymerase (article No. F122L), Mg2+, dNTPS is purchased from
ThermoFisher company, other biochemical reagents are that import packing or domestic analysis are pure；Fluorescence detector is ParaDNA.
Reaction system: 15 μ L of total system, 13 μ L of buffer, the polymerization including 0.5-0.75U/ μ L is prepared according to following operation
Enzyme, the dNTP of 1mM, 10-105Copy/μ L DNA profiling, the final of upstream primer use concentration for 500nM, and downstream primer is most
It the use of concentration is eventually 1 μM, every the final of probe is respectively 200nM using concentration.
Kit is divided into A pipe and two reaction tubes of B pipe, and A pipe is containing shown in SEQ ID NO.1-32,61-62 in upper table 1
SEQ ID NO.63-78 in primer and upper table 2, probe shown in 93, B pipe is containing shown in SEQ ID NO.33-62 in upper table 1
Probe shown in SEQ ID NO.79-93 in primer and upper table 2.
PCR pipe is put into fluorescence quantitative PCR instrument, selects FAM, VIC, CY5, ROX as reporter group, response procedures are such as
Under: 98 DEG C, 60s, (98 DEG C, 5s, 58 DEG C, 5s, 72 DEG C, 5s, 49 circulations)；Solubility curve analysis: 98 DEG C, 60s, 35 DEG C,
60s, drop rate are 1.0 DEG C/s；80 DEG C, 5s, liter rate is 0.5 DEG C/s, which collects fluorescence.
If blank control, positive control and positive internal reference are set up, testing result is effective；
If A pipe FAM fluorescence channel has Tm value be 58 DEG C of corresponding dissolution peak curves if determine to wish in sample containing large intestine angstrom
Salmonella；If A pipe FAM fluorescence channel has Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain kerekou pneumonia primary in sample
Bacterium；If A pipe FAM fluorescence channel have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain Acinetobacter bauamnnii in sample；
If A pipe FAM fluorescence channel have Tm value be 69 DEG C of corresponding dissolution peak curves if determine to contain pseudomonas aeruginosa in sample；If A
It is that 58 DEG C of corresponding dissolution peak curves then determine to contain serratia marcescens in sample that pipe VIC fluorescence channel, which has Tm value,；If A is managed
VIC fluorescence channel has 62 DEG C of Tm value corresponding dissolution peak curves then to determine to contain enterobacter cloacae in sample；If A pipe VIC fluorescence
It is that 66 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-1 in sample that, which there is Tm value in channel,；If A pipe VIC fluorescence channel has Tm
Value is that 70 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-10 in sample；If it is 58 that A pipe CY5 fluorescence channel, which has Tm value,
DEG C corresponding dissolution peak curve then determines to contain blaIMP-4 in sample；If A pipe CY5 fluorescence channel have 61 DEG C of Tm value it is corresponding molten
Solution peak curve then determines to contain blaIMP-6 in sample；If it is 65 DEG C of corresponding dissolution peak curves that A pipe CY5 fluorescence channel, which has Tm value,
Then determine to contain blaIMP-8 in sample；Sample is determined if A pipe CY5 fluorescence channel there are 69 DEG C of Tm value corresponding dissolution peak curves
In contain blaTEM；If A pipe ROX fluorescence channel have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain in sample
blaVEB；If A pipe ROX fluorescence channel have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain blaSHV in sample；If A
It is that 65 DEG C of corresponding dissolution peak curves then determine to contain blaVIM in sample that pipe ROX fluorescence channel, which has Tm value,；If A pipe ROX fluorescence
It is that 69 DEG C of corresponding dissolution peak curves then determine to contain blaCTX in sample that, which there is Tm value in channel,；If A pipe ROX fluorescence channel has Tm value
Then it is determined as in the positive that Quality Control is qualified for 69 DEG C of corresponding dissolution peak curves；
If B pipe FAM fluorescence channel have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain aadA1 in sample；If B
Pipe FAM fluorescence channel has 61 DEG C of Tm value corresponding dissolution peak curves then to determine to contain aadA2 in sample；If B pipe FAM fluorescence channel
Having Tm value is that 65 DEG C of corresponding dissolution peak curves then determine to contain aadA3 in sample；If it is 69 that B pipe FAM fluorescence channel, which has Tm value,
DEG C corresponding dissolution peak curve then determines to contain aadA4 in sample；If it is 65 corresponding dissolutions that B pipe VIC fluorescence channel, which has Tm value,
Peak curve then determines to contain sull in sample；Sample is determined if B pipe VIC fluorescence channel there are 69 DEG C of Tm value corresponding dissolution peak curves
Contain sul3 in product；If B pipe VIC fluorescence channel have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain in sample
aadA5；If B pipe VIC fluorescence channel have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain aadA6 in sample；If B is managed
It is that 58 DEG C of corresponding dissolution peak curves then determine to contain sut1 in sample that CY5 fluorescence channel, which has Tm value,；If B pipe CY5 fluorescence channel
Having Tm value is that 61 DEG C of corresponding dissolution peak curves then determine to contain sat1 in sample；If it is 65 DEG C that B pipe CY5 fluorescence channel, which has Tm value,
Corresponding dissolution peak curve then determines to contain dfrA1 in sample；If B pipe ROX fluorescence channel has 58 DEG C of Tm value corresponding dissolution peaks
Curve then determines to contain dfrA14 in sample；If B pipe ROX fluorescence channel have Tm value be 65 DEG C of corresponding dissolution peak curves if determine
Contain dfrA17 in sample；If B pipe ROX fluorescence channel have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain in sample
dfrA15；If B pipe ROX fluorescence channel has Tm value be 69 DEG C of corresponding dissolution peak curves if be determined as Quality Control qualification in the positive；
If determining sample containing at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA6 in sample
Product have Gram-negative bacteria aminoglycoside resistant；If in sample containing blaOXA-1, blaOXA-10, blaIMP-4,
At least one of blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSHV, blaVIM and blaCTX then determine sample
With Gram-negative bacteria penicillin cephalosporins drug resistance；If in sample containing in sull, sul3, sut1 and sat1 extremely
Few one kind then determines that sample has Gram-negative bacteria sulfamido drug resistance；If containing dfrA1, dfrA14, dfrA17 in sample
At least one of with dfrA15, then determine that sample has Gram-negative bacteria trimethoprim class drug resistance.
4, specificity verification
Select Shigella, sramana, proteus, pertussis, citric acid bacillus, small intestine Yersinia ruckeri, thermophilic malt narrow
Monad, citric acid bacillus (Chinese Center for Disease Control and Prevention bacterial disease prevention and control are provided) are eaten as specificity assessment sample
This, after the samplers sample anus swab sample in system detection, the reaction condition established and optimized using early period is existed
It is detected on ParaDNA.
As the result is shown under conditions of positive control is set up, dissolution peak of the target to be checked without specificity shows the disclosure
Nucleic acid reagent can effective district sorting survey target and non-detection target, there is preferable specificity.
5, minimum detectability is verified
Assessment is with detecting sample: selecting the escherichia coli containing drug resistant gene to represent strain, by the thin of 5 strains
Bacteria suspension is separately adjusted to angularly 108CFU/mL extracts bacterial genomes DNA respectively.By template respectively gradient dilution at being equivalent to
107CFU/mL, 106CFU/mL, 105CFU/mL, 104CFU/mL, 103CFU/mL, 102The detection sample of CFU/mL, 10CFU/mL.
The minimum detectability of disclosure kit detection target bacteria and drug resistant gene reaches 10 as the result is shown2CFU/
ML, the minimum detectability of kit totality are the target molecule of each reaction detection 1 copy.
6, coverage is verified
Anus swab sample is selected to use template as coverage assessment.Reaction system and response procedures carry out as described above
Test.
All sample standard deviations can be covered as the result is shown and detected.
7, the storage life test of kit
Strong positive 10 is taken respectively5CFU/mL and weakly positive 103The mould of the Klebsiella Pneumoniae containing drug resistant gene of CFU/mL
Plate is that assessment was distributed into 9 parts and frozen in -70 DEG C of refrigerators with detection sample at the 0th day.The kit finished will be set up to put
- 20 DEG C of preservations are placed in, 0,10,15,30,60,90,120,150,180 and 360 day kit is taken to carry out storage life examination respectively
It tests.
Disclosure kit is stored in -20 DEG C of refrigerators as the result is shown, is the positive in the detection of different storage lives, shows the examination
The storage life of agent box is at least 1 year.
Comparative example
1, primer, probe synthesis
According to primer, probe sequence shown in table 3 and table 4, sequent synthesis is carried out.
Table 3
Table 4
2, specificity verification carries out specificity verification according to the method for embodiment.The results show that the primer of comparative example, probe
Reaction result be feminine gender.
3, minimum detectability verifying carries out minimum detectability verifying according to the method for embodiment.Embodiment and comparative example are most
Low detection limits comparison such as the following table 5.
Table 5
As shown in Table 5, for the kerekou pneumonia primary of trace in sample, escherichia coli, pseudomonas aeruginosa, cloaca bar
Bacterium, serratia marcesens, Acinetobacter bauamnnii and its carrying drug resistant gene nucleic acid, disclosure kit has stronger than comparative example
Detectability.
4, coverage is verified
Coverage verifying is carried out according to the method for embodiment.The coverage of embodiment and comparative example comparison such as the following table 6.
Table 6
| Detect target | Embodiment | Comparative example |
| Escherichia coli | 120 plants of total positives | 118 plants of positives |
| Klebsiella Pneumoniae | 80 plants of total positives | 77 plants of total positives |
| Acinetobacter bauamnnii | 100 plants of total positives | 90 plants of total positives |
| Pseudomonas aeruginosa | 90 plants of total positives | 86 plants of total positives |
| Serratia marcescens | 80 plants of total positives | 74 plants of total positives |
| Enterobacter cloacae | 120 plants of total positives | 109 plants of positives |
| aadA1 | 89 plants of total positives | 72 plants of total positives |
| aadA2 | 120 plants of total positives | 118 plants of positives |
| aadA3 | 82 plants of total positives | 77 plants of total positives |
| aadA4 | 87 plants of total positives | 78 plants of total positives |
| aadA5 | 90 plants of total positives | 86 plants of total positives |
| aadA6 | 80 plants of total positives | 74 plants of total positives |
| blaOXA-1 | 113 plants of total positives | 109 plants of positives |
| blaOXA-10 | 76 plants of total positives | 72 plants of total positives |
| blaIMP-4 | 120 plants of total positives | 118 plants of positives |
| blaIMP-6 | 80 plants of total positives | 80 plants of total positives |
| blaIMP-8 | 111 plants of total positives | 108 plants of total positives |
| blaTEM | 90 plants of total positives | 86 plants of total positives |
| blaVEB | 82 plants of total positives | 80 plants of total positives |
| blaSHV | 120 plants of total positives | 109 plants of positives |
| blaVIM | 89 plants of total positives | 76 plants of total positives |
| blaCTX | 120 plants of total positives | 119 plants of positives |
| sull | 80 plants of total positives | 77 plants of total positives |
| sul3 | 100 plants of total positives | 90 plants of total positives |
| sut1 | 90 plants of total positives | 86 plants of total positives |
| Sat1 | 80 plants of total positives | 75 plants of total positives |
| dfrA1 | 120 plants of total positives | 118 plants of positives |
| dfrA14 | 82 plants of total positives | 73 plants of total positives |
| dfrA15 | 119 plants of total positives | 118 plants of positives |
| dfrA17 | 40 plants of total positives | 38 plants of total positives |
As shown in Table 6, the detection coverage of disclosure kit is far longer than the detection coverage of comparative example.
The disclosure can disposably detect kerekou pneumonia primary, large intestine it can be seen from the comparison of embodiment and comparative example
6 kinds of pathogen such as angstrom uncommon bacterium, pseudomonas aeruginosa, bacillus cloacae, serratia marcesens, Acinetobacter bauamnnii 6, can also identify simultaneously
The multidrug resistant gene that above-mentioned 6 kinds of pathogen carry, specificity is high, and minimum detectability is lower, and coverage is wider.
The preferred embodiment of the disclosure is described in detail above, still, during the disclosure is not limited to the above embodiment
Detail a variety of simple variants can be carried out to the technical solution of the disclosure in the range of the technology design of the disclosure, this
A little simple variants belong to the protection scope of the disclosure.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, for example, A pipe detection target and B pipe detection target can be with
It exchanges.In order to avoid unnecessary repetition, no further explanation will be given to various combinations of possible ways for the disclosure.
In addition, any combination can also be carried out between a variety of different embodiments of the disclosure, as long as it is without prejudice to originally
Disclosed thought equally should be considered as disclosure disclosure of that.
Sequence table
<110>biotech inc Beijing Zhuo Cheng Hui Sheng
<120>for detect the nucleic acid reagent of Gram-negative bacteria and/or Gram-negative bacteria drug resistance, kit, system and
Method
<130> 12326ABT
<160> 186
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tttcaccgaa gttcatgcc 19
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggaatttcgc cgattttgc 19
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aggccaacaa gaagtacaac c 21
<210> 4
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tcaacccaac gatcctg 17
<210> 5
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ccctagaccc tcaatatgca c 21
<210> 6
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
accgagtcca caacgat 17
<210> 7
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
caacctgaag gacgatt 17
<210> 8
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ccttcttggc cttgtcgag 19
<210> 9
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
atgcaggccg cgtttc 16
<210> 10
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
acagattcac gtccggctc 19
<210> 11
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tgcgtcagat cgtgtcca 18
<210> 12
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cttcagttgc cgcgttgt 18
<210> 13
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gcagcaaaga tgaaatc 17
<210> 14
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
aggtggttgt aaataatgtt 20
<210> 15
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
aataaaagat gaaaaatgat gaa 23
<210> 16
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
cttctttact cgcctttatc gg 22
<210> 17
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
atgttatcag tttcaata 18
<210> 18
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
ggaaaccatc ttcgtatttt agatggg 27
<210> 19
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
tcgggcaatg tagacagt 18
<210> 20
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
caccattggc ttcggtca 18
<210> 21
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
tgacactcca tttacggcta 20
<210> 22
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
tagttaattc agacgcatac gtg 23
<210> 23
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
gacactccat ttacggcta 19
<210> 24
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
gatagatcga gaattaagcc act 23
<210> 25
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
cgcctatctg attgacactc c 21
<210> 26
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
ttgttaattc agatgcatac gtg 23
<210> 27
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ggacagttgg gtgcac 16
<210> 28
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
gcagcaaaga tgaaatc 17
<210> 29
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
actatcgcca gcaggatct 19
<210> 30
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
agttcgccga ccgtca 16
<210> 31
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
ccgactttac cagattgcc 19
<210> 32
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
tcacggacaa tgagacca 18
<210> 33
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
agtatcgact caactatcag a 21
<210> 34
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
tgtggcttca ggccgccatc c 21
<210> 35
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
agcaacgatg ttacgcagca g 21
<210> 36
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
cattgccaat aacccgattg g 21
<210> 37
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
gcaagtagcg tatgcgctca c 21
<210> 38
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
gcccgaggca tagactgtac a 21
<210> 39
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
gtgggtcgat gtttgatgtt a 21
<210> 40
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
atattttcaa tttaactccc 20
<210> 41
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
gatgttatgg agcagcaacg at 22
<210> 42
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
caaatcgcaa atatgcagta 20
<210> 43
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
gcaacgatgt tacgcagcag g 21
<210> 44
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
agctagaatt ttgtgtatca a 21
<210> 45
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
attttatctc tncggtgttn aatat 25
<210> 46
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
gaatggatgt agacactcga g 21
<210> 47
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
ttcaaggcat ctgataaaga c 21
<210> 48
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
agcaccaact cttgcagcag a 21
<210> 49
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
gatcgacaac ttaaacgcga t 21
<210> 50
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
tatctttagg gttcagtttt g 21
<210> 51
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
gtgacccgac gcctgcgcag c 21
<210> 52
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
cgtacgccgc ccaggccgac a 21
<210> 53
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
ggagcagcaa cgatgttacg c 21
<210> 54
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
atatagctac cattagtgat a 21
<210> 55
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
atgagaacct tgaaagtatc a 21
<210> 56
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
tgctcccctt tcgcggacca g 21
<210> 57
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
gttacgcagc agggcagtcg c 21
<210> 58
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
atatctgggc catttccgat a 21
<210> 59
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
ggagcagcaa cgatgttacg c 21
<210> 60
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
gacactgcag aaatcaatga t 21
<210> 61
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
gattcatggc tcagaacgaa c 21
<210> 62
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
cgctttactc atcccgttg 19
<210> 63
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
ccctttctgt tacgccaa 18
<210> 64
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
agaagacaac cgcattatta cccgctc 27
<210> 65
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
cttgccgttc aggcgaattg at 22
<210> 66
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
ctgcgctgct gcgtcgctt 19
<210> 67
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
ctgctgtcta cgcccggtg 19
<210> 68
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
atgtacctcg cccgcttcac gtt 23
<210> 69
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
aatatccttc aaagacgtat tcaaa 25
<210> 70
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
ctggcgcgct ccgaacgtgt aactta 26
<210> 71
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
ccacctatcc gcccgtgct 19
<210> 72
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
ccgcccggct gcgctat 17
<210> 73
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
ccgcgcccac aaacaattga ct 22
<210> 74
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
gggtacacga actggat 17
<210> 75
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
tctggggaca ctcgccggtc a 21
<210> 76
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
ttgtgaacag caaacgccgg gttatt 26
<210> 77
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
tagccgcgcc atcaacgac 19
<210> 78
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
gagagcagtc aaattctt 18
<210> 79
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 79
cgccactcga acgacgttg 19
<210> 80
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 80
gccaatgaac cggaatcag 19
<210> 81
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 81
cctgaccgaa gcagcggtg 19
<210> 82
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 82
acgagcaggg agtcgccct 19
<210> 83
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 83
gtcagaaaag gcgaatcggt agtgg 25
<210> 84
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 84
aaagtaggcc gcaggacac 19
<210> 85
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 85
ggtgacattt taatttacct gggt 24
<210> 86
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 86
gcggagaaat cgcaccttt 19
<210> 87
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 87
gcatatttaa caattgaaa 19
<210> 88
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 88
cgtacgatcg cctctcgtg 19
<210> 89
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 89
caaaacaaag taacctctg 19
<210> 90
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 90
tgcgaagcga aaaaggcgt 19
<210> 91
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 91
accccaagga atatcgtga 19
<210> 92
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 92
gtcgcctaaa acaaagttag 20
<210> 93
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 93
ctttccgcrc gcttgcat 18
<210> 94
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 94
cgcaactggc tgacgat 17
<210> 95
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 95
tcgggacgga ttatagttat g 21
<210> 96
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 96
gtcccggcgc cggcggagcc 20
<210> 97
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 97
tcacctgctg cacgcggtgg gc 22
<210> 98
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 98
aatgacattg caagcaattg ct 22
<210> 99
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 99
tgcttgtccg ttcaggcgca att 23
<210> 100
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 100
gaggaaggca tccgcgaagt gatgagc 27
<210> 101
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 101
acgacgaagg cgacggcggt ccgg 24
<210> 102
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 102
ctttaaaagc ctgctttcgc agg 23
<210> 103
<211> 14
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 103
ctggcgtttc agca 14
<210> 104
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 104
gttgctggtg gcggcgttgc 20
<210> 105
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 105
tcggtattct ggacccaact aaagt 25
<210> 106
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 106
gagcgccatc tcgaaccgac g 21
<210> 107
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 107
ggtcaccgta accagcaaat c 21
<210> 108
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 108
ttagccatat gaactcggaa t 21
<210> 109
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 109
aatcttctgc tcacccggaa t 21
<210> 110
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 110
ccagaacctt gaccgaacgc a 21
<210> 111
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 111
gcgtaacgcg cttgctgctt g 21
<210> 112
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 112
tgatgttatg gagcagcaac g 21
<210> 113
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 113
ccactaccga ttacgccatt t 21
<210> 114
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 114
taagggagtt aaattgaaaa t 21
<210> 115
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 115
gagtagttgc tcacctttta c 21
<210> 116
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 116
aacgatgtta cgcagcaggg c 21
<210> 117
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 117
ggcagatttc gctcatctgc c 21
<210> 118
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 118
ttgccacgtg tcaaacgaga a 21
<210> 119
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 119
gcaatgagct catccacaga c 21
<210> 120
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 120
gaaaacaaaa gttggaatgc t 21
<210> 121
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 121
gctattggga attttaaagg t 21
<210> 122
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 122
agcattgcta ccgcagcaga g 21
<210> 123
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 123
caaccaaacc atgtttagga a 21
<210> 124
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 124
tttccatagc gacagcacag g 21
<210> 125
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 125
atgcatacgt gggaatagat t 21
<210> 126
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 126
ggagcggctt tgcctgattt a 21
<210> 127
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 127
gagtgtcaat cagataggcg t 21
<210> 128
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 128
tacatcgaac tggatctcaa c 21
<210> 129
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 129
accgagttgc tcttgcccgg c 21
<210> 130
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 130
tgctcagagg agcatgacgt a 21
<210> 131
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 131
attttccata caccagattt c 21
<210> 132
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 132
gaattgtgaa tcagcaaaac g 21
<210> 133
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 133
cagcggcagg gtggctaaca g 21
<210> 134
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 134
cgcatatcgc aacgcagtcg 20
<210> 135
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 135
ccgcgagaag tgccgctgtg t 21
<210> 136
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 136
aatcagcgcg ttgaaattaa g 21
<210> 137
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 137
ccgcactaag ctcagccagc g 21
<210> 138
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 138
tgtttggtct gtgtttatta c 21
<210> 139
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 139
gggtcccctc ccccacttgg g 21
<210> 140
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 140
acttaacatc atgggtgcgg a 21
<210> 141
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 141
agtgtcacgg aaatcattct t 21
<210> 142
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 142
tttcaattga ttaaataatg c 21
<210> 143
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 143
aacagaaggt aggtggcagg g 21
<210> 144
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 144
gtgcctgctc gtgccgttcg g 21
<210> 145
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 145
cggtgtccgc gatggcgtcg c 21
<210> 146
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 146
tggtagctat atcgaagaat g 21
<210> 147
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 147
cttgcgtcca accaacagcc a 21
<210> 148
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 148
cataccctgg tccgcgaaag g 21
<210> 149
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 149
tggggagtgc gcccatagat t 21
<210> 150
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 150
aagtatcgtg aaactatcac taat 24
<210> 151
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 151
ccaaagggga acaattactc ttca 24
<210> 152
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 152
attgaaaata tcattgattt c 21
<210> 153
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 153
cttttactga ccacgggata t 21
<210> 154
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 154
acgaacgctg gcggcgtgcc taacac 26
<210> 155
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 155
ggaaccaaag ggggcgagcg ta 22
<210> 156
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 156
atgggtacag ggaggat 17
<210> 157
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 157
ggcccgcgcg gcggcgcgtc ag 22
<210> 158
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 158
acgcaaactt ggtgtagata ttgataa 27
<210> 159
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 159
cggcagcctg cccgccgaag aggtg 25
<210> 160
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 160
cactgtaagg cgacaacgac gacaacgccc c 31
<210> 161
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 161
gcgcgcaatg gaagcgcctc t 21
<210> 162
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 162
tccgcagtgg atggcggcct g 21
<210> 163
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 163
tctcgttgct gcgatgggag c 21
<210> 164
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 164
acggcgcagt ggcggttttc a 21
<210> 165
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 165
cgcagcaggg cagtcgccct a 21
<210> 166
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 166
cagaaaatgg cgtaatcggt a 21
<210> 167
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 167
gacacaacgc aggtcacatt g 21
<210> 168
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 168
ctcttcaaat gacgtattca a 21
<210> 169
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 169
gcgtagttgt gctctggaat g 21
<210> 170
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 170
tgatgaaggc gtttatgttc a 21
<210> 171
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 171
tggtttgtgg agcgcggcta t 21
<210> 172
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 172
gtgtttatgt tcatacatcg t 21
<210> 173
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 173
tcgccccgaa gaacgttttc c 21
<210> 174
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 174
gatagccgct aacattaata a 21
<210> 175
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 175
cgggttattc ttatttgtcg c 21
<210> 176
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 176
ccgtccaatg gtctcattgt c 21
<210> 177
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 177
acagagcgag agcgataagc a 21
<210> 178
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 178
tactgtcctt gcctgttcta g 21
<210> 179
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 179
atacatttct gctgcaagag t 21
<210> 180
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 180
tcccgatctc aaaactgaac c 21
<210> 181
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 181
ggcctgggcg gcgtacgggc t 21
<210> 182
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 182
ccctgatatt ccatggagtg c 21
<210> 183
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 183
attgacctac aatcagtggc t 21
<210> 184
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 184
gagttatcgg aaatggccca gatattccat 30
<210> 185
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 185
agaaaatggc gtaatcggta g 21
<210> 186
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 186
cgtgggtaat ctaccctacg gatcggg 27
Claims (10)
1. a kind of for detecting the nucleic acid reagent of Gram-negative bacteria and/or Gram-negative bacteria drug resistance, wherein the nucleic acid
Reagent includes storage or mutually primer and SEQ ID shown in the SEQ ID NO.1-60 of any mixed storage independently of one another respectively
Probe shown in NO.63-92.
2. nucleic acid reagent according to claim 1, wherein primer shown in the SEQ ID NO.1 relative to 0.5 μM, point
The content of the not primer as shown in SEQ ID NO.2-60 be respectively 0.9~1.1M, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~
0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~
1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~
0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.3~0.6 μM, 0.9~
1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~1.1 μM, 0.3~
0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~
1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.3~
0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM, 0.9~1.1 μM, 0.4~0.8 μM, 0.9~
1.1 μM, 0.3~0.6 μM, 0.9~1.1 μM, 0.3~0.5 μM, 0.9~1.1 μM, 0.5~1 μM and 0.9~1.1 μM, respectively by
The content of probe shown in SEQ ID NO.63-92 is each independently 0.1~0.3 μM.
3. nucleic acid reagent according to claim 1, wherein the nucleic acid reagent further includes Quality Control in the positive；
Quality Control contains probe shown in primer shown in SEQ ID NO.61-62 and SEQ ID NO.93 in the positive.
4. nucleic acid reagent according to claim 3, wherein the nucleic acid reagent includes A pipe and B pipe；A pipe contains SEQ ID
Probe shown in primer shown in NO.1-32,61-62 and SEQ ID NO.63-78,93；B pipe contains SEQ ID NO.33-62
Shown in probe shown in primer and SEQ ID NO.79-93.
5. nucleic acid reagent according to claim 4, wherein probe shown in SEQ ID NO.63-66,79-82 has the
One fluorescent marker；Probe shown in SEQ ID NO.67-70,83-86 has the second fluorescent marker；SEQ ID NO.71-74,
Probe shown in 87-89 has third fluorescent marker；Probe shown in SEQ ID NO.75-78,90-93 has the 4th fluorescence mark
Note；The each not phase of first fluorescent marker, second fluorescent marker, the third fluorescent marker and the 4th fluorescent marker
One of together, and be each independently selected from FAM fluorescent marker, VIC fluorescent marker, CY5 fluorescent marker and ROX fluorescent marker.
6. nucleic acid reagent described according to claim 1~any one of 5, wherein the Gram-negative bacteria includes pneumonia
In Cray primary, escherichia coli, pseudomonas aeruginosa, enterobacter cloacae, serratia marcescens and Acinetobacter bauamnnii extremely
Few one kind, the Gram-negative bacteria drug resistance include Gram-negative bacteria aminoglycoside resistant, Gram-negative bacteria blueness
In mycin cephalosporins drug resistance, Gram-negative bacteria sulfamido drug resistance and Gram-negative bacteria trimethoprim class drug resistance
At least one.
7. a kind of for detecting the kit of Gram-negative bacteria and/or Gram-negative bacteria drug resistance, which, which contains, has the right
Benefit require any one of 1~6 described in nucleic acid reagent, and optionally, the kit also contain reaction system buffer,
At least one of archaeal dna polymerase, DMSO, magnesium ion, BSA, dNTP and water.
8. nucleic acid reagent described in any one of claim 1~6 is blue for detecting Gram-negative bacteria and/or leather in preparation
Purposes in the kit of family name's negative bacterium drug resistance.
9. a kind of system for detecting Gram-negative bacteria and/or Gram-negative bacteria drug resistance, which includes having A pipe
The PCR instrument of detector and B pipe detector, computing device and output device, the A pipe detector and B pipe detector are respectively to fill
It is loaded with the nucleic acid reagent tank of nucleic acid reagent described in any one of claim 4~6, the PCR instrument includes first
Fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel, it is first fluorescence channel, described second glimmering
Optical channel, the third fluorescence channel and the 4th fluorescence channel are different, and be each independently FAM fluorescence channel,
VIC fluorescence channel, CY5 fluorescence channel or ROX fluorescence channel；The computing device includes memory and processor, the storage
Computer program is stored in device, the processor is configured to the computer program stored in the memory is executed, with reality
Now following differentiation:
If blank control, positive control and positive internal reference are set up, testing result is effective；
If the first fluorescence channel of A pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain escherichia coli in sample
Bacterium；If the first fluorescence channel of A pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain Klebsiella Pneumoniae in sample；
If the first fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain Acinetobacter bauamnnii in sample；If A
It is that 69 DEG C of corresponding dissolution peak curves then determine to contain pseudomonas aeruginosa in sample that the first fluorescence channel of pipe, which has Tm value,；If A is managed
It is that 58 DEG C of corresponding dissolution peak curves then determine to contain serratia marcescens in sample that second fluorescence channel, which has Tm value,；If A pipe
Two fluorescence channels have 62 DEG C of Tm value corresponding dissolution peak curves then to determine to contain enterobacter cloacae in sample；If the second fluorescence of A pipe
It is that 66 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-1 in sample that, which there is Tm value in channel,；If the second fluorescence channel of A pipe has
Tm value is that 70 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-10 in sample；If A pipe third fluorescence channel has the Tm value to be
58 DEG C of corresponding dissolution peak curves then determine to contain blaIMP-4 in sample；If A pipe third fluorescence channel has 61 DEG C of correspondences of Tm value
Dissolution peak curve then determine to contain blaIMP-6 in sample；If it is 65 DEG C of corresponding dissolutions that A pipe third fluorescence channel, which has Tm value,
Peak curve then determines to contain blaIMP-8 in sample；If A pipe third fluorescence channel has 69 DEG C of Tm value corresponding dissolution peak curves
Determine to contain blaTEM in sample；If the 4th fluorescence channel of A pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine sample
In contain blaVEB；If the 4th fluorescence channel of A pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain in sample
blaSHV；If the 4th fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain blaVIM in sample；If
It is that 69 DEG C of corresponding dissolution peak curves then determine to contain blaCTX in sample that the 4th fluorescence channel of A pipe, which has Tm value,；If A pipe the 4th is glimmering
It is that 69 DEG C of corresponding dissolution peak curves are then determined as in the positive that Quality Control is qualified that optical channel, which has Tm value,；
If the first fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain aadA1 in sample；If B is managed
First fluorescence channel has 61 DEG C of Tm value corresponding dissolution peak curves then to determine to contain aadA2 in sample；If the first fluorescence channel of B pipe
Having Tm value is that 65 DEG C of corresponding dissolution peak curves then determine to contain aadA3 in sample；If it is 69 that the first fluorescence channel of B pipe, which has Tm value,
DEG C corresponding dissolution peak curve then determines to contain aadA4 in sample；If it is 65 corresponding dissolutions that the second fluorescence channel of B pipe, which has Tm value,
Peak curve then determines to contain sull in sample；Determine if the second fluorescence channel of B pipe there are 69 DEG C of Tm value corresponding dissolution peak curves
Contain sul3 in sample；If the second fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain in sample
aadA5；If the second fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain aadA6 in sample；If B
It is that 58 DEG C of corresponding dissolution peak curves then determine to contain sut1 in sample that pipe third fluorescence channel, which has Tm value,；If B pipe third fluorescence
It is that 61 DEG C of corresponding dissolution peak curves then determine to contain sat1 in sample that, which there is Tm value in channel,；If B pipe third fluorescence channel has Tm value
Then determine to contain dfrA1 in sample for 65 DEG C of corresponding dissolution peak curves；If the 4th fluorescence channel of B pipe have 58 DEG C of Tm value it is corresponding
Dissolution peak curve then determines to contain dfrA14 in sample；If it is that 65 DEG C of corresponding dissolution peaks are bent that the 4th fluorescence channel of B pipe, which has Tm value,
Line then determines to contain dfrA17 in sample；If the 4th fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine
Contain dfrA15 in sample；If the 4th fluorescence channel of B pipe have Tm value be 69 DEG C of corresponding dissolution peak curves if be determined as in the positive
Quality Control is qualified；
If determining that sample has containing at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA6 in sample
There is Gram-negative bacteria aminoglycoside resistant；If in sample containing blaOXA-1, blaOXA-10, blaIMP-4,
At least one of blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSHV, blaVIM and blaCTX then determine sample
With Gram-negative bacteria penicillin cephalosporins drug resistance；If in sample containing in sull, sul3, sut1 and sat1 extremely
Few one kind then determines that sample has Gram-negative bacteria sulfamido drug resistance；If containing dfrA1, dfrA14, dfrA17 in sample
At least one of with dfrA15, then determine that sample has Gram-negative bacteria trimethoprim class drug resistance.
10. a kind of method for detecting Gram-negative bacteria and/or Gram-negative bacteria drug resistance, wherein this method comprises:
Using nucleic acid reagent described in any one of claim 4~6, PCR amplification is carried out to the DNA of sample to be tested；Progress described in
The PCR instrument of PCR amplification includes the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel；Described
One fluorescence channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are different, and respectively
It independently is FAM fluorescence channel, VIC fluorescence channel, CY5 fluorescence channel or ROX fluorescence channel；And carry out following differentiation:
If blank control, positive control and positive internal reference are set up, testing result is effective；
If the first fluorescence channel of A pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain escherichia coli in sample
Bacterium；If the first fluorescence channel of A pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain Klebsiella Pneumoniae in sample；
If the first fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain Acinetobacter bauamnnii in sample；If A
It is that 69 DEG C of corresponding dissolution peak curves then determine to contain pseudomonas aeruginosa in sample that the first fluorescence channel of pipe, which has Tm value,；If A is managed
It is that 58 DEG C of corresponding dissolution peak curves then determine to contain serratia marcescens in sample that second fluorescence channel, which has Tm value,；If A pipe
Two fluorescence channels have 62 DEG C of Tm value corresponding dissolution peak curves then to determine to contain enterobacter cloacae in sample；If the second fluorescence of A pipe
It is that 66 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-1 in sample that, which there is Tm value in channel,；If the second fluorescence channel of A pipe has
Tm value is that 70 DEG C of corresponding dissolution peak curves then determine to contain blaOXA-10 in sample；If A pipe third fluorescence channel has the Tm value to be
58 DEG C of corresponding dissolution peak curves then determine to contain blaIMP-4 in sample；If A pipe third fluorescence channel has 61 DEG C of correspondences of Tm value
Dissolution peak curve then determine to contain blaIMP-6 in sample；If it is 65 DEG C of corresponding dissolutions that A pipe third fluorescence channel, which has Tm value,
Peak curve then determines to contain blaIMP-8 in sample；If A pipe third fluorescence channel has 69 DEG C of Tm value corresponding dissolution peak curves
Determine to contain blaTEM in sample；If the 4th fluorescence channel of A pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine sample
In contain blaVEB；If the 4th fluorescence channel of A pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain in sample
blaSHV；If the 4th fluorescence channel of A pipe have Tm value be 65 DEG C of corresponding dissolution peak curves if determine to contain blaVIM in sample；If
It is that 69 DEG C of corresponding dissolution peak curves then determine to contain blaCTX in sample that the 4th fluorescence channel of A pipe, which has Tm value,；If A pipe the 4th is glimmering
It is that 69 DEG C of corresponding dissolution peak curves are then determined as in the positive that Quality Control is qualified that optical channel, which has Tm value,；
If the first fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain aadA1 in sample；If B is managed
First fluorescence channel has 61 DEG C of Tm value corresponding dissolution peak curves then to determine to contain aadA2 in sample；If the first fluorescence channel of B pipe
Having Tm value is that 65 DEG C of corresponding dissolution peak curves then determine to contain aadA3 in sample；If it is 69 that the first fluorescence channel of B pipe, which has Tm value,
DEG C corresponding dissolution peak curve then determines to contain aadA4 in sample；If it is 65 corresponding dissolutions that the second fluorescence channel of B pipe, which has Tm value,
Peak curve then determines to contain sull in sample；Determine if the second fluorescence channel of B pipe there are 69 DEG C of Tm value corresponding dissolution peak curves
Contain sul3 in sample；If the second fluorescence channel of B pipe have Tm value be 58 DEG C of corresponding dissolution peak curves if determine to contain in sample
aadA5；If the second fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine to contain aadA6 in sample；If B
It is that 58 DEG C of corresponding dissolution peak curves then determine to contain sut1 in sample that pipe third fluorescence channel, which has Tm value,；If B pipe third fluorescence
It is that 61 DEG C of corresponding dissolution peak curves then determine to contain sat1 in sample that, which there is Tm value in channel,；If B pipe third fluorescence channel has Tm value
Then determine to contain dfrA1 in sample for 65 DEG C of corresponding dissolution peak curves；If the 4th fluorescence channel of B pipe have 58 DEG C of Tm value it is corresponding
Dissolution peak curve then determines to contain dfrA14 in sample；If it is that 65 DEG C of corresponding dissolution peaks are bent that the 4th fluorescence channel of B pipe, which has Tm value,
Line then determines to contain dfrA17 in sample；If the 4th fluorescence channel of B pipe have Tm value be 61 DEG C of corresponding dissolution peak curves if determine
Contain dfrA15 in sample；If the 4th fluorescence channel of B pipe have Tm value be 69 DEG C of corresponding dissolution peak curves if be determined as in the positive
Quality Control is qualified；
If determining that sample has containing at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA6 in sample
There is Gram-negative bacteria aminoglycoside resistant；If in sample containing blaOXA-1, blaOXA-10, blaIMP-4,
At least one of blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSHV, blaVIM and blaCTX then determine sample
With Gram-negative bacteria penicillin cephalosporins drug resistance；If in sample containing in sull, sul3, sut1 and sat1 extremely
Few one kind then determines that sample has Gram-negative bacteria sulfamido drug resistance；If containing dfrA1, dfrA14, dfrA17 in sample
At least one of with dfrA15, then determine that sample has Gram-negative bacteria trimethoprim class drug resistance.
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| CN110438247A (en) * | 2019-08-20 | 2019-11-12 | 中国医学科学院北京协和医院 | For detecting the nucleic acid reagent, kit, system and method for Escherichia coli and Klebsiella pneumoniae and its toxicity and drug resistance |
| CN111349721A (en) * | 2020-03-19 | 2020-06-30 | 北京卓诚惠生生物科技股份有限公司 | Nucleic acid reagent, kit, system and method for detecting respiratory tract infection pathogen |
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